Codon-optimized nucleotide sequence encoding the AP-1 transcription factor

Abstract

Disclosed herein are polynucleotides comprising a nucleotide sequence encoding an AP-1 transcription factor (i.e., c-Jun). In some embodiments, the nucleotide sequence is codon optimized. In some embodiments, the polynucleotide comprises one or more additional nucleotide sequences encoding a linker, a signal peptide, an antigen-binding domain, a spacer, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, a truncated EGFR, and combinations thereof. Also disclosed herein are cells, vectors, and pharmaceutical compositions comprising such polynucleotides. Use of such polynucleotides, cells, vectors, and pharmaceutical compositions for treating a disease or disorder (e.g., cancer) is also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 153,879, filed February 25, 2021, No. 63 / 263,231, filed October 28, 2021, and No. 63 / 309,380, filed February 11, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] References to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (Name: 4385_068PC03_Seqlisting_ST25.txt, Size: 99,232 bytes, and Creation Date: February 22, 2022) are incorporated by reference in their entirety into this specification.

[0003] Field of the Disclosure The subject matter of the present disclosure generally relates to polynucleotides, polypeptides, cells, vectors, uses, and kits relating to codon-optimized nucleotide sequences encoding AP-1 transcription factors. [Background technology]

[0004] Cancer immunotherapy relies on utilizing T cells (the immune system's main killer cells of infected and diseased cells) to attack and kill tumor cells. However, the ability of immune cells to target and kill tumor cells is suppressed by the presence of various inhibitors of immune response present in the tumor microenvironment. Thus, although CAR T cells have had varying success in treating certain cancers (e.g., KYMRIAH™ (tisagenlecleucel, Novartis) and YESCARTA™ (axicabtageneciloleucel, Kite / Gilead) are FDA approved), problems remain. For example, the success of CAR T cell immunotherapy is often limited by the extent of CAR T proliferation in the recipient's body, which generally requires large amounts of cell infusion. Furthermore, exhaustion and loss of persistence of transplanted CAR T cells are observed, leading to reduced clinical efficacy and possible relapse.

[0005] Therefore, there remains a need for new and improved treatment options with acceptable safety profiles and high efficacy in cancer patients. Summary of the Invention

[0006] a) a nucleotide sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1; b) a nucleotide sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2; c) a nucleotide sequence having at least d) a nucleotide sequence having at least about 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:5; a nucleotide sequence having 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6; e) a nucleotide sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6; f) a nucleotide sequence having 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6; 7; g) a nucleotide sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8;h) a nucleotide sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:9; or i) a polynucleotide comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:10, wherein the nucleotide sequence encodes an AP-1 transcription factor.

[0007] Provided herein is a polynucleotide comprising a nucleotide sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1, wherein the nucleotide sequence encodes an AP-1 transcription factor. In certain embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO:1.

[0008] Also provided herein is a polynucleotide comprising a nucleotide sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2, wherein the nucleotide sequence encodes an AP-1 transcription factor. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO:2.

[0009] Provided herein is a polynucleotide comprising a nucleotide sequence having at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 4, wherein the nucleotide sequence encodes an AP-1 transcription factor. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 4.

[0010] The present disclosure further provides a polynucleotide comprising a nucleotide sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 5, wherein the nucleotide sequence encodes an AP-1 transcription factor. In certain embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO:5.

[0011] Provided herein is a polynucleotide comprising a nucleotide sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6, wherein the nucleotide sequence encodes an AP-1 transcription factor. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO:6.

[0012] Provided herein is a polynucleotide comprising a nucleotide sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7, wherein the nucleotide sequence encodes an AP-1 transcription factor. In certain embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 7.

[0013] Provided herein is a polynucleotide comprising a nucleotide sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:8, wherein the nucleotide sequence encodes an AP-1 transcription factor. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO:8.

[0014] Provided herein is a polynucleotide comprising a nucleotide sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 9, wherein the nucleotide sequence encodes an AP-1 transcription factor. In certain embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO:9.

[0015] Also provided herein is a polynucleotide comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10, wherein the nucleotide sequence encodes an AP-1 transcription factor. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 10.

[0016] In some embodiments, the AP-1 transcription factor encoded by any one of the above polynucleotides comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:13.

[0017] In some embodiments, the polynucleotide of the present disclosure further comprises a nucleotide sequence encoding a ligand-binding protein. In certain embodiments, the ligand-binding protein comprises a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric antibody-T cell receptor (caTCR), a chimeric signal transduction receptor (CSR), a T cell receptor mimic (TCR mimic), or a combination thereof. In some embodiments, the CAR is designed as a standard CAR, a split CAR, an off-switch CAR, an on-switch CAR, a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR.

[0018] In some embodiments, the ligand binding protein comprises an antigen binding domain, a transmembrane domain, a costimulatory domain, and / or an intracellular signaling domain. In certain embodiments, the antigen binding domain is selected from the group consisting of AFP (alpha-fetoprotein), αvβ6 or another integrin, BCMA, Braf, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (CC motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD79c, CD79d, CD79e, CD79f, CD79g, CD79h, CD79i, CD79i, CD79m ... D98, CD123, CD138, CD171, CD352, CEA (carcinoembryonic antigen), claudin 18.2, claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrin B2, EPHa2 (ephrin receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP-α (fibroblast growth factor receptor α) Blast activation protein alpha), fetal AchR (fetal acetylcholine receptor), FBP (folate binding protein), FCRL5, FR-α (folate receptor alpha), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gp100 (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G protein-coupled receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukocyte antigen A1), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight melanoma-associated antigen), IGF1R (insulin-like growth factor 1 receptor), Ig kappa, Ig lambda, IL-22Ra (IL-22 receptor alpha), IL-13Ra2 (IL-13 receptor alpha 2), KDR (kinase insert domain receptor), LI cell adhesion molecule (LI-CAM), Liv-1, LRRC8A (leucine-rich repeat containing 8 family member A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1 (melan A),Murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complexes (e.g., HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), nectin-4, NKG2D (natural killer group 2 member D) ligand, NY-ESO, oncofetal antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen in melanoma), progesterone receptor, PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), ROR1, ROR2, SIRPα (signal regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (six transmembrane epithelial antigen of the prostate 1), survivin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens derived from HIV, HBV, HCV, HPV, and other pathogens, and any combination thereof.

[0019] In some embodiments, the antigen binding domain specifically binds to ROR1. In certain embodiments, the antigen binding domain specifically binds to GPC2.

[0020] In some embodiments, the costimulatory domain of the ligand binding protein comprises an interleukin-2 receptor (IL-2R), an interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, or any combination thereof. In some embodiments, the costimulatory domain comprises a 4-1BB / CD137 costimulatory domain.

[0021] In some aspects, the transmembrane domain of the ligand binding protein described herein is selected from the group consisting of KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG The transmembrane domain region of AM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C, CD19, or any combination thereof. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0022] In some embodiments, the intracellular signaling domain of the ligand binding domain comprises an intracellular signaling domain region from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, CD32, DAP10, DAP12, or any combination thereof. In certain embodiments, the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain.

[0023] In some embodiments, the ligand binding domain encoded by the polynucleotide of the present disclosure comprises a TCR, and the TCR specifically binds to a tumor antigen / MHC complex. In certain embodiments, the tumor antigen is selected from the group consisting of AFP, CD19, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5 , HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-Al, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant (e.g., HRAS, KRAS, NRAS), hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, neoantigens, or any combination thereof.

[0024] In some embodiments, the AP-1 transcription factor is linked to the ligand binding protein by a linker. In certain embodiments, the linker comprises a cleavable linker. In some embodiments, the linker is a P2A linker, a T2A linker, an F2A linker, an E2A linker, a furin cleavage site, or any combination thereof. In certain embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 14-18. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0025] In some embodiments, the polynucleotides described herein further comprise a nucleic acid sequence encoding a truncated EGFR (EGFRt). In certain embodiments, the EGFRt comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the EGFRt comprises the amino acid sequence set forth in SEQ ID NO: 23 or 24.

[0026] In some embodiments, the EGFRt is linked to the AP-1 transcription factor and / or the chimeric binding protein by a linker. In certain embodiments, the linker comprises a cleavable linker. In some embodiments, the linker is a P2A linker, a T2A linker, an F2A linker, an E2A linker, a furin cleavage site, or any combination thereof. In certain embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 14-18. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0027] Provided herein is a vector comprising any of the polynucleotides and regulatory elements described herein. In some aspects, the vector is a polycistronic expression vector. In certain aspects, the regulatory element comprises a promoter. In some aspects, the promoter comprises a dl587rev primer binding site displacement (MND) promoter, an EF1a promoter, a ubiquitin promoter, or a combination thereof.

[0028] In some embodiments, the vector comprises a viral vector, a mammalian vector, or a bacterial vector.In certain embodiments, the vector is an adenovirus vector, a lentivirus, a Sendai virus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, a hybrid vector, or an adeno-associated virus (AAV) vector.In some embodiments, the vector is a lentivirus.

[0029] Also provided herein are compositions comprising the polynucleotides or vectors described herein. The disclosure also provides kits comprising the polynucleotides, vectors, or compositions described herein. Provided herein are polypeptides comprising an AP-1 transcription factor encoded by the polynucleotides, vectors, or compositions described herein. The disclosure further provides sets of polypeptides (e.g., AP-1 transcription factors), ligand binding proteins encoded by the polynucleotides of the disclosure, and / or polypeptides comprising the EGFRt described herein.

[0030] Also provided herein is a cell comprising the polynucleotide, vector, composition, polypeptide, or set of polypeptides described herein.In some embodiments, the cell comprises an immune cell.In certain embodiments, the cell comprises a T cell, a B cell, a regulatory T cell (Treg), a natural killer (NK) cell, a natural killer T (NKT) cell, a stem cell, or an induced pluripotent stem cell.

[0031] Provided herein is a pharmaceutical composition comprising a polynucleotide, vector, polypeptide, set of polypeptides, or cell described herein and a pharma- ceutically acceptable carrier.

[0032] In some embodiments, the cells or pharmaceutical compositions described herein are for treating a subject in need of treatment. In certain embodiments, the polynucleotides, vectors, compositions, polypeptides, sets of polypeptides, cells, or pharmaceutical compositions described herein are for reducing or preventing cell exhaustion useful in therapy.

[0033] Provided herein is the use of a cell or pharmaceutical composition described herein for the manufacture of a medicament in treating or preventing a disease or condition in a subject in need thereof. In some embodiments, the disease or condition is cancer.

[0034] Provided herein is the use of a polynucleotide, vector, composition, polypeptide, set of polypeptides, cell, or pharmaceutical composition described herein to prevent or reduce cellular exhaustion, useful in therapy.

[0035] In some aspects, the methods of treating or preventing a disease or condition in a subject in need thereof provided herein comprise administering to the subject any of the cells or pharmaceutical compositions disclosed herein. In certain aspects, the disease or condition is cancer.

[0036] Also provided herein is a method for increasing a c-Jun polypeptide in a cell, comprising modifying the cell to contain any of the polynucleotides, vectors, compositions, polypeptides or sets of polypeptides provided herein, wherein after modification, expression of a c-Jun polypeptide in the cell is increased compared to a corresponding cell modified with a wild-type c-Jun polynucleotide.

[0037] In some embodiments, expression of c-Jun polypeptide is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more as compared to a corresponding cell. In some embodiments, expression of c-Jun polypeptide is increased by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold or about 10-fold compared to the corresponding cell. [Brief description of the drawings]

[0038] [Figure 1] 1 shows a graphical representation of thirteen different anti-ROR1 CAR constructs described herein. The constructs comprise nucleotide sequences encoding one or more of: (i) c-Jun protein ("cJun"); (ii) truncated CD19 ("CD19t"); (iii) P2A linker ("P2A"); (iv) human Igκ signal peptide ("hIgK"); (v) anti-ROR1 scFv ("R12"); and (vi) truncated EGFR ("EGFRt"). Constructs #3-#12 comprise the codon-optimized c-Jun sequences set forth in SEQ ID NOs: 1-10, respectively. Construct #13 comprises the wild-type (i.e., non-codon-optimized) c-Jun sequence (SEQ ID NO: 11). [Figure 2A] Figure 1 shows the transduction efficiency of various anti-ROR1 constructs. Transduction efficiency is shown as a percentage of EGFRt+cJun+ cells. As indicated, each anti-ROR1 construct was used to transduce cells at the following multiplicity of infection (MOI): 20 (first bar of each construct), 10 (second bar of each construct), 5 (third bar of each construct), and 2 (last bar of each construct). The anti-ROR1 CAR constructs tested are shown in Figure 1 and included (i) construct #1 (i.e., lacking c-Jun); (ii) construct #11 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO: 9); (iii) construct #2 (i.e., lacking c-Jun and containing truncated CD19); (iv) construct #3 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO: 1); and (v) construct #12 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO: 10). [Figure 2B]Figure 1 shows the transduction efficiency of various anti-ROR1 constructs. Transduction efficiency is shown as a percentage of EGFRt+R12+ cells. As indicated, each anti-ROR1 construct was used to transduce cells at the following multiplicity of infection (MOI): 20 (first bar of each construct), 10 (second bar of each construct), 5 (third bar of each construct), and 2 (last bar of each construct). The anti-ROR1 CAR constructs tested are shown in Figure 1 and included (i) construct #1 (i.e., lacking c-Jun); (ii) construct #11 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO: 9); (iii) construct #2 (i.e., lacking c-Jun and containing truncated CD19); (iv) construct #3 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO: 1); and (v) construct #12 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO: 10). [Figure 2C] Figure 1 shows transgene expression levels for various anti-ROR1 constructs. Gene expression levels per cell are shown as the geometric mean (gMFI) of c-Jun expression among EGFRt+ cells. As indicated, each anti-ROR1 construct was used to transduce cells at the following multiplicity of infection (MOI): 20 (first bar for each construct), 10 (second bar for each construct), 5 (third bar for each construct), and 2 (last bar for each construct). The anti-ROR1 CAR constructs tested are shown in Figure 1 and included: (i) construct #1 (i.e., lacking c-Jun); (ii) construct #11 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:9); (iii) construct #2 (i.e., lacking c-Jun and containing a truncated CD19); (iv) construct #3 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:1); and (v) construct #12 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:10). [Figure 2D]Figure 1 shows transgene expression levels of various anti-ROR1 constructs. Gene expression levels per cell are shown as the geometric mean (gMFI) of anti-ROR1(R12)scFv expression among EGFRt+ cells. As indicated, each anti-ROR1 construct was used to transduce cells at the following multiplicity of infection (MOI): 20 (first bar of each construct), 10 (second bar of each construct), 5 (third bar of each construct), and 2 (last bar of each construct). Anti-ROR1 CAR constructs tested are shown in Figure 1. The anti-ROR1 CAR constructs tested are shown in Figure 1 and included (i) construct #1 (i.e., lacking c-Jun); (ii) construct #11 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:9); (iii) construct #2 (i.e., lacking c-Jun and containing a truncated CD19); (iv) construct #3 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:1); and (v) construct #12 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:10). [Figure 3A] Figure 1 shows the transduction efficiency of anti-ROR1 CAR constructs containing various codon-optimized c-Jun sequences. Transduction efficiency is shown as the percentage of EGFRt+R12CAR+ cells (y-axis) at various MOIs (x-axis). Results are shown for the following anti-ROR1 CAR constructs: (i) construct #3 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO: 1) (circles); (ii) construct #5 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO: 3) (squares); (iii) construct #4 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO: 2) (triangles); and (iv) construct #2 (i.e., lacking c-Jun and containing truncated CD19) (diamonds). Circled data points represent the titers used for comparison between the various constructs as shown in the table to the right of the graph. [Figure 3B]Figure 2 shows transgene expression levels of anti-ROR1 CAR constructs containing different codon-optimized c-Jun sequences. Transduction efficiency is shown as the percentage of EGFRt+R12CAR+ cells (y-axis) at different MOIs (x-axis). Results are shown for the following anti-ROR1 CAR constructs: (i) construct #3 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:1) (small circle); (ii) construct #6 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:4) (small square); (iii) construct #7 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:5) (triangle); (iv) construct #8 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:6) (inverted triangle); (v) construct #13 (i.e., containing the wild-type c-Jun sequence) (diamond); (vi) construct #9 (i.e., containing the codon-optimized c-Jun sequence set forth in SEQ ID NO:7) (large circle); and (vii) construct #2 (i.e., lacking c-Jun and containing truncated CD19) (large square). Circled data points represent the titers used for comparison between the various constructs as shown in the table to the right of the graph. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] overview The present disclosure relates to polynucleotides (e.g., isolated polynucleotides) comprising a nucleotide sequence encoding the Jun proto-oncogene, AP-1 transcription factor subunit ("c-Jun"; also referred to herein as "AP-1") protein. As described herein, the polynucleotides disclosed herein comprise one or more features that render the polynucleotide different (e.g., structurally and / or functionally) from a reference polynucleotide occurring in nature. For example, as further described elsewhere in this disclosure, the nucleotide sequence is codon-optimized (i.e., synthetic). In some aspects, a polynucleotide of the present disclosure (e.g., comprising a codon-optimized AP-1 nucleotide sequence) further comprises one or more additional nucleotide sequences encoding one or more of the following: a linker, a signal peptide, an antigen-binding domain, a spacer, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, a truncated EGFR, and combinations thereof.

[0040] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0041] It should be noted that an entity designated with the term "a" or "an" refers to one or more of that entity, e.g., "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, "a" (or "an"), as well as "one or more" and "at least one" may be used interchangeably in this disclosure. It should also be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate to the use of exclusive language, such as "only" or "only" in connection with the recitation of claim elements, or to the use of a negative limitation.

[0042] As used herein, the term "and / or" should be considered as a specific disclosure of each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (only), B (only), and C (only).

[0043] Whenever an embodiment is described herein using the term "comprising," it is understood that otherwise similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0044] As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest, unless otherwise specified or clear from the context (unless such number would be greater than 100% of the possible values), refers to a value that is similar to the stated reference value and within a range of values ​​that are 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less than) the stated reference value. When the term "approximately" or "about" is applied to a particular value herein, the value without the term "approximately" or "about" is also disclosed herein.

[0045] As described herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0046] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM", respectively.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary for many of the terms used in this disclosure.

[0048] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted format. Numerical ranges are intended to be inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of the disclosure, which can be had by reference to the specification in its entirety. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0049] As used herein, the term "immune cell" refers to a cell of the immune system. In some aspects, the immune cell is selected from a T lymphocyte ("T cell"), a B lymphocyte ("B cell"), a natural killer (NK) cell, a natural killer T (NKT) cell, a macrophage, an eosinophil, a mast cell, a dendritic cell, or a neutrophil. As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocyte produced or processed by the thymus. Non-limiting classes of T cells include effector T cells and Th cells (CD4 + or CD8 + In some embodiments, the immune cells are Th1 cells. In some embodiments, the immune cells are Th2 cells. In some embodiments, the immune cells are Tc17 cells. In some embodiments, the immune cells are Th17 cells. In some embodiments, the immune cells are tumor infiltrating cells (TILs). In some embodiments, the immune cells are T reg As used herein, "immune cell" also refers to a pluripotent cell that can differentiate into an immune cell, such as a stem cell (e.g., an embryonic stem cell or a hematopoietic stem cell) or an induced pluripotent stem cell.

[0050] In some embodiments, the T cells are memory T cells. As used herein, the term "memory" T cells refers to T cells that have previously encountered and responded to their cognate antigen (e.g., in vivo, in vitro, or ex vivo) or T cells that have been stimulated (e.g., in vitro or ex vivo) with, for example, an anti-CD3 antibody. Immune cells that have a "memory-like" phenotype upon secondary exposure, such memory T cells can replicate to mount a more rapid and potent immune response than during primary exposure. In some embodiments, memory T cells are referred to as central memory T cells (T CM cells), effector memory T cells (T EM cells), tissue-resident memory T cells (T RM cells), stem cell-like memory T cells (T SCM cells), or any combination thereof.

[0051] In some embodiments, the T cells are stem cell-like memory T cells. As used herein, "stem cell-like memory T cells," "T memory stem cells," or "T SCM The term "memory T cells" refers to memory T cells that express CD95, CD45RA, CCR7, and CD62L and are endowed with stem cell-like capacity to self-renew and pluripotency to reconstitute the entire range of memory and effector subsets.

[0052] In some embodiments, the T cells are central memory T cells. As used herein, "central memory T cells" or "T CM The term "central memory T cells" refers to memory T cells that express CD45RO, CCR7, and CD62L. Central memory T cells are generally found in lymph nodes and the peripheral circulation.

[0053] In some embodiments, the T cells are effector memory T cells. As used herein, "effector memory T cells" or "T EM The term "memory T cells" refers to memory T cells that express CD45RO but lack expression of CCR7 and CD62L. Because effector memory T cells lack lymph node homing receptors (e.g., CCR7 and CD62L), these cells are generally found in the peripheral circulation and non-lymphoid tissues.

[0054] In some embodiments, the T cells are tissue-resident memory T cells. As used herein, "tissue-resident memory T cells" or "T RM The term "memory T cells" refers to memory T cells that are not circulating and are resident in peripheral tissues such as the skin, lungs, and gastrointestinal tract. In certain aspects, tissue-resident memory T cells are also effector memory T cells.

[0055] In some embodiments, the T cells are naive T cells. As used herein, "naive T cells" or "T NThe term "T cell" refers to a T cell that expresses CD45RA, CCR7, and CD62L, but does not express CD95. N T cells are the most primitive cells of the T cell lineage. N Activated T cells are generated by interactions with antigen-presenting cells (APCs). EFF In some embodiments, T cells are differentiated into effector T (T eff ) cells.

[0056] As used herein, "cell engineering" refers to targeted modification of a cell, e.g., an immune cell, as disclosed herein. In some embodiments, cell engineering includes viral genetic engineering, non-viral genetic engineering, introduction of a receptor that allows tumor-specific targeting (e.g., a chimeric binding protein), introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve immune cell, e.g., T cell, function (e.g., a codon-optimized AP-1 nucleotide sequence provided herein), or any combination thereof.

[0057] As used herein, the term "cytokine" refers to small secreted proteins released by cells that have specific effects on cell-cell interactions and communication. Non-limiting examples of cytokines include interleukins (e.g., interleukin (IL)-1, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-10, IL-20, IL-14, IL-16, IL-17, IL-21, and IL-23), interferons (IFNs, e.g., IFNα, IFNβ, and IFNγ), tumor necrosis factor (TNF) family members, and transforming growth factor (TGF) family members. In certain aspects of the present disclosure, the interaction between TGFβ and the chimeric activating receptor disclosed herein in host immune cells leads to increased expression of one or more cytokines. In some aspects, the cytokine is an interleukin. In some embodiments, the cytokine is selected from IL-2, IL-7, IL-15, IL-21, and any combination thereof. In certain embodiments, the cytokine is IL-2. IL-2 (UniProtKB-P60568) is produced by T cells in response to antigenic or mitogenic stimulation. IL-2 is known to stimulate T cell proliferation and other activities important in controlling immune responses.

[0058] In some embodiments, the cytokine is an interferon. In some embodiments, the interferon is selected from IFNα, IFNβ, and IFNγ. In certain embodiments, the interferon is IFNγ. IFNγ (UniProtKB-P01579) is produced by activated lymphocytes to promote immune cell function.

[0059] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. As used herein, the term "alloantigen" refers to an antigen that is recognized by an immune cell (e.g., a T cell) and thereby induces immune cell activation (e.g., induces effector functions such as cytokine production and / or induces intracellular signals for cell proliferation). In certain embodiments, the antigen comprises a tumor antigen. In some embodiments, the antigen comprises a neoantigen.

[0060] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors, which may invade adjacent tissues and metastasize through the lymphatic system or bloodstream to distant parts of the body. As used herein, "cancer" refers to primary cancers, metastatic cancers, and recurrent cancers.

[0061] As used herein, the term "immune response" refers to a biological response in a vertebrate to foreign agents that protects the organism from these agents and the diseases that result from them. An immune response involves the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, NKT cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver that selectively target, bind to, damage, destroy, and / or eliminate from the vertebrate body an invading pathogen, a pathogen-infected cell or tissue, a cancer or other abnormal cell, or, in the case of autoimmunity or pathological inflammation, a normal human cell or tissue. Immune responses include, for example, T cells, e.g., effector T cells or Th cells, e.g., CD4 + Or CD8 +The activation or inhibition of T cells, etc., or the inhibition of Treg cells. As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocyte produced or processed by the thymus. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a NKT cell.

[0062] As used herein, the term "anti-tumor immune response" refers to an immune response against tumor antigens.

[0063] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein. As used herein, the phrase "subject in need thereof" includes subjects, such as mammalian subjects, who would benefit from administration of immune cells, e.g., T cells, as described herein, for example, to inhibit tumor growth.

[0064] The term "effective amount" or "effective dosage" refers to an amount of an agent (e.g., immune cells transduced with a polynucleotide comprising a nucleotide sequence encoding a chimeric binding protein and a codon-optimized c-Jun sequence) that produces a desired biological, therapeutic, and / or prophylactic result. The result can be a reduction, amelioration, alleviation, reduction, delay, and / or amelioration of one or more of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce the rate of tumor growth (e.g., inhibit tumor growth) or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor formation. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. Those skilled in the art will understand that a therapeutically effective amount, etc., may be administered in a single dose or may be achieved by administration of multiple doses (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses). The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of disease can be evaluated using a variety of methods known to the skilled practitioner, for example, in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0065] An effective amount of a composition (e.g., an immune cell as described herein) can, for example, (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, slow to some extent, or stop cancer cell invasion into peripheral organs, (iv) inhibit (i.e., slow to some extent or stop) tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay tumor onset and / or recurrence, and / or (vii) relieve to some extent one or more of the symptoms associated with cancer.

[0066] The terms "effective" and "efficacy" with respect to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the compositions disclosed herein (e.g., immune cells described herein) to promote cancer regression in patients. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organism level that result from administration of the compositions disclosed herein (e.g., immune cells described herein).

[0067] The terms "chimeric antigen receptor" and "CAR" as used herein refer to a set of polypeptides, generally two polypeptides in their simplest form, which, in the case of immune effector cells, provide the cell with specificity for a target cell, generally a cancer cell, and intracellular signal generation. In some embodiments, the CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below. In some embodiments, the set of polypeptides is on the same polypeptide chain, e.g., comprises a chimeric fusion protein. In some embodiments, the set of polypeptides is not adjacent to each other, e.g., is on a different polypeptide chain. In some embodiments, the set of polypeptides comprises a dimerization switch that allows the polypeptides to be linked to each other upon the presence of a dimerization molecule, e.g., allows the antigen binding domain to be linked to the intracellular signaling domain. In some embodiments, the stimulatory molecule of the CAR is a zeta chain associated with the T cell receptor complex (e.g., CD3 zeta). In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28.

[0068] In some embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule, the antigen binding domain and the transmembrane domain being linked by a CAR spacer. In some embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding domain linked to a transmembrane domain by a CAR spacer and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino terminus (N-terminus) of the CAR. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the antigen binding domain, which is optionally cleaved from the antigen binding domain (e.g., scFv) during cellular processes and localization of the CAR to the cell membrane.

[0069] Chimeric antigen receptors are distinguished from other antigen-binding agents by their ability to both bind MHC-independent antigens and transduce activating signals through their intracellular domains.

[0070] The antigen-specific extracellular domain of the chimeric antigen receptor recognizes and specifically binds to an antigen, typically a surface-expressed antigen of a malignant tumor. The antigen-specific extracellular domain has an affinity constant or affinity of interaction (K) of, for example, about 0.1 pM to about 10 μM, e.g., about 0.1 pM to about 1 μM or about 0.1 pM to about 100 nM. D ) specifically binds to an antigen. Methods for determining the affinity of the interaction are known in the art. The antigen-specific extracellular domain suitable for use in the CAR of the present disclosure can be any antigen-binding polypeptide, and a wide variety of polypeptides are known in the art. In some embodiments, the antigen-binding domain is a single chain Fv (scFv). Other antibody-based recognition domains, such as cAb VHH (camelid antibody variable domain) and humanized versions thereof, IgNAR VH (shark antibody variable domain) and humanized versions thereof, sdAb VH (single domain antibody variable domain), and "camelized" antibody variable domains, are also suitable for use in the CAR of the present disclosure.

[0071] In some embodiments, T cell receptor (TCR)-based recognition domains, such as single chain TCRs (scTvs, i.e., single chain two domain TCRs containing VαVβ), are also suitable for use in the chimeric binding proteins of the present disclosure.

[0072] The terms "nucleic acid", "nucleic acid molecule", "nucleotide", "nucleotide(s) sequence", and "polynucleotide" can be used interchangeably and refer to ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule") in phosphate polymeric form, either in single-stranded form or double-stranded helices, or any phosphate analogs thereof, such as phosphorothioates and thioesters. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The terms nucleic acid molecule, and particularly DNA or RNA molecule, refer only to the primary and secondary structure of the molecule and are not limited to any particular tertiary form. Thus, the term includes double-stranded DNA found in, among others, linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When describing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the usual convention of providing only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the present disclosure includes one or more nucleic acids described herein. As described herein, in some embodiments, a polynucleotide of the present disclosure may include a single nucleotide sequence (e.g., a codon-optimized c-Jun nucleotide sequence) that encodes a single protein ("monocistronic"). In some embodiments, a polynucleotide of the present disclosure is polycistronic (i.e., includes two or more cistrons). In certain embodiments, each of the cistrons of a polycistronic polynucleotide may encode a protein disclosed herein (e.g., an AP-1 transcription factor, a chimeric binding protein, or an EGFRt). In some embodiments, each of the cistrons may be translated independently of the others.

[0073] As used herein, the term "polypeptide", unless otherwise indicated, encompasses both peptides and proteins.

[0074] As used herein, a "coding region," "coding sequence," or "translatable sequence" is a portion of a polynucleotide that consists of codons translatable into amino acids. A "stop codon" (TAG, TGA, or TAA) is not normally translated into an amino acid but can be considered part of the coding region. However, all flanking sequences, e.g., promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of a coding region are generally determined by a start codon at the 5' end, which encodes the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end, which encodes the carboxy terminus of the resulting polypeptide.

[0075] The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each containing a nucleobase sequence) related to one another by Watson-Crick base pairing rules, or between an oligomer and a target gene. For example, the nucleobase sequence "TGA(5' to 3')" is complementary to the nucleobase sequence "ACT(3' to 5')". Complementarity may be "partial", in which fewer than all of the nucleobases of a given nucleobase sequence match another nucleobase sequence according to the base pairing rules. For example, in some embodiments, the complementarity between a given nucleobase sequence and another nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Thus, in certain aspects, the term "complementarity" refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity or complementarity with a target nucleic acid sequence (e.g., a c-Jun-encoding nucleic acid sequence). Alternatively, as examples, "perfect" or "complete" (100%) complementarity may exist between a given nucleobase sequence and another nucleobase sequence. In some aspects, the degree of complementarity between nucleobase sequences significantly affects the efficiency and strength of hybridization between the sequences.

[0076] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, e.g., AP-1 transcription factor. Expression includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA) and translation of mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcription product. Gene products as described herein further include nucleic acids having post-transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides having post-translational modifications, e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0077] As used herein, the term "identity" refers to the overall monomer conservation between polymer molecules, e.g., between polynucleotide molecules. The term "identical" without any additional modifiers, e.g., "polynucleotide A is identical to polynucleotide B," means that the polynucleotide sequences are 100% identical (100% sequence identity). For example, describing two sequences as "70% identical" is equivalent to describing them as having, e.g., "70% sequence identity." A "reference nucleotide sequence," as used herein as a comparison to a nucleotide sequence of the present disclosure, refers to a polynucleotide sequence that is essentially identical to the nucleotide sequence of the present disclosure, except that the sequence is not optimized. For example, in some embodiments, the reference nucleotide sequence comprises the wild-type JUN nucleic acid sequence set forth in SEQ ID NO:11.

[0078] The calculation of the percent identity (%) of two polypeptide or polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second polypeptide or polynucleotide sequences for optimal alignment, and non-identical sequences can be ignored for comparison). In certain embodiments, the length of the sequence aligned for comparison is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the full length of the reference sequence. The amino acids at corresponding amino acid positions, or bases in the case of polynucleotides, are then compared.

[0079] When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percent identity between two sequences can be accomplished using a mathematical algorithm.

[0080] Suitable software programs that can be used to align different sequences (e.g., polynucleotide sequences) are available from a variety of sources. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST package of programs available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm to perform a comparison between two sequences. BLASTN is used to compare nucleic acid sequences, whereas BLASTP is used to compare amino acid sequences. Other suitable programs include, for example, Needle, Stretcher, Water, or Matcher, which are part of the bioinformatics program package EMBOSS, also available from the European Bioinformatics Institute (EBI) at worldwideweb.ebi.ac.uk / Tools / psa.

[0081] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, and the like.

[0082] Different regions in a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own sequence identity percentage.Please note that the sequence identity percentage is rounded to the nearest tenth.For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2.Please also note that the length value is always an integer.

[0083] In certain embodiments, the percent identity (%ID) or identity of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID=100×(Y / Z), where Y is the number of amino acid residues (or nucleic acid bases) that are evaluated as perfect matches in an alignment of the first and second sequences (aligned by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is greater than the length of the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0084] Those skilled in the art will understand that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence comparisons performed solely by primary sequence data. It will also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., protein crystal structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available at worldwidewebtcoffee.org or, for example, from EBI. It will also be understood that the final alignments used to calculate percent sequence identity can be curated either automatically or manually.

[0085] As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof are used interchangeably and refer to a preparation of a desired composition of the disclosure, e.g., the state of an engineered cell expressing a codon-optimized polynucleotide or a protein encoded by a codon-optimized polynucleotide, that has undergone one or more purification processes. In some aspects, isolation or purification, as used herein, is a process of removing, partially removing (e.g., a fraction of), a composition of the disclosure, e.g., a codon-optimized polynucleotide of the disclosure, from a sample containing contaminants.

[0086] In some embodiments, the isolated composition has no detectable undesirable activity, or alternatively, the level or amount of undesirable activity is below an acceptable level or amount. In other embodiments, the isolated composition has an amount and / or concentration of the desired composition of the present disclosure that is above an acceptable amount and / or concentration and / or activity. In other embodiments, the isolated composition is enriched compared to the starting material from which the composition is obtained. This enrichment can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or more than 99.9999% compared to the starting material.

[0087] In some embodiments, the isolated preparation is substantially free of residual biological products. In some embodiments, the isolated preparation is 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, or at least about 90% free of any contaminating biological material. The residual biological products may include non-biological materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.

[0088] As used herein, the term "linked" refers to a first amino acid sequence or polynucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence may be directly linked or juxtaposed to the second amino acid or polynucleotide sequence, or alternatively, an intervening sequence may be covalently linked from the first sequence to the second sequence. The term "linked" does not only mean the fusion of the first polynucleotide sequence to the second polynucleotide sequence at the 5' or 3' end, but also includes the insertion of the entire first polynucleotide sequence (or the second polynucleotide sequence, respectively) into any two nucleotides in the second polynucleotide sequence (or the first polynucleotide sequence). The first polynucleotide sequence may be linked to the second polynucleotide sequence by a phosphodiester bond or a linker. The linker may be, for example, a polynucleotide.

[0089] "Administering" refers to the physical introduction of a therapeutic agent (e.g., a nucleic acid molecule described herein) into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration include intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, oral, rectal, topical, epithelial, mucosal, intranasal, vaginal, rectal, sublingual administration, and combinations thereof. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0090] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or biochemical manifestations associated with a disease.

[0091] As used herein, the term "optimized" with respect to a nucleotide sequence refers to a polynucleotide sequence that encodes a polypeptide and has been mutated to improve the properties of the polynucleotide sequence. In some embodiments, optimization is performed to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease binding of regulatory proteins such as general transcription factors, increase or decrease splicing, or increase the yield of the polypeptide produced by the polynucleotide sequence. Examples of modifications that can be made to a polynucleotide sequence to optimize the polynucleotide sequence include codon optimization, G / C content optimization, removal of repetitive sequences, removal of AT-rich elements, removal of cryptic splice sites, removal of cis-acting elements that repress transcription or translation, addition or removal of poly-T or poly-A sequences, addition of sequences near the transcription start site that increase transcription, such as Kozak consensus sequences, removal of sequences that would form stem-loop structures, removal of destabilizing sequences, and combinations of two or more thereof. Further disclosure related to such optimization is provided throughout this disclosure.

[0092] As used herein, the term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. A promoter may be derived in its entirety from a native gene, or may be composed of different elements derived from different promoters found in nature, or even may include synthetic DNA segments. Those skilled in the art will appreciate that different promoters can induce expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. A promoter that causes a gene to be expressed in most cell types most of the time is generally referred to as a "constitutive promoter." A promoter that causes a gene to be expressed in a particular cell type is generally referred to as a "cell-specific promoter" or "tissue-specific promoter." A promoter that causes a gene to be expressed at a particular stage of development or cell differentiation is generally referred to as a "development-specific promoter" or "cell differentiation-specific promoter." A promoter that is induced to express a gene following exposure or treatment of cells with a drug, biological molecule, chemical, ligand, light, etc. that induces the promoter is generally referred to as an "inducible promoter" or "regulatable promoter." It will be further recognized that in many cases the exact boundaries of regulatory sequences have not been completely defined, such that DNA fragments of different lengths may have the same promoter activity.

[0093] Various aspects of the disclosure are described in further detail in the following subsections.

[0094] c-Jun expression nucleotide sequence c-Jun (also referred to herein as "AP-1 transcription factor" or "AP-1") is an oncogenic transcription factor belonging to the activator protein-1 (AP-1) family. It interacts with various proteins (e.g., c-Fos) to form dimeric complexes that regulate various cell signaling pathways, including cell proliferation and tumor progression. Accordingly, increased c-Jun expression has been identified in certain cancers, and there has been great interest in developing c-Jun antagonists to treat such cancers. See, e.g., Brennan, A., et al., J Exp Clin Cancer Res 39(1):184 (Sep.2020).

[0095] In humans, the c-Jun protein is encoded by the JUN gene, which is located on chromosome 1 (nucleotides 58,780,791 to 58,784,047, minus-strand orientation, GenBank accession number NC_000001.11). Known synonyms for the JUN gene and its encoded protein include "Jun proto-oncogene," "AP-1 transcription factor subunit," "v-Jun avian sarcoma virus 17 oncogene homolog," "transcription factor AP-1," "Jun oncogene," "AP-1," "Jun activation domain binding protein," "p39," and "enhancer binding protein AP1." The wild-type human c-Jun protein sequence is 331 amino acids long. The amino acid and nucleic acid sequences of wild-type human c-Jun are shown in Tables 1 and 2, respectively. [Table 1] [Table 2-1] [Table 2-2]

[0096] Codon Optimization As described herein, the polynucleotide of the present disclosure comprises a nucleotide sequence encoding an AP-1 transcription factor, and the nucleotide sequence is codon-optimized. Thus, in some embodiments, the nucleotide sequence encoding the c-Jun protein described herein differs from that of the wild-type c-Jun nucleotide sequence (e.g., SEQ ID NO: 11).

[0097] In some embodiments, a nucleotide sequence encoding an AP-1 transcription factor has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-10. The nucleotide sequence encoding an AP-1 transcription factor comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 1-10.

[0098] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 1.

[0099] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 2.

[0100] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the nucleotide sequence encoding the AP-1 transcription factor described herein has at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 3.

[0101] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the nucleotide sequence has at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 4.

[0102] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 5.

[0103] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 80%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 6.

[0104] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 7.

[0105] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO:8.

[0106] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 9.

[0107] In some embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the nucleotide sequence encoding the AP-1 transcription factor has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 10.

[0108] Exemplary codon-optimized AP-1 nucleotide sequences are shown in Table 3 (below). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0109] The AP-1 nucleotide sequences disclosed herein may be codon-optimized using any method known in the art. For example, in certain embodiments, the codons of the AP-1 nucleotide sequences disclosed herein are optimized to modify (e.g., increase or decrease) one or more of the following parameters compared to the wild-type nucleotide sequence (e.g., SEQ ID NO: 11): (i) codon accommodation index (i.e., codon usage bias); (ii) guanine-cytosine (GC) nucleotide content; (iii) mRNA secondary structure and instability motifs; (iv) repetitive sequences (e.g., direct repeats, inverted repeats, diad repeats); (v) restriction enzyme recognition sites; or (vi) combinations thereof.

[0110] Without being bound to any one theory, in some embodiments, such codon optimization can increase expression of the protein (e.g., c-Jun) encoded by the nucleotide sequence. Thus, in some embodiments, the codon-optimized AP-1 nucleotide sequences of the present disclosure, when transfected, transduced, or otherwise introduced into a human cell, e.g., a human T cell, can increase expression of the encoded AP-1 transcription factor compared to the corresponding expression in a cell transfected with a wild-type nucleotide sequence (e.g., SEQ ID NO:11). In some embodiments, the expression of the AP-1 transcription factor is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, compared to the corresponding expression in a cell transfected, transduced, or otherwise genetically modified to express the wild-type nucleotide sequence (e.g., SEQ ID NO:11) (i.e., a "reference cell"). , at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more. In some embodiments, expression of a c-Jun polypeptide is increased by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold as compared to a reference cell.

[0111] In some embodiments, increased expression of AP-1 transcription factor may improve and / or enhance one or more properties of the transfected cells (e.g., immune cells, e.g., T cells, such as CD4+ and / or CD8+ T cells). Non-limiting examples of such properties include resistance to exhaustion (e.g., as indicated by reduced expression of exhaustion markers such as PD-1, CD39, TIM-3, and / or LAG-3; increased survival; and / or increased cytokine production), persistence / survival, increased expansion / proliferation, improved effector function (e.g., cytokine production upon antigen stimulation, lysis of cells expressing the target antigen, or both), or a combination thereof.

[0112] Useful assays for determining exhaustion, cell phenotype, persistence, cytotoxicity and / or killing, proliferation, cytokine production / release, and gene expression profiles are known in the art and include, for example, flow cytometry, intracellular cytokine staining (ICS), INCUCYTE® immune cell killing assay, Meso Scale Discovery (MSD) or similar assays, sustained antigen stimulation assays, bulk and single cell RNAseq (see, e.g., Fron Genet. 2020;11:220;2019 Bioinformatics 35:i436-445;2019 Annual Review of Biomed. Data Sci. 2:139-173), cytotoxicity / killing assays, ELISA, Western blot, and other standard molecular and cell biology methods, for example, as described herein or in, for example, Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, Inc., 1999-2021) or elsewhere.

[0113] In some embodiments, increasing expression of the AP-1 transcription factor increases the resistance of cells to exhaustion. In some embodiments, resistance to exhaustion is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more as compared to a reference cell (e.g., a corresponding cell that has not been modified to increase AP-1 expression).

[0114] In some embodiments, overexpression of AP-1 transcription factor can reduce exhaustion in exhausted cells.In certain embodiments, increasing expression of AP-1 transcription factor can reduce exhaustion by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to reference cells (e.g., corresponding exhausted cells that are not modified to increase AP-1 expression).

[0115] In some embodiments, increasing expression of the AP-1 transcription factor can, for example, increase the persistence / survival of cells when administered to a subject in vivo. In some embodiments, the persistence / viability of the cells is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more as compared to a reference cell (e.g., a corresponding cell that has not been modified to increase AP-1 expression).

[0116] In some embodiments, increasing expression of the AP-1 transcription factor can increase cell expansion / proliferation, for example, upon antigenic stimulation. In some embodiments, the expansion / proliferation of the cells is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more as compared to a reference cell (e.g., a corresponding cell that has not been modified to increase AP-1 expression).

[0117] In some aspects, increased expression of the AP-1 transcription factor can enhance cellular effector functions, for example, increased cytokine production, granzyme release, and / or cytotoxicity. In some embodiments, the effector function of a cell is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more as compared to a reference cell (e.g., a corresponding cell that has not been modified to increase AP-1 expression).

[0118] Overexpression of c-Jun in T cells helps maintain the active state of the cells, for example, by reducing or preventing T cell dysfunction (e.g., T cell exhaustion). The codon-optimized c-Jun described herein can be used to engineer immune cells, such as T cells, which then exhibit sustained potent cytotoxicity against desired target cells (e.g., targets of endogenous TCR or targets of chimeric binding proteins described herein). Compared to T cells that do not overexpress c-Jun, engineered T cells that overexpress the codon-optimized c-Jun disclosed herein show less signs of T cell exhaustion.

[0119] Codon Usage Bias Despite an ever-increasing knowledge of expression systems and recombinant DNA technology, there are significant obstacles to expressing a particular gene (e.g., foreign or synthetic) in a selected cell. For example, expression of a synthetic gene, even when linked to a strong promoter, often occurs with a much lower efficiency or rate than expected. The same is often true for an exogenous gene that is exogenous to a cell. In some embodiments, this lower than expected expression efficiency is due to the protein coding region of the gene having a codon usage pattern that does not resemble the codon usage pattern in the cell in which the protein is expressed.

[0120] Thus, in some embodiments, the codon-optimized AP-1 nucleotide sequence disclosed herein has an increased human codon adaptation index compared to the wild-type nucleotide sequence (e.g., SEQ ID NO: 11). As used herein, the term "codon adaptation index" refers to a measure of codon usage bias. The codon adaptation index (CAI) evaluates the deviation of a given protein-coding gene sequence relative to a reference set of genes (Sharp PM and Li WH, Nucleic Acids Res. 15(3): 1281-95 (1987)). CAI is calculated by taking the geometric mean of the weights associated with each codon over the entire length of the gene sequence (determined by codon).

number

[0121] For each amino acid, the weight of each of its codons in the CAI is calculated as the ratio between the observed frequency of the codon (fi) and the frequency of the synonymous codons (fj) for that amino acid.

number

[0122] In some embodiments, the codon-optimized AP-1 nucleotide sequences disclosed herein have a codon-optimized AP-1 nucleotide sequence that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81%), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about 0.87 (87%), at least about 0.89 (88%), at least about 0.90 (90%), at least about 0.91 (91%), at least about 0.92 (92%), at least about 0.93 (93%), at least about 0.94 (94%), at least about 0.95 (95%), at least about 0.96 (96%), at least about 0.97 (97%), at least about 0.98 (98%), at least about 0.99 (99%), at least about 100 (10 ... 0.95 (95%), at least about 0.96 (96%), at least about 0.97 (97%), at least about 0.98 (98%), or at least about 0.99 (99%).

[0123] G / C content optimization Human genes are highly heterogeneous in G / C content, with some genes having a G / C content as low as 20% and others as high as 95%. In general, genes that are G / C rich are more highly expressed. In fact, it has been shown that increasing the G / C content of a gene can lead to increased gene expression, primarily due to increased transcription and higher steady-state mRNA levels. See Kudla et al., PLoS Biol 4(6):e180 (2006). As used herein, the term "G / C content" (or guanine-cytosine content) or "percentage of G / C nucleotides" refers to the percentage of nitrogenous bases in a DNA molecule that are either guanine or cytosine. G / C content can be calculated using the following formula:

number

[0124] In some embodiments, the codon-optimized AP-1 nucleotide sequences disclosed herein contain a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in the wild-type nucleotide sequence (e.g., SEQ ID NO: 11). In some embodiments, the codon-optimized AP-1 nucleotide sequences described herein have a G / C content that is at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, or at least about 65%. In certain embodiments, the codon-optimized AP-1 nucleotide sequences disclosed herein have a G / C content of at least about 0.60 (60%) (e.g., SEQ ID NO: 1). In some embodiments, the codon-optimized AP-1 nucleotide sequences disclosed herein have a G / C content of at least about 0.61 (61%) (e.g., SEQ ID NO:2). In some embodiments, the codon-optimized AP-1 nucleotide sequences have a G / C content of at least about 0.59 (59%) (e.g., SEQ ID NO:3). In some embodiments, the codon-optimized AP-1 nucleotide sequences have a G / C content of at least about 0.52 (52%) (e.g., SEQ ID NO:4). In some embodiments, the codon-optimized AP-1 nucleotide sequences of the present disclosure have a G / C content of at least about 0.56 (56%) (e.g., SEQ ID NO:5). In some embodiments, the codon-optimized AP-1 nucleotide sequences described herein have a G / C content of at least about 0.59 (59%) (e.g., SEQ ID NO:6). In some embodiments, the codon-optimized AP-1 nucleotide sequences have a G / C content of at least about 0.61 (61%) (e.g., SEQ ID NO:7). In some embodiments, the codon-optimized AP-1 nucleotide sequence has a G / C content of at least about 0.63 (63%) (e.g., SEQ ID NO: 8). In some embodiments, the codon-optimized AP-1 nucleotide sequence has a G / C content of at least about 0.56 (56%) (e.g., SEQ ID NO: 9).In some embodiments, the codon-optimized AP-1 nucleotide sequence has a G / C content of at least about 0.62 (62%) (eg, SEQ ID NO:10).

[0125] Additional translatable sequences In some embodiments, the polynucleotides described herein (e.g., expression constructs comprising a nucleotide sequence encoding an AP-1 transcription factor) may further comprise one or more nucleotide sequences encoding additional proteins of interest. Thus, in some embodiments, the polynucleotides of the present disclosure can be used to express an AP-1 transcription factor in combination with any suitable protein of interest. Non-limiting examples of such additional translatable sequences are provided below.

[0126] Ligand-binding proteins In some embodiments, the polynucleotides provided herein (e.g., expression constructs comprising codon-optimized AP-1 nucleotide sequences) further comprise a nucleotide sequence encoding a ligand-binding protein. As used herein, the term "ligand-binding protein" refers to any protein capable of binding to a molecule of interest (i.e., a ligand) (e.g., an antigen or peptide / MHC complex expressed on a tumor cell). In certain embodiments, the ligand-binding protein is a chimeric binding protein. As used herein, the term "chimeric binding protein" refers to a protein capable of binding to one or more ligands (e.g., an antigen (e.g., comprising an antigen-binding moiety)) and created by the linkage of two or more polynucleotide sequences that originally encode separate proteins. Unless otherwise indicated, the terms may be used interchangeably in this disclosure.

[0127] Non-limiting examples of ligand binding proteins (e.g., chimeric binding proteins) include chimeric antigen receptors (CARs), T cell receptors (TCRs), chimeric antibody-T cell receptors (caTCRs), chimeric signal transducing receptors (CSRs), T cell receptor mimetics (TCR mimetics), and combinations thereof.

[0128] In some embodiments, the chimeric binding protein comprises a CAR.

[0129] In some embodiments, the CAR (e.g., may be expressed in combination with AP-1 transcription factor) is designed as a standard CAR. In a "standard CAR", different components (e.g., extracellular targeting domain, transmembrane domain, and intracellular signaling / activation domain) are linearly assembled into a single fusion protein. In some embodiments, the CAR is designed as a first generation CAR. A "first generation" CAR is composed of an extracellular binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains. All first generation CARs contain a CD3 zeta chain domain as the intracellular signaling domain. In some embodiments, the CAR is designed as a second generation CAR. A "second generation" CAR further contains a costimulatory domain (e.g., CD28 or 4-1BB). In some embodiments, the CAR is designed as a third generation CAR. A "third generation" CAR is similar to a second generation CAR except that it contains multiple costimulatory domains (e.g., CD28-4-1BB or CD28-OX40). In some embodiments, the CAR is designed as a fourth generation CAR. "Fourth generation" CARs (also known as TRUCK or armed CARs) further comprise additional factors that can further improve function. For example, in some embodiments, fourth generation CARs further comprise cytokines that can be released upon CAR signaling in the target tumor tissue. In certain embodiments, fourth generation CARs comprise one or more additional elements, such as homing and suicide genes, that may serve to further regulate the activity of the CAR. In some embodiments, the CAR is designed as a split CAR. In a "split CAR" system, one or more components of the CAR (e.g., an extracellular targeting domain, a transmembrane domain, and an intracellular signaling / activation domain) are split into two or more parts to depend on multiple inputs that promote the assembly of a complete functional receptor. In some embodiments, the CAR is designed as a switchable CAR. With a "switchable CAR," the CAR can be switched (e.g., transiently) on (on-switch CAR) or off (off-switch CAR) in the presence of a stimulus.Further examples of CARs that can be used in the present disclosure are described, for example, in US2020 / 0172879A1 and US2019 / 0183932A1, each of which is incorporated by reference in its entirety.

[0130] In some embodiments, the constructs herein encode engineered T cell receptors (TCRs) (also referred to in the art as "transgenic" TCRs). TCRs are molecules found on the surface of T cells and are involved in recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules. TCRs are heterodimers composed of two different protein chains. In some embodiments, the TCR consists of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively). In some embodiments, the TCR consists of a gamma and delta (γ / δ) chain (encoded by TRG and TRD, respectively). When the TCR binds to an antigen peptide presented by an MHC molecule (peptide / MHC), it signals the T lymphocyte to become activated. In some embodiments, the TCR is an engineered (transgenic) TCR. As used herein, the term "engineered TCR" or "engineered T cell receptor" refers to a T cell receptor (TCR) that has been isolated or engineered to specifically bind a major histocompatibility complex (MHC) / peptide target antigen with a desired affinity and introduced into a population of immune cells, e.g., T cells, NK cells, and / or TILs.

[0131] In some embodiments, the chimeric binding protein comprises a chimeric antibody-T cell receptor (caTCR). As used herein, a "chimeric antibody-T cell receptor" or "caTCR" comprises (i) an antibody portion that specifically binds to an antigen of interest and (ii) a T cell receptor module that can recruit at least one TCR-associated signaling molecule. In some embodiments, the antibody portion and the T cell receptor module are fused together. In some embodiments, the chimeric binding protein comprises a chimeric signaling receptor (CSR). A "chimeric signaling receptor" or "CSR" comprises a ligand binding domain that specifically binds to a target ligand and a costimulatory signaling domain that can provide a stimulatory signal to an immune cell that expresses the CSR. Non-limiting examples of caTCRs and CSRs are further described in US 10,822,413 B2, which is incorporated herein by reference in its entirety.

[0132] In some embodiments, the chimeric binding protein comprises a T cell receptor mimic (TCR mimic). As used herein, the term "T cell receptor mimic" or "TCR mimic" refers to an antibody (or a fragment thereof) that is engineered to recognize a tumor antigen, which is presented in the context of an HLA molecule. As will be apparent to those skilled in the art, these antibodies can mimic the specificity of a TCR. Non-limiting examples of TCR mimics are provided, for example, in US2009 / 0226474A1 and US2019 / 0092876A1, each of which is incorporated herein by reference in its entirety.

[0133] In some embodiments, the chimeric binding protein can be associated with a gene editing tool (e.g., a CRISPR-Cas system), and activation of the chimeric binding protein can induce activation of the gene editing tool such that expression and / or activity of one or more genes is regulated in the cell. For example, in some embodiments, a cell (e.g., a T cell) described herein is modified to include a chimeric binding protein (e.g., a CAR) linked to a protease and a single guide RNA that targets a regulatory region (e.g., a promoter) of a gene of interest. In some embodiments, the cell is further modified to include a T cell activation linker (LAT), e.g., complexed to the gene editing tool via a linker. Activation of the chimeric binding protein (e.g., by antigen stimulation) allows for release of the gene editing tool for nuclear localization and regulation of gene expression. Further embodiments of such chimeric binding proteins are presented elsewhere in this disclosure. See also Pietrobon et al., Int J Mol Sci 22(19):10828 (Oct. 2021), which is incorporated herein by reference in its entirety.

[0134] As described herein, the chimeric binding proteins useful in the present disclosure include an antigen-binding domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, or a combination thereof. In certain embodiments, the antigen-binding domain recognizes and specifically binds to an antigen. Non-limiting examples of antigens include AFP (alpha-fetoprotein), αvβ6 or another integrin, BCMA, Braf, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (CC motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD123, CD138, CD171, CD352, CEA (carcinoembryonic antigen), claudin 18.2, claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrin B2, EPHa2 (ephrin receptor A2), ERBB dimer, estrogen receptor, ETBR ( Endothelin B receptor), FAP-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (folate binding protein), FCRL5, FR-α (folate receptor alpha), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gp100 (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G protein-coupled receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA -A1 (human leukocyte antigen A1), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight melanoma-associated antigen), IGF1R (insulin-like growth factor 1 receptor), Ig kappa, Ig lambda, IL-22Ra (IL-22 receptor alpha), IL-13Ra2 (IL-13 receptor alpha 2), KDR (kinase insert domain receptor), LI cell adhesion molecule (LI-CAM), Liv-1, LRRC8A (leucine-rich repeat containing 8 family member A), Lewis Y,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1 (Melan-A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complex (e.g., AFP, KRAS, HPV (e.g., HPV E6 or E7), NY-ESO, MAGE-A, and HLA-A complexed with peptides derived from WT1), NCAM (neural cell adhesion molecule), Nectin-4, NKG2D (natural killer group 2 member D) ligand, NY-ESO, oncofetal antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen in melanoma), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), ROR1, ROR2, SIRPα (signal regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (six transmembrane epithelial antigen 1 of the prostate), survivin, TAG72 (tumor associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens derived from HIV, HBV, HCV, HPV, and other pathogens, or combinations thereof. In some embodiments, the antigen binding domain of the chimeric binding protein described herein specifically binds to ROR1. In some embodiments, the antigen binding domain of the chimeric binding protein specifically binds to GPC2. In some embodiments, the antigen binding domain of the chimeric binding protein specifically binds to a tumor antigen, the tumor antigen being selected from the group consisting of alpha fetoprotein (AFP), CD19, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR,NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-Al, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, surviving, telomerase, PCTA-1 / galectin 8, MelanA / MARTl, Ras mutant (e.g., HRAS, KRAS, NRAS), hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, neoantigens, or any combination thereof.

[0135] As further described elsewhere in this disclosure, the antigen-binding domain of the chimeric binding protein may be any polypeptide capable of binding to one or more antigens. In some embodiments, the antigen-binding domain comprises or is derived from an antigen-binding region derived from an antibody capable of specifically binding to any of the following: Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single chain variable fragment (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, intrabody, disulfide stabilized Fv protein (dsFv), unibody, nanobody, and a protein of interest, ligand, receptor, receptor fragment, peptide aptamer, or combinations thereof. In some embodiments, the antigen-binding domain is a single chain Fv (scFv).

[0136] In some embodiments, the chimeric binding proteins described herein comprise an intracellular signaling domain that transmits an effector function signal upon binding of the extracellular domain to an antigen, allowing the cell expressing the chimeric binding protein (e.g., a T cell) to perform a specialized function. Non-limiting examples of intracellular signaling domains include intracellular signaling domain regions from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, CD32, DAP10, DAP12, or any combination thereof. In certain embodiments, the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain. In some embodiments, the chimeric binding protein comprises the entire intracellular domain of a protein disclosed herein. In some embodiments, the intracellular domain is truncated. A truncated portion of the intracellular domain can be used in place of the entire chain so long as it still transmits the effector function signal, and thus the term intracellular domain is meant to include any truncated portion of the intracellular domain that is sufficient to transmit the effector function signal.

[0137] In some embodiments, the chimeric binding protein that may be encoded by the polynucleotides described herein (e.g., including codon-optimized AP-1 nucleotide sequences) further comprises a transmembrane domain. In certain embodiments, the antigen binding domain is linked to the intracellular domain of the chimeric binding protein by a transmembrane domain. In some embodiments, the antigen binding domain is linked to the transmembrane domain of the chimeric binding protein (e.g., CAR) by a linker. In certain embodiments, the inclusion of a linker between the antigen binding domain and the transmembrane domain can affect the flexibility of the antigen binding domain, thereby improving chimeric binding protein function.

[0138] Any transmembrane domain known in the art can be used in the chimeric binding proteins (e.g., CARs) described herein. In some embodiments, the transmembrane domain can be artificial (e.g., an engineered transmembrane domain). In some embodiments, the transmembrane domain is derived from a naturally occurring polypeptide. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a naturally occurring polypeptide. Non-limiting examples of transmembrane domains include KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG Examples of the transmembrane domain include AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C, the transmembrane domain region of CD19, or any combination thereof. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0139] As described herein, in some embodiments, the chimeric binding protein (e.g., CAR) comprises one or more costimulatory domains (e.g., second and third generation CARs). Without being bound by any theory, these costimulatory domains can further enhance proliferation, activation, memory, persistence, and / or effector function of cells engineered to express the chimeric binding protein (e.g., in combination with an AP-1 transcription factor). In some embodiments, the transmembrane domain is fused to a costimulatory domain, and optionally, the costimulatory domain is fused to a second costimulatory domain, and the costimulatory domain is fused to a signaling domain, including but not limited to CD3ζ. Non-limiting examples of costimulatory domains include interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, or any combination thereof. In certain embodiments, the costimulatory domain comprises a 4-1BB / CD137 costimulatory domain.

[0140] Linker In some aspects, the polynucleotides described herein (e.g., including codon-optimized AP-1 nucleotide sequences) further comprise a nucleotide sequence encoding a linker. The linker may be between any of the various components of the polynucleotides provided herein. For example, in certain aspects, the polynucleotide comprises a first nucleotide sequence encoding an AP-1 transcription factor, a second nucleotide sequence encoding a linker, and a third nucleotide sequence encoding a chimeric binding protein (e.g., a CAR), the second nucleotide sequence being between the first and third nucleotide sequences such that the AP-1 transcription factor is linked to the chimeric binding protein by the linker. In some aspects, the polynucleotides of the present disclosure may comprise multiple nucleotide sequences (e.g., at least two separate nucleotide sequences) encoding linkers. In certain aspects, the multiple linkers are the same. In some aspects, the multiple linkers are different.

[0141] In certain embodiments, the linker is a peptide linker. In some embodiments, the linker comprises at least about 1 amino acid, at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, or at least about 30 amino acids. In some embodiments, the linker is rich in glycine (e.g., for flexibility). In some embodiments, the linker comprises serine and / or threonine (e.g., for solubility). In some embodiments, the linker is a Gly / Ser linker.

[0142] In some aspects, the linker is a non-cleavable linker such that the linker and various components of the polynucleotides provided herein (e.g., AP-1 transcription factor and chimeric binding protein) are expressed as a single polypeptide. In certain aspects, the linker is a cleavable linker. As used herein, the term "cleavable linker" refers to a linker that includes a cleavage site such that it can be selectively cleaved to produce two or more products when expressed. In some aspects, the linker is selected from a P2A linker, a T2A linker, an F2A linker, an E2A linker, a Furin cleavage site, or any combination thereof (see Table 4 below). In some aspects, the linker further comprises a GSG linker sequence. In some aspects, the linker useful in the present disclosure includes an internal ribosome entry site (IRES) such that separate polypeptides encoded by the first and second genes are produced upon translation. Further description of linkers that may be used in the present disclosure is provided, for example, in WO2020 / 223625A1 and US2019 / 0276801A1, each of which is incorporated by reference in its entirety. [Table 4]

[0143] In some embodiments, the linker comprises a P2A linker. In some embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0144] In some embodiments, the linker comprises a T2A linker. In some embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0145] In some embodiments, the linker comprises an F2A linker. In some embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0146] In some embodiments, the linker comprises an E2A linker. In some embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0147] In some embodiments, the linker comprises an amino acid sequence that includes a furin cleavage site. In some embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0148] Truncated EGFR In some aspects, a polynucleotide of the disclosure (e.g., comprising a codon-optimized AP-1 nucleotide sequence) comprises an additional nucleotide sequence encoding a truncated epidermal growth factor receptor (EGFRt), such that the EGFRt comprises only a partial sequence of the full-length EGFR protein (e.g., SEQ ID NO: 19). In some aspects, the EGFRt comprises EGFR extracellular domains III and IV and the EGFR transmembrane domain, but lacks EGFR extracellular domains I and II and the EGFR intracellular sequence.

[0149] EGFR is a 180 kDa monomeric glycoprotein that contains a large extracellular region, a single transmembrane domain, an intracellular juxtamembrane region, a tyrosine kinase domain, and a C-terminal regulatory region. The extracellular region contains four domains, domains I and III are homologous ligand binding domains, and domains II and IV are cysteine-rich domains (Ferguson, Annu Rev Biophys. (2008) 37:353-3). Unless otherwise indicated, EGFR as used herein refers to human EGFR. Due to alternative splicing, there are at least four known isoforms of human EGFR. The sequences for the various EGFR isoforms are shown in Table 5 (below). [Table 5-1] [Table 5-2]

[0150] In the canonical sequence of EGFR above (i.e., isoform 1), the various EGFR domains are described as follows: the signal peptide spans amino acids 1-24; the extracellular sequence spans amino acids 25-645, with domain I, domain II, domain III, and domain IV spanning amino acids 25-188, 189-333, 334-504, and 505-645, respectively; the transmembrane domain spans amino acids 646-668; the intracellular domain spans amino acids 669-1,210, the juxtamembrane domain spans amino acids 669-703, and the tyrosine kinase domain spans amino acids 704-1,210.

[0151] In some embodiments, EGFRt useful in the present disclosure comprises an amino acid sequence having at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:19.

[0152] In some embodiments, the EGFRt that may be used in the present disclosure comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the EGFRt comprises an amino acid sequence set forth in SEQ ID NO: 23 (see Table 6). In some embodiments, the EGFRt that may be used in the present disclosure comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the EGFRt comprises an amino acid sequence set forth in SEQ ID NO: 24 (see Table 6). [Table 6]

[0153] In some embodiments, the EGFRt described herein further comprises a juxtamembrane domain.As used herein, the term "juxtamembrane domain" refers to the intracellular portion of a cell surface protein (e.g., EGFR) that is immediately C-terminal to the transmembrane domain.Without being bound by any theory, in some embodiments, the addition of the juxtamembrane domain can increase the expression of the protein encoded by the polynucleotide of the present disclosure.

[0154] In some embodiments, the juxtamembrane domain may be about 1 to about 20 (e.g., 2-20, 3-20, 4-20, 5-20, 2-18, 3-18, 4-18, or 5-18) amino acids long. In certain embodiments, the juxtamembrane domain may be longer than 20 amino acids. In some embodiments, the first one or more (e.g., the first 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids of the juxtamembrane domain are net neutral or net positive charged sequences (e.g., the number of arginine and lysine residues is equal to or greater than the number of aspartic acid and glutamic acid residues). In certain embodiments, these first amino acids comprise more than about 30% (e.g., more than 40, 50, 60, 70, 80, or 90%) hydrophilic amino acids. Non-limiting examples of juxtamembrane domains useful in the present disclosure are shown in Table 7 (below). [Table 7]

[0155] In some aspects, juxtamembrane domains that may be used in the present disclosure may be derived from the juxtamembrane region of a naturally occurring cell surface protein, e.g., the juxtamembrane region (e.g., the entire or partial sequence of the first 20 juxtamembrane amino acids) of a human receptor tyrosine kinase that interacts with phosphatidylcholine (PC), phosphatidylserine (PS), or phosphatidylinositol-4,5-bisphosphate (PIP2) (see, e.g., Hedger et al., Sci Rep. (2015) 5:9198). Non-limiting examples of receptor tyrosine kinases include ERBB1 (EGFR), ERBB2 (HER2), ERBB3 (HER3), ERBB4 (HER4), INSR, IGF1R, INSRR, PGFRA, PGFRB, KIT, CSF1R, FLT3, VGFR1, VGFR2, VGFR3, FGFR1, FGFR2, FGFR3, FGFR4, PTK7, NTRK1, NTRK2, NTRK3, ROR1, ROR2, ROR3, ROR4, ROR5, ROR6, ROR7, ROR8, ROR9, ROR11, ROR12, ROR13, ROR14, ROR15, ROR16, ROR17, ROR18, ROR19, ROR21, ROR22, ROR23, ROR24, ROR25, ROR36, ROR37, ROR48, ROR49, ROR50, ROR51, ROR52, ROR53, ROR54, ROR55, ROR56, ROR57, ROR58, ROR59, ROR61, ROR62, ROR73, ROR74, ROR75, ROR76, ROR77, ROR78, ROR79, ROR81, ROR95, ROR96, ROR111, ROR122, ROR133, ROR140, ROR151, ROR162, ROR173, ROR184, ROR195, ROR196, ROR2197, ROR225, ROR234, ROR245, ROR255, ROR366, ROR377, ROR40, ROR41, ROR42, ROR43, ROR44, ROR55, ROR56, ROR57, ROR 2, MUSK, MET, RON, UFO, TYRO3, MERTK, TIE1, TIE2, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHAA, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, RET, RYK, DDR1, DDR2, ROS1, LMTK1, LMTK2, LMTK3, LTK, ALK, and STYK1. In certain aspects, the juxtamembrane domain may contain one or more mutations (e.g., substitutions or deletions) that remove residues known to be phosphorylated to avoid any unintended signaling capabilities of the proteins encoded by the polynucleotides of the present disclosure.

[0156] In some embodiments, the juxtamembrane domain is derived from the juxtamembrane region of EGFR. Non-limiting examples of juxtamembrane domains from EGFR include one of the sequences shown in Table 8 (below). In certain embodiments, the juxtamembrane domain comprises the amino acid sequence RRR. In certain embodiments, an EGFRt comprising such a juxtamembrane domain comprises the sequence set forth in SEQ ID NO:22. [Table 8]

[0157] As is evident from the present disclosure, the inclusion of a nucleotide sequence encoding EGFRt provides certain advantages to the polynucleotides of the present disclosure (e.g., including a codon-optimized c-Jun nucleotide sequence and a nucleotide sequence encoding a chimeric binding protein). For example, in some embodiments, EGFRt may function as a kill switch. When the engineered cells (e.g., CAR T cells transduced with a polynucleotide described herein) are no longer needed in the body, a pharmaceutical grade anti-EGFR antibody, such as cetuximab, panitumumab, nimotuzumab, or necitumumab, can be administered to the subject that received the engineered cells, thereby removing the engineered cells, for example, by antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular phagocytosis (ADCP).

[0158] In some aspects, the polynucleotide of the present disclosure comprises a first nucleotide sequence encoding an AP-1 transcription factor, a second nucleotide sequence encoding a chimeric binding protein (e.g., CAR), and a third nucleotide sequence encoding EGFRt. In certain aspects, the polynucleotide further comprises a fourth nucleotide sequence encoding a first linker, the fourth nucleotide sequence being between the first nucleotide sequence and the second nucleotide sequence, such that the AP-1 transcription factor is linked to the chimeric binding protein by the first linker. In certain aspects, the polynucleotide further comprises a fifth nucleotide sequence encoding a second linker, the fifth nucleotide sequence being between the second nucleotide sequence and the third nucleotide sequence, such that the chimeric binding protein is linked to the EGFRt by the second linker. In some aspects, the first linker and the second linker are the same. In certain aspects, the first linker and the second linker are different. Any linker described herein (e.g., P2A linker) can be used as the second linker.

[0159] Spacer In some aspects, a polynucleotide of the disclosure (e.g., including a codon-optimized AP-1 nucleotide sequence) comprises a nucleotide sequence encoding a spacer. As used herein, the term "spacer" refers to a polypeptide sequence that can covalently link two spaced apart moieties together (e.g., a P2A linker and a chimeric binding protein).

[0160] In certain embodiments, the spacer is derived from an immunoglobulin (e.g., from a hinge or loop region). In some embodiments, the spacer comprises an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge region, a fragment thereof (alone or capped with additional sequences, e.g., CH1 or CH2 region sequences), or a combination of fragments of an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge region (also referred to herein as "spacers derived from hinge regions"). In some embodiments, the spacer comprises an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM constant domain loop region, fragments thereof (alone or capped with additional sequence, e.g., from an adjacent β-strand), or combinations of fragments of IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM loop regions (also referred to herein as "loop region derived spacers"). In certain embodiments, the spacer comprises a hinge region derived spacer, a loop region derived spacer, or both (e.g., two or more linked hinge region derived spacers and loop region derived spacers).

[0161] Thus, in some aspects, a polynucleotide of the disclosure encodes a polypeptide that includes: (i) an AP-1 transcription factor, (ii) a first linker (e.g., a P2A linker), (iii) a signal peptide (e.g., hIgκ), (iv) an antigen binding domain (e.g., an scFv), (v) a second linker (e.g., GGGSG; SEQ ID NO:40), (vi) a spacer (e.g., a spacer derived from an IgG2 hinge), (vii) a transmembrane domain (e.g., CD28), (viii) a costimulatory domain (e.g., 4-1BB), (ix) an intracellular signaling domain (e.g., CD3ζ), (x) a third linker (e.g., a P2A linker), and (xi) an EGFRt.

[0162] In some embodiments, spacers useful in the present disclosure include human IgA1 (Uniprot: P01876, IGHA1_HUMAN, immunoglobulin heavy constant alpha 1, SEQ ID NO: 41), human IgA2 (Uniprot P01877, IGHA2_HUMAN, immunoglobulin heavy constant alpha 2, SEQ ID NO: 42), mouse IgG2A (Uniprot P01665, GCAM_MOUSE, immunoglobulin gamma 2 A chain C region, SEQ ID NO: 43), human IgG1 (Uniprot P01857, IGHG1_HUMAN, immunoglobulin heavy constant gamma 1, SEQ ID NO: 44), human IgG2 (Uniprot P01859, IGHG2_HUMAN, immunoglobulin heavy constant gamma 2, SEQ ID NO: 45), human IgG3 (Uniprot P01860, IGHG3_HUMAN, immunoglobulin heavy constant gamma 3, SEQ ID NO: 46), human IgG4 (Uniprot P01861, IGHG4, immunoglobulin heavy constant gamma 4, SEQ ID NO: 47), human IgD (Uniprot P01880, IGHD_HUMAN, immunoglobulin heavy constant delta, SEQ ID NO: 48), human IgE (Uniprot P01854, IGHE_HUMAN, immunoglobulin heavy constant epsilon, SEQ ID NO: 49), or IgM (Uniprot P01871, IGHM_HUMAN, immunoglobulin heavy constant mu, SEQ ID NO: 50), wherein the subsequence comprises a CH1-CH2 hinge region or a portion thereof. In some embodiments, the subsequence further comprises a contiguous portion of the CH1 and / or CH2 constant domains.

[0163] In some embodiments, the spacer is selected from the group consisting of human IgA1 (Uniprot: P01876, IGHA1_HUMAN, immunoglobulin heavy constant alpha 1, SEQ ID NO: 41), human IgA2 (Uniprot P01877, IGHA2_HUMAN, immunoglobulin heavy constant alpha 2, SEQ ID NO: 42), mouse IgG2A (Uniprot P01665, GCAM_MOUSE, immunoglobulin gamma 2 A chain C region, SEQ ID NO: 43), human IgG1 (Uniprot P01857, IGHG1_HUMAN, immunoglobulin heavy constant gamma 1, SEQ ID NO: 44), human IgG2 (Uniprot P01859, IGHG2_HUMAN, immunoglobulin heavy constant gamma 2, SEQ ID NO: 45), human IgG3 (Uniprot P01860, IGHG3_HUMAN, immunoglobulin heavy constant gamma 3, SEQ ID NO: 46), human IgG4 (Uniprot P01861, IGHG4, immunoglobulin heavy constant gamma 4, SEQ ID NO: 47), human IgD (Uniprot P01880, IGHD_HUMAN, immunoglobulin heavy constant delta, SEQ ID NO: 48), human IgE (Uniprot P01854, IGHE_HUMAN, immunoglobulin heavy constant epsilon, SEQ ID NO: 49), or IgM (Uniprot P01871, IGHM_HUMAN, immunoglobulin heavy constant mu, SEQ ID NO: 50), wherein the subsequence comprises a loop region of the constant domain or a portion thereof. In some embodiments, the subsequence further comprises a contiguous portion of a beta chain.

[0164] In some embodiments, the spacer useful in the present disclosure is derived from IgG, e.g., IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the spacer is derived from an IgG2 hinge. In some embodiments, the spacer derived from an IgG2 hinge comprises at least 5, 6, or 7 consecutive amino acids of SEQ ID NO:51 (KPCPPCKCP). In some embodiments, the spacer comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence set forth in SEQ ID NO:51 (KPCPPCKCP). In certain embodiments, the spacer comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO:51 (KPCPPCKCP). In another embodiment, the spacer comprises the sequence set forth in SEQ ID NO:51 (KPCPPCKCP), except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the amino acid substitutions include at least one non-conservative amino acid substitution.

[0165] In some embodiments, a spacer of the present disclosure is comprised of the sequence set forth in SEQ ID NO:51, where the spacer sequence further comprises an optional flexible linker (e.g., a GGGSG (SEQ ID NO:40) linker). Thus, in some embodiments, a spacer of the present disclosure comprises a spacer sequence (e.g., SEQ ID NO:51) and an optional C-terminal or N-terminal flexible linker. In some embodiments, any of the non-required flexible linkers disclosed herein (e.g., gly / ser-rich linkers) may be added to the C-terminus and / or N-terminus of the spacer.

[0166] Signal peptide As described herein, in some aspects, a polynucleotide of the disclosure (e.g., including a codon-optimized AP-1 nucleotide sequence) also includes a nucleotide sequence encoding a signal peptide that can facilitate cell surface expression of the encoded protein, which can then be subsequently cleaved from the mature protein.

[0167] Any suitable signal peptide known in the art can be used in the present disclosure. Non-limiting examples of signal peptides are shown in Table 9 (below). In certain embodiments, the signal peptide is derived from human Ig kappa. In some embodiments, the signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 54 (MVLQTQVFISLLLWISGAYG). In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 54 (MVLQTQVFISLLLWISGAYG). In some embodiments, the signal peptide is derived from GM-CSF. In certain embodiments, such a signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53 (MLLLVTSLLLCELPHPAFLLIP). In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 53 (MLLLVTSLLLCELPHPAFLLIP). [Table 9]

[0168] In some embodiments, the polynucleotides described herein comprise a single signal peptide (e.g., SEQ ID NO: 53 or 54). In some embodiments, the polynucleotides comprise multiple signal peptides (e.g., at least 2, 3, 4 or more). Thus, in some aspects, a polynucleotide of the disclosure encodes a polypeptide that includes: (i) an AP-1 transcription factor, (ii) a first linker (e.g., a P2A linker), (iii) a first signal peptide (e.g., hIgκ), (iv) an antigen binding domain (e.g., an scFv), (v) a second linker (e.g., GGGSG; SEQ ID NO: 40), (vi) a spacer (e.g., a spacer derived from an IgG2 hinge), (vii) a transmembrane domain (e.g., CD28), (viii) a costimulatory domain (e.g., 4-1BB), (ix) an intracellular signaling domain (e.g., CD3ζ), (x) a third linker (e.g., a P2A linker), and (xi) a second signal peptide (e.g., GM-CSF), and (xii) EGFRt.

[0169] vector In some aspects, provided herein are vectors (e.g., expression vectors) that include a polynucleotide described herein (e.g., including a nucleotide sequence encoding an AP-1 transcription factor, e.g., a codon-optimized nucleotide sequence). In some aspects, the vectors described herein include a plurality (e.g., two, three, or four or more) of polynucleotides, each of which encodes a protein described herein (e.g., an AP-1 transcription factor, a ligand binding protein (e.g., a chimeric binding protein, e.g., CAR), or EGFRt). Thus, in certain aspects, the vector comprises a polycistronic vector (e.g., a bicistronic or tricistronic vector). In certain aspects, the polynucleotides described herein are included on the same vector (e.g., a multicistronic expression vector). In certain aspects, the polynucleotides encoding the proteins described herein (e.g., an AP-1 transcription factor, a ligand binding protein (e.g., a chimeric binding protein, e.g., CAR), or EGFRt) are provided on one or more separate vectors.

[0170] As described herein, such vectors are useful for recombinant expression in host cells and cells targeted for therapeutic intervention. The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked, or an element that contains such a nucleic acid molecule capable of transporting another nucleic acid. In some embodiments, a vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In some embodiments, a vector is a viral vector, and additional DNA segments can be ligated into the viral genome. Certain vectors, or polynucleotides that are part of a vector, are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Additionally, certain vectors are capable of inducing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids. In this disclosure, "plasmid" and "vector" can be used interchangeably depending on the context, as the plasmid is the most commonly used form of vector, although other forms of expression vectors, such as viral vectors (e.g., lentiviruses, replication defective retroviruses, poxviruses, herpes viruses, baculoviruses, adenoviruses, and adeno-associated viruses) that serve equivalent functions are also disclosed herein.

[0171] In some aspects, the vector comprises a polynucleotide described herein (e.g., comprising a codon-optimized AP-1 nucleotide sequence) and a regulatory element. For example, in certain aspects, the vector comprises a polynucleotide described herein (e.g., comprising a codon-optimized AP-1 nucleotide sequence) operably linked to a promoter. In some aspects, the vector can comprise multiple (e.g., at least two, at least three, at least four, at least five or more) promoters. For example, in some aspects, a nucleotide sequence encoding an AP-1 transcription factor (e.g., a codon-optimized AP-1 nucleotide sequence) can be under the control of a first promoter, and a nucleotide sequence encoding one or more additional components of the polynucleotide (e.g., a chimeric binding protein) can be under the control of a second promoter. In certain aspects, each of the multiple promoters is the same. In some aspects, one or more of the multiple promoters are different.

[0172] Any suitable promoter known in the art can be used in the present disclosure.In some embodiments, the promoter useful in the present disclosure comprises a mammalian or viral promoter, such as a constitutive or inducible promoter.In some embodiments, the promoter for the present disclosure comprises at least one constitutive promoter and at least one inducible promoter, such as a tissue-specific promoter.

[0173] Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, promoters from cytomegalovirus (CMV), simian viruses (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus long terminal repeat (LTR), and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. As described herein, in some aspects, the promoter that may be used in the present disclosure is an inducible promoter. An inducible promoter is expressed in the presence of an inducer. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. When multiple inducible promoters are present, they can be induced by the same inducer molecule or different inducers.

[0174] In some embodiments, the promoter comprises a myeloproliferative sarcoma virus enhancer, a negative regulatory region deletion, a dl587rev primer binding site displacement (MND) promoter, an EF1a promoter, or both.

[0175] In some embodiments, a vector comprising any polynucleotide of the present disclosure (e.g., comprising a codon-optimized AP-1 nucleotide sequence) further comprises one or more additional regulatory elements. Non-limiting examples of regulatory elements include a translational enhancer element (TEE), a translational initiation sequence, a microRNA binding site or its seed, a 3' tailing region of linked nucleosides, an AU-rich element (ARE), a post-transcriptional modulator, a 5'UTR, a 3'UTR, a localization sequence (e.g., a membrane localization sequence, a nuclear localization sequence, a nuclear exclusion sequence, or a proteasome targeting sequence), a post-translational modification sequence (e.g., ubiquitination, phosphorylation, or dephosphorylation), or a combination thereof.

[0176] In some embodiments, the vector may further comprise a transposable element. Thus, in certain embodiments, the vector comprises a polynucleotide described herein (e.g., comprising a codon-optimized AP-1 nucleotide sequence), which is flanked by at least two transposon-specific inverted terminal repeats (ITRs). In some embodiments, the transposon-specific ITRs are recognized by DNA transposons. In some embodiments, the transposon-specific ITRs are recognized by retrotransposons. Any transposon system known in the art can be used to introduce a nucleic acid molecule into the genome of a host cell, e.g., an immune cell. In some embodiments, the transposon is selected from hAT-like Tol2, Sleeping Beauty (SB), Frog Prince, piggyBac (PB), and any combination thereof. In some embodiments, the transposon comprises Sleeping Beauty. In some embodiments, the transposon comprises piggyBac. See, e.g., Zhao et al., Transl. Lung Cancer Res. 5(1):120-25 (2016), which is incorporated by reference in its entirety.

[0177] In some embodiments, the vector is a transfer vector. The term "transfer vector" refers to a composition that includes an isolated nucleic acid (e.g., a polynucleotide of the present disclosure) and can be used to deliver the isolated nucleic acid inside a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes self-replicating plasmids or viruses. The term should also be construed to further include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acid into cells, such as polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0178] In some aspects, the vector is an expression vector. The term "expression vector" refers to a vector that contains a recombinant polynucleotide (e.g., a polypeptide of the present disclosure) that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0179] In some aspects, the vector is a viral vector, a mammalian vector, or a bacterial vector. In some aspects, the vector is selected from the group consisting of an adenovirus vector, a lentivirus, a Sendai virus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, a hybrid vector, and an adeno-associated virus (AAV) vector.

[0180] In some embodiments, the adenoviral vector is a third generation adenoviral vector. ADEASY™ is by far the most popular method for making adenoviral vector constructs. This system is composed of two types of plasmids: shuttle (or transfer) vector and adenoviral vector. The transgene of interest is cloned into the shuttle vector, verified, and linearized with the restriction enzyme PmeI. This construct is then transformed into ADEASIER-1 cells, which are BJ5183 E. coli cells containing PADEASY™. PADEASY™ is an approximately 33Kb adenoviral plasmid that contains the adenoviral genes necessary for virus production. The shuttle vector and adenoviral plasmid have corresponding left and right homology arms that facilitate the homologous recombination of the transgene into the adenoviral plasmid. Standard BJ5183 can also be co-transformed with supercoiled PADEASY™ and shuttle vector, but this method results in a high background of non-recombinant adenoviral plasmid. The recombinant adenoviral plasmid is then verified for size and appropriate restriction digestion product pattern to determine that the transgene has been inserted into the adenoviral plasmid and that no other patterns of recombination have occurred. Once verified, the recombinant plasmid is linearized with PacI to create a linear dsDNA construct flanked by ITRs. 293 or 911 cells are transfected with the linear construct and virus can be harvested approximately 7-10 days later. In addition to this method, methods for making adenoviral vector constructs well known in the art at the time of filing of this application can be used to practice the methods disclosed herein.

[0181] In some embodiments, the viral vector is a retroviral vector, e.g., a lentiviral vector (e.g., a third or fourth generation lentiviral vector). The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and are one of the most efficient methods of gene delivery vectors because they can deliver a significant amount of genetic information into the DNA of the host cell. HIV, SIV, and FIV are all examples of lentiviruses. The term "lentiviral vector" refers to vectors derived from at least a portion of the lentiviral genome, including, among others, the self-inactivating lentiviral vectors provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX™ vector system, and the like. Non-clinical types of lentiviral vectors are also available and will be recognized by those skilled in the art.

[0182] Lentiviral vectors are usually produced in a transient transfection system in which a cell line is transfected with three separate plasmid expression systems. These include a transfer vector plasmid (part of the HIV provirus), a packaging plasmid or construct, and a plasmid carrying the heterologous envelope gene (env) of a different virus. The three plasmid components of the vector are placed into a packaging cell, which is then inserted into the HIV shell. The viral portion of the vector contains an insertion sequence so that the virus cannot replicate in the cell line. Current third generation lentiviral vectors only encode three of the nine HIV-1 proteins (Gag, Pol, Rev), which are expressed from separate plasmids to avoid recombination-related formation of replication-competent viruses. In fourth generation lentiviral vectors, the retroviral genome has been further reduced (see, for example, TAKARA® LENTI-X™ fourth generation packaging system).

[0183] In some embodiments, non-viral methods can be used to deliver the polynucleotides of the present disclosure (e.g., including the codon-optimized AP-1 nucleotide sequence and the nucleotide sequence encoding the chimeric binding protein) to cells or tissues of a subject. In some embodiments, non-viral methods include the use of transposons. In some embodiments, the use of non-viral delivery methods allows for the reprogramming of cells, e.g., T or NK cells, and direct injection of cells into a subject. In some embodiments, the polynucleotides of the present disclosure (e.g., including the codon-optimized AP-1 nucleotide sequence and the nucleotide sequence encoding the chimeric binding protein) can be inserted into the genome of a target cell (e.g., a T cell) or a host cell (e.g., a cell for recombinant expression of an encoded protein) by using the CRISPR / Cas system and genome editing alternatives, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases (MNs).

[0184] In some aspects, a vector (e.g., a lentiviral vector) disclosed herein comprises a polynucleotide comprising one or more nucleotide sequences encoding (i) an AP-1 transcription factor (e.g., a codon-optimized AP-1 nucleotide sequence) and (ii) an antigen-binding domain (e.g., an anti-ROR1 scFv). In some aspects, a vector comprises a polynucleotide comprising one or more nucleotide sequences encoding (i) an AP-1 transcription factor (e.g., a codon-optimized AP-1 nucleotide sequence), (ii) an antigen-binding domain (e.g., an anti-ROR1 scFv), and (iii) an EGFRt. In some aspects, a vector comprises a polynucleotide comprising one or more nucleotide sequences encoding (i) an AP-1 transcription factor (e.g., a codon-optimized AP-1 nucleotide sequence), (ii) an antigen-binding domain (e.g., an anti-ROR1 scFv), (iii) a transmembrane domain (e.g., CD28), (iv) a costimulatory domain (4-1BB), (v) an intracellular signaling domain (CD3ζ), and (vi) an EGFRt. In certain embodiments, the one or more nucleotide sequences further encode a linker, a spacer, a signal peptide, or a combination thereof.For example, in some embodiments, the vectors described herein comprise (from 5' to 3') (i) a first nucleotide sequence encoding an AP-1 transcription factor (e.g., a codon-optimized AP-1 nucleotide sequence), (ii) a second nucleotide sequence encoding a first linker (e.g., a P2A linker), (iii) a third nucleotide sequence encoding a first signal peptide (e.g., hIgκ), (iv) a third nucleotide sequence encoding an antigen binding domain (e.g., an anti-ROR1 (v) a fourth nucleotide sequence encoding a second linker (e.g., GGGSG; SEQ ID NO: 40), (vi) a sixth nucleotide sequence encoding a spacer (e.g., a spacer derived from an IgG2 hinge), (vii) a seventh nucleotide sequence encoding a transmembrane domain (e.g., CD28), (viii) an eighth nucleotide sequence encoding a costimulatory domain (e.g., 4-1BB), (ix) a ninth nucleotide sequence encoding an intracellular signaling domain (e.g., CD3ζ), (x) a tenth nucleotide sequence encoding a third linker (e.g., a P2A linker), (xi) an eleventh nucleotide sequence encoding a second signal peptide (e.g., GM-CSF), and (xii) a twelfth nucleotide sequence encoding EGFRt.

[0185] In some aspects, the polynucleotides disclosed herein (including, e.g., codon-optimized AP-1 nucleotide sequences) are DNA (e.g., DNA molecules or combinations thereof), RNA (e.g., RNA molecules or combinations thereof), or any combination thereof. In some aspects, the polynucleotides disclosed herein include nucleic acid sequences including single-stranded or double-stranded RNA or DNA (e.g., ssDNA or dsDNA) in genomic or cDNA form, or DNA-RNA hybrids, each of which may be chemically or biochemically modified, non-natural, or derivatized nucleotide bases. As described herein, such nucleic acid sequences may include additional sequences useful for facilitating expression and / or purification of the encoded polypeptide, including, but not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export and secretion signals, nuclear localization signals, and cell membrane localization signals. Based on the teachings herein, it will be apparent to one of skill in the art which nucleotide sequences encode the various polypeptides described herein (e.g., AP-1 transcription factors or chimeric binding proteins).

[0186] cell In some aspects, provided herein are cells (e.g., immune cells, e.g., T cells) that are modified (e.g., genetically) to include any of the polynucleotides, vectors, compositions, polypeptides, or sets of polypeptides described herein. Thus, in some aspects, the modified cells described herein overexpress AP-1 transcription factor. As described elsewhere in this disclosure, overexpression of AP-1 transcription factor can improve and / or increase one or more properties of the cells (e.g., resistance to exhaustion, persistence / survival, expansion / proliferation, effector function (e.g., cytokine production upon antigen stimulation, lysis of cells expressing a target antigen, or both), or a combination thereof). In some aspects, the modified cells further express one or more proteins (e.g., chimeric binding proteins, linkers, EGFRt, spacers, signal peptides, or a combination thereof) encoded by additional translation sequences described herein.

[0187] In some aspects, modified cells disclosed herein are transfected with a polynucleotide or vector described herein. The term "transfected" (or the equivalent terms "transformed" and "transduced") refers to the process by which an exogenous nucleic acid, e.g., a polynucleotide or vector described herein, is transferred or introduced into the genome of a host cell, e.g., a T cell. A "transfected" cell is one that has been transfected, transformed, or transduced with an exogenous nucleic acid, e.g., a polynucleotide or vector described herein. Cells include the primary subject cell and its progeny.

[0188] In some aspects, the cells described herein are modified with a transcription activator capable of inducing and / or increasing endogenous expression of a protein of interest (e.g., c-Jun) in the cell. As used herein, the term "transcription activator" refers to a protein that increases transcription of a gene or set of genes (e.g., by binding to an enhancer or promoter-proximal element of a nucleic acid sequence, thereby inducing its transcription). Non-limiting examples of such transcription activators that may be used in the present disclosure include: transcription activator-like effector (TALE)-based transcription activators, zinc finger protein (ZFP)-based transcription activators, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein (Cas) system-based transcription activators, or combinations thereof. See, e.g., Kabadi et al., Methods 69(2):188-197 (Sep. 2014), which is incorporated herein by reference in its entirety.

[0189] In some embodiments, the cells described herein are modified with a CRISPR / Cas system-based transcription activator, such as CRISPR Activator (CRISPRa). See, e.g., Nissim et al., Molecular Cell 54:1-13 (May 2014), which is incorporated herein by reference in its entirety. CRISPRa is a type of CRISPR tool that involves the use of a modified Cas protein that lacks endonuclease activity but retains its ability to bind to guide RNA and target DNA nucleic acid sequences. Non-limiting examples of such modified Cas proteins that may be used in the present disclosure are known in the art. See, e.g., Pandelakis et al., Cell Systems 10(1):1-14 (Jan. 2020), which is incorporated herein by reference in its entirety. In some embodiments, the modified Cas protein comprises a modified Cas9 protein (also referred to in the art as "dCas9"). In some embodiments, the modified Cas protein comprises a modified Cas12a protein. In some embodiments, an engineered Cas protein useful in the present disclosure is associated with a guide polynucleotide (e.g., a small guide RNA) (an "engineered Cas-guide complex"), where the guide polynucleotide comprises a recognition sequence complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun). In some embodiments, one or more transcriptional activators are associated with the engineered Cas-guide complex (e.g., to the N-terminus and / or C-terminus of the engineered Cas protein) such that, when the engineered Cas-guide complex is introduced into a cell, the one or more transcriptional activators can bind to regulatory elements of the nucleic acid sequence, thereby inducing and / or increasing expression of the encoded protein (e.g., c-Jun). In some embodiments, one or more transcriptional repressors (e.g., Kruppel-associated box domains (KRAB)) may be attached to the engineered Cas-guide complex (e.g., to the N-terminus and / or C-terminus of the engineered Cas protein) such that, upon introduction into a cell, the one or more transcriptional repressors can repress or reduce transcription of genes, such as, for example, genes that may interfere with the expression of c-Jun (e.g., Bach2).See, e.g., US20200030379A1 and Yang et al., J Transl Med 19:459 (2021), each of which is incorporated by reference in its entirety. In some embodiments, engineered Cas proteins useful in the present disclosure may be bound to both one or more transcriptional activators and one or more transcriptional repressors.

[0190] Without being bound by any theory, in some embodiments, the use of such engineered Cas proteins can allow for conditional transcription and expression of a gene of interest. For example, in some embodiments, a cell (e.g., a T cell) is engineered to contain a chimeric antigen receptor (CAR) (e.g., an anti-ROR1 CAR) linked to a protease (e.g., a tobacco etch virus (TEV)) and a single guide RNA (sgRNA) targeting the promoter region of c-Jun. In some embodiments, the cell is further engineered to contain a T cell activation linker (LAT) complexed with an engineered Cas protein (e.g., dCas9-VP64-p65-Rta transcription activator (VPR)) linked to a transcription activator via a linker (e.g., a TEV cleavable linker). Upon activation of the CAR, the engineered Cas protein is released for nuclear localization and conditionally and reversibly induces the expression of c-Jun. Yang et al., J Immunother Cancer 9(Suppl2):A164(2021), incorporated herein by reference in its entirety.

[0191] As will be apparent to those skilled in the art, in some embodiments, the cells described herein are modified using a combination of approaches. For example, in some embodiments, the cells are modified to include the codon-optimized c-Jun nucleotide sequence described herein, for example, alone or in combination with one or more nucleotide sequences encoding other proteins of interest (e.g., ligand-binding proteins). In some embodiments, such cells are further modified with an exogenous transcriptional activator (e.g., CRISPRa) that can increase the expression of endogenous c-Jun protein. Without being bound by any theory, in some embodiments, such a combination approach may enable immune cells to have a higher level of c-Jun protein expression (e.g., encoded by an exogenous nucleotide sequence and endogenously expressed by the immune cell).

[0192] Unless otherwise indicated, one or more exogenous nucleotide sequences and / or transcriptional activators can be introduced into cells using any suitable method known in the art. Non-limiting examples of suitable methods for delivering one or more exogenous nucleotide sequences into cells include transfection (also known as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof.

[0193] In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is isolated from a human subject. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a Th1 cell, a Th2 cell, a Th17 cell, a Tc17 cell, or a combination thereof. In some embodiments, the immune cell is a NK cell. In some embodiments, the immune cell is a tumor infiltrating lymphocyte (TIL). In some embodiments, the immune cell is a regulatory T cell (Treg). In some embodiments, the immune cell is a natural killer T (NKT) cell. In some embodiments, the immune cell is a B cell.

[0194] In some embodiments, the cell is an immune effector cell. As used herein, the term "immune effector cell" refers to a cell that is involved in an immune response, e.g., promoting an immune effector response. "Immune effector function" or "immune effector response" refers to a function or response of an immune effector cell, e.g., enhancing or promoting an immune attack of a target cell. For example, an immune effector function or response refers to a property of a T or NK cell that promotes killing or inhibiting the growth or proliferation of a target cell. For T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0195] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. The intracellular signaling domain of a chimeric binding protein (e.g., CAR) can generate a signal that promotes immune effector function of a cell, e.g., a CAR T cell, that contains the chimeric binding protein. For example, in a CAR T cell, examples of immune effector function include cytolytic activity and helper activity, including secretion of cytokines. In some embodiments, the intracellular signaling domain is the part of the chimeric binding protein (e.g., CAR) that transmits an effector function signal and induces the cell to perform a specialized function. Although the entire intracellular signaling domain can be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the complete chain, so long as it transmits an effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0196] In some embodiments, the immune cells are differentiated from pluripotent or multipotent progenitor cells. Thus, as used herein, "immune cells" further includes pluripotent or multipotent cells that can give rise to mature immune cells. In some embodiments, the cells (e.g., immune cells) are induced pluripotent stem cells (IPSCs). In some embodiments, the cells (e.g., immune cells) are embryonic stem cells. In some embodiments, the cells are hematopoietic stem cells.

[0197] In some embodiments, the T cells are CD4 + In some embodiments, the T cells are CD8 + In some embodiments, the T cells are naive T cells (T N In some embodiments, the T cells are CD95 - / CD45RA + / CD62L + / CCR7 + In some embodiments, the T cells are + / CD45RA + / CD62L + / CCR7 + In some embodiments, the T cells are CD45RO + / CCR7 + / CD62L + In some embodiments, the T cells are CD45RO + / CCR7 - / CD62L - It is.

[0198] As is evident from the present disclosure, in some aspects, the cells (e.g., immune cells) with increased AP-1 expression further express a chimeric binding protein. In some aspects, the chimeric binding protein is a chimeric antigen receptor (CAR). Thus, in some aspects, the cells (e.g., immune cells) described herein are engineered to express an AP-1 transcription factor and a CAR from a single polynucleotide (e.g., as described herein). In some aspects, the cells (e.g., immune cells) described herein are engineered to express an AP-1 transcription factor from a first polynucleotide and a CAR from a second polynucleotide. Any CAR known in the art can be used in the cells of the present disclosure, such as those described in section 5.5.1. In certain aspects, the CAR specifically binds to ROR1. In some aspects, the CAR specifically binds to GPC2. In some aspects, the CAR-expressing cells are CAR T cells, e.g., mono-CAR T cells, genome-edited CAR T cells, dual CAR T cells, or tandem CAR T cells. Examples of such CAR T cells are provided in International Application No. PCT / US2019 / 044195 (corresponding to US20210299223A1), which is incorporated by reference in its entirety.

[0199] In some aspects, the chimeric binding protein comprises a TCR (e.g., an engineered TCR). In some aspects, the cells (e.g., immune cells) described herein are engineered to express an AP-1 transcription factor and a TCR from a single polynucleotide (e.g., as described herein). In some aspects, the cells (e.g., immune cells) described herein are engineered to express an AP-1 transcription factor from a first polynucleotide and a TCR from a second polynucleotide. Any TCR known in the art can be used in the cells of the present disclosure, such as those described in Section 5.4.1.

[0200] In some aspects, the chimeric binding protein comprises a chimeric antibody-T cell receptor (CaTCR). In some aspects, the cells (e.g., immune cells) described herein are engineered to express an AP-1 transcription factor and a caTCR from a single polynucleotide (e.g., as described herein). In some aspects, the cells (e.g., immune cells) described herein are engineered to express an AP-1 transcription factor from a first polynucleotide and a caTCR from a second polynucleotide. Any caTCR known in the art can be used in the cells of the present disclosure, such as those described in Section 5.4.1.

[0201] In some aspects, the chimeric binding protein comprises a chimeric signal transduction receptor (CSR). In some aspects, the cells described herein (e.g., immune cells) are engineered to express an AP-1 transcription factor and a CSR from a single polynucleotide (e.g., as described herein). In some aspects, the cells described herein (e.g., immune cells) are engineered to express an AP-1 transcription factor from a first polynucleotide and a CSR from a second polynucleotide. Any CSR known in the art can be used in the cells of the present disclosure, such as those described in Section 5.4.1.

[0202] In some aspects, the chimeric binding protein comprises a TCR mimetic. In some aspects, the cells described herein (e.g., immune cells) are engineered to express an AP-1 transcription factor and a TCR mimetic from a single polynucleotide (e.g., as described herein). In some aspects, the cells described herein (e.g., immune cells) are engineered to express an AP-1 transcription factor from a first polynucleotide and a TCR mimetic from a second polynucleotide. Any TCR mimetic known in the art can be used in the cells of the present disclosure, such as those described in Section 5.4.1.

[0203] In some aspects, the cells described herein (e.g., expressing an AP-1 transcription factor and a chimeric binding protein) may be modified to further express one or more proteins encoded by additional translation sequences described herein (see, e.g., Section 5.4).

[0204] Certain aspects of the present disclosure relate to methods of producing the chimeric binding proteins disclosed herein by transfecting a cell, e.g., an immune cell, with a polynucleotide disclosed herein (e.g., a polycistronic polynucleotide comprising a codon-optimized AP-1 nucleotide sequence and a nucleotide sequence encoding the chimeric binding protein) and culturing the cell under suitable conditions.

[0205] composition In some aspects, the disclosure further includes a composition comprising any of the polynucleotides, vectors, cells, polypeptides, or sets of polypeptides described herein. In certain aspects, the composition comprises a protein encoded by a polynucleotide described herein, e.g., a chimeric binding protein (e.g., a CAR) and a T cell engineered to express an AP-1 transcription factor (e.g., a CAR T cell, e.g., an anti-ROR1 CAR T cell). In some aspects, the composition is a pharmaceutical composition. Thus, disclosed herein is a pharmaceutical composition comprising: (i) a cell (e.g., an immune cell) modified to express an AP-1 transcription factor, e.g., expression of the AP-1 transcription factor is increased compared to expression in a corresponding cell not transduced with a polynucleotide comprising a codon-optimized AP-1 nucleotide sequence, and (ii) a pharma- ceutically acceptable carrier. In some aspects, provided herein is a pharmaceutical composition comprising: (i) a cell (e.g., an immune cell) modified to express an AP-1 transcription factor and a chimeric binding protein (e.g., a CAR), e.g., expression of the AP-1 transcription factor is increased compared to expression in a corresponding cell not transduced with a polynucleotide comprising a codon-optimized AP-1 nucleotide sequence; and (ii) a pharmaceutically acceptable carrier. As described herein, such pharmaceutical compositions can be used to prevent and / or treat a disease or disorder (e.g., cancer). In some aspects, the cell present in the pharmaceutical composition disclosed herein is a T cell or a NK cell.

[0206] As used herein, the term "pharmaceutical composition" refers to one or more compounds described herein, such as, for example, engineered T cells that include a chimeric binding protein (e.g., CAR) and overexpress an AP-1 transcription factor, mixed or admixed with or suspended in one or more other chemical components, such as pharma- ceutically acceptable carriers and excipients. Without wishing to be bound by any theory, in some aspects, the purpose of a pharmaceutical composition is to facilitate administration to a subject of, for example, a preparation of cells expressing an AP-1 transcription factor and a chimeric binding protein (e.g., CAR) of the present disclosure.

[0207] The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound, e.g., any polynucleotide, vector, cell, polypeptide, or set of polypeptides described herein.

[0208] The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", and grammatical variations thereof, include any agent approved by a regulatory agency of the U.S. Federal government for use in animals, including humans, or listed in the U.S. Pharmacopeia, as well as any carrier or diluent that does not produce undesirable physiological effects to an extent that would prohibit administration of the composition to a subject, and does not inhibit the biological activity and properties of the compound being administered. Included are excipients and carriers that are generally safe, non-toxic, and desirable, and that are useful in preparing pharmaceutical compositions.

[0209] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol alcohol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; These include proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0210] The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of routes of administration include intramuscular, subcutaneous, ophthalmic, intravenous, intraperitoneal, intradermal, intraorbital, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, or intratumoral. Parenteral administration, characterized by injection, either subcutaneous, intramuscular, or intravenous, is also contemplated herein. Injections can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injections, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, or glycerol. In addition, if necessary, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, pH buffers, stabilizers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0211] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), as well as solutions containing thickening agents and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0212] Compositions used for in vivo administration (e.g., engineered T cells containing a chimeric binding protein and overexpressing an AP-1 transcription factor) can be sterile, which can be accomplished, for example, by filtration through sterile filtration membranes.

[0213] kit Also disclosed herein are kits comprising any of the polynucleotides, vectors, compositions, cells (e.g., CAR T cells overexpressing AP-1 transcription factor), polypeptides, or sets of polypeptides described herein. In some embodiments, the kits are for use in immunotherapy against a disease or disorder (e.g., cancer) and / or for use in treating or reducing the risk of a disease or disorder (e.g., cancer). In certain embodiments, the kits comprise one or more containers comprising any of the polynucleotides, vectors, compositions, polypeptides, or sets of polypeptides described herein.

[0214] In some embodiments, the kit includes instructions for use according to any method described herein.For example, the included instructions may include instructions for administering the pharmaceutical composition described herein to treat, delay the onset of, or alleviate the targeted disease.In some embodiments, the instructions include instructions for administering the composition described herein to a subject at risk of the targeted disease / disorder (e.g., cancer).

[0215] In some embodiments, the instructions include dosage information, dosing schedules, and routes of administration. In some embodiments, the containers are unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. In some embodiments, the instructions are written instructions on a label or package insert (e.g., a paper sheet included in the kit). In some embodiments, the instructions are machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk).

[0216] In some embodiments, the label or package insert indicates that the compositions disclosed herein are used for treating, delaying the onset of, and / or ameliorating a disease or disorder associated with cancer, such as those described herein. Instructions for practicing any of the methods described herein can be provided.

[0217] In some embodiments, the kits described herein are in suitable packaging. In some embodiments, suitable packaging includes vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), or combinations thereof. In some embodiments, the packaging includes packaging for use in combination with a particular device, for example, an infusion device, such as an inhaler, a nasal administration device (e.g., an atomizer), or a minipump. In some embodiments, the kit includes a sterile access port (e.g., the container can be an intravenous fluid bag or vial with a stopper that can be pierced by a hypodermic needle). In some embodiments, the container can also have a sterile access port (e.g., the container can be an intravenous fluid bag or vial with a stopper that can be pierced by a hypodermic needle). In some embodiments, at least one active agent is a composition described herein (e.g., engineered T cells that include a chimeric binding protein and overexpress AP-1 transcription factor).

[0218] In some aspects, the kit further comprises additional components such as buffers and instructional information. In some aspects, the kit comprises a container and a label or package insert(s) on or associated with the container. In some aspects, the disclosure provides an article of manufacture comprising the contents of the kit described herein.

[0219] therapeutic use Certain aspects of the disclosure relate to methods of administering any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides (e.g., AP-1 transcription factor and chimeric binding protein) described herein. Certain aspects of the disclosure relate to methods of treating a disease or disorder in a subject in need of such treatment, comprising administering to the subject any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides (e.g., AP-1 transcription factor and chimeric binding protein) described herein. For example, in certain aspects, disclosed herein are methods of treating a disease or disorder in a subject in need of such treatment, comprising administering to the subject cells that express a chimeric binding protein (e.g., CAR) and are engineered to overexpress an AP-1 transcription factor. In some aspects, the disease or condition comprises a tumor, i.e., cancer. In some embodiments, the method comprises stimulating a T cell-mediated immune response against a target cell population or tissue in a subject comprising administering an effective amount of any polynucleotide, vector, composition, cell, polypeptide, or set of polypeptides described herein (e.g., engineered cells that contain a chimeric binding protein and overexpress an AP-1 transcription factor). In some embodiments, the target cell population comprises a tumor. In some embodiments, the tumor is a solid tumor.

[0220] In some embodiments, administration of any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that contain chimeric binding proteins and overexpress AP-1 transcription factors) reduces tumor volume in a subject compared to a reference tumor volume. In some embodiments, the reference tumor volume is the tumor volume in the subject before administration. In further embodiments, the reference tumor volume is the tumor volume in a corresponding subject that does not receive administration. In some embodiments, the tumor volume in the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after administration compared to the reference tumor volume.

[0221] In some embodiments, treating a tumor comprises reducing the tumor weight of the subject. In certain embodiments, administration of any polynucleotide, vector, composition, cell, polypeptide, or set of polypeptides described herein (e.g., engineered cells that contain chimeric binding proteins and overexpress AP-1 transcription factor) can reduce the tumor weight of the subject when administered to the subject. In some embodiments, the tumor weight is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after administration compared to the reference tumor weight. In some embodiments, the reference tumor weight is the tumor weight in the subject before administration. In further embodiments, the reference tumor weight is the tumor weight of a corresponding subject that does not receive administration.

[0222] In some aspects, administration of any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that comprise a chimeric binding protein and overexpress an AP-1 transcription factor) to a subject, e.g., suffering from a tumor, increases the number of TILs (e.g., CD4 + or CD8 + In certain embodiments, the number and / or percentage of TILs in the tumor and / or TME can be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 ... In some embodiments, the increase is at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% or more.

[0223] In some embodiments, administration of any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that contain a chimeric binding protein and overexpress an AP-1 transcription factor) to a subject, e.g., suffering from a tumor, can extend the duration of an immune response in the subject as compared to the duration of an immune response in a corresponding non-treated subject (e.g., treated with a corresponding cell that lacks AP-1 transcription factor expression). In certain embodiments, the duration of the immune response is extended by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 1000% or more as compared to a reference (e.g., a corresponding non-treated subject). In certain embodiments, the duration of the immune response is extended by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold or more as compared to a reference (e.g., a corresponding non-administered subject).

[0224] As described herein, any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that contain the chimeric binding protein and overexpress the AP-1 transcription factor) can be used to treat a variety of cancers. Non-limiting examples of cancers that can be treated include adrenal cortical carcinoma, advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastases, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary site, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma family, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung cancer, tinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, gastric cancer, testicular cancer, throat cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, secondary cancers caused by cancer treatment, and combinations thereof. In some embodiments, the tumor is associated with a solid tumor.

[0225] In some embodiments, any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides (e.g., engineered cells that contain chimeric binding proteins and overexpress AP-1 transcription factors) described herein are used in combination with other therapeutic agents (e.g., anti-cancer agents and / or immunomodulatory agents). Thus, in certain embodiments, the methods of treating a disease or disorder (e.g., tumor) disclosed herein include administering any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides (e.g., engineered cells that contain chimeric binding proteins and overexpress AP-1 transcription factors) described herein in combination with one or more additional therapeutic agents. Such agents can include, for example, chemotherapeutic agents, targeted anti-cancer therapies, oncolytic agents, cytotoxic agents, immune-based therapies, cytokines, surgery, radiation therapy, activators of costimulatory molecules, immune checkpoint inhibitors, vaccines, cellular immunotherapy, or any combination thereof.

[0226] In some aspects, any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that contain chimeric binding proteins and overexpress AP-1 transcription factors) are administered to a subject before or after administration of an additional therapeutic agent. In other aspects, any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that contain chimeric binding proteins and overexpress AP-1 transcription factors) are administered to a subject simultaneously with an additional therapeutic agent. In certain aspects, any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that contain chimeric binding proteins and overexpress AP-1 transcription factors) and an additional therapeutic agent may be administered simultaneously as a single composition in a pharma- ceutically acceptable carrier. In other aspects, any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that contain chimeric binding proteins and overexpress AP-1 transcription factors) and an additional therapeutic agent are administered simultaneously as separate compositions.

[0227] In some aspects, any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that contain chimeric binding proteins and overexpress AP-1 transcription factors) are used in combination with standard treatments (e.g., surgery, radiation, and chemotherapy). The methods described herein can also be used as maintenance therapy, e.g., therapy aimed at preventing tumor development or recurrence.

[0228] In some embodiments, any of the polynucleotides, vectors, compositions, cells, polypeptides, or sets of polypeptides described herein (e.g., engineered cells that contain the chimeric binding protein and overexpress the AP-1 transcription factor) are used in combination with one or more anti-cancer drugs such that multiple elements of the immune pathway can be targeted. Non-limiting examples of such combinations include therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, imiquimod), therapies that inhibit negative immune regulation, e.g., by inhibition of the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or by depleting or blocking Tregs or other immune suppressive cells (e.g., myeloid-derived suppressor cells), therapies that stimulate positive immune regulation, e.g., using agonists that stimulate the CD-137, OX-40, and / or CD40 or GITR pathways and / or stimulate T cell effector function, therapies that increase the frequency of anti-tumor T cells systemically, e.g., using antagonists of CD25 (e.g., daclizumab) or ex vivo. These include therapies that deplete or inhibit Tregs, such as Tregs in the tumor by anti-CD25 bead depletion, therapies that affect the function of suppressor myeloid cells in the tumor, therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines), adoptive T cell or NK cell transfer including genetically engineered cells, e.g., cells engineered to express chimeric antigen receptors (CAR-T therapy), therapies that inhibit metabolic enzymes, such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase, therapies that reverse / prevent T cell anergy or exhaustion, therapies that induce innate immune activation and / or inflammation at the tumor site, administration of immune stimulatory cytokines, blockade of immune suppressive cytokines, or any combination thereof.

[0229] In some embodiments, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through a particular immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the methods of the present invention include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies), or combinations thereof. Non-limiting examples of such immune checkpoint inhibitors include: anti-PD1 antibodies (e.g., nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI 754091, SHR-1210, and combinations thereof); anti-PD-L1 antibodies (e.g., atezolizumab (TECENTRIQ®; RG7446; MPDL3280A; RO5541267), durvalumab (MEDI4736, IMFINZI®), BMS-936559, avelumab (BAVENCIO®), LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105, and combinations thereof); and anti-CTLA-4 antibodies (e.g., ipilimumab (YERVOY®), tremelimumab (ticilimumab; CP-675,206), AGEN-1884, ATOR-1015, and combinations thereof).

[0230] In some embodiments, the anti-cancer agent comprises an immune checkpoint activator (i.e., promotes signaling through a particular immune checkpoint pathway). In certain embodiments, the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX40 antibody), a LAG-3 agonist (e.g., an anti-LAG-3 antibody), a 4-1BB (CD137) agonist (e.g., an anti-CD137 antibody), a GITR agonist (e.g., an anti-GITR antibody), a TIM3 agonist (e.g., an anti-TIM3 antibody), or a combination thereof.

[0231] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of one of ordinary skill in the art, and such techniques are fully explained in the literature. For example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DNGlover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. Synthesis; Mullis et al. U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press)(1986);Perbal (1984)A Practical Guide To Molecular Cloning;the treatise,Methods In Enzymology(Academic Press,Inc.,NY);Miller and Calos eds.(1987)Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory);Wu et al.,eds.,Methods In Enzymology,Vols.154 and 155;Mayer and Walker,eds.(1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986);); Crooks, Antisense drug Technology: Principles, strategies and applications,2. nd Ed. CRC Press (2007) and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0232] All references cited above, and all references and amino acid or nucleotide sequences (e.g., GenBank numbers and / or UniProt numbers) cited herein are hereby incorporated by reference in their entirety.

[0233] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0234] Example 1 – Generation of constructs with codon-optimized c-Jun sequences To increase transgene expression, R12-EGFRt constructs containing codon-optimized c-Jun sequences (i.e., containing anti-ROR1 scFv + truncated EGFR) were generated using different optimization approaches. The codon-optimized c-Jun sequences are shown in SEQ ID NOs: 1-10. As a control, a bicistronic R12-EGFRt construct lacking the c-Jun sequence was used (construct #1 in Figure 1). As an additional control, a tricistronic R12-EGFRt construct containing truncated CD19 (CD19t) instead of c-Jun was also used (construct #2 in Figure 1). In SEQ ID NO: 1, the codon optimization was adjusted to mimic codon usage bias in human cells (construct #3 in Figure 1), and in SEQ ID NO: 2, the codon optimization was adjusted to mimic codon usage bias in human T cells (construct #4 in Figure 1).

[0235] To evaluate whether codon-optimized c-Jun can improve the expression of c-Jun in anti-ROR1 constructs, T cells were transduced at different multiplicities of infection (MOI 2, 5, 10, and 20) with a series of anti-ROR1 constructs shown in Figure 1. The expression levels of c-Jun and R12 CAR and EGFRt in the various constructs were then determined using flow cytometry.

[0236] As shown in FIG. 2A, anti-ROR1 constructs 3 and 12, which contain the c-Jun codon-optimized sequences shown in SEQ ID NOs: 1 and 10, respectively, showed the highest percentage of EGFRt expression compared to the control anti-ROR1 constructs (i.e., R12-EGFRt and CD19t-R12-EGFRt). + c-Jun +As expected, cells transduced with anti-ROR1 constructs lacking c-Jun (i.e., R12-EGFRt and CD19t-R12-EGFRt) had a negligible percentage of cells expressing both EGFRt and c-Jun. When transduced cells were analyzed for expression of both EGFRt and anti-ROR1 scFv (R12), the percentage of EGFRt+R12+ cells was much higher when cells were transduced with the codon-optimized c-Jun anti-ROR1 constructs, constructs 3 and 12, compared to the anti-ROR1 constructs containing construct 11, which contains the codon-optimized c-Jun sequence of SEQ ID NO: 9 (FIG. 2B) and the control CD19t-R12-EGFRt construct.

[0237] At the single cell level, cells transduced with the codon-optimized c-Jun-anti-ROR1 constructs Construct 3 and Construct 12 also had substantially higher c-Jun expression compared to the anti-ROR1 construct containing the codon-optimized c-Jun sequence of SEQ ID NO: 9, Construct 11 (FIG. 2C). Furthermore, cells transduced with the codon-optimized c-Jun-anti-ROR1 constructs Construct 3 and Construct 12 also had higher anti-ROR1 CAR expression compared to the anti-ROR1 construct containing the codon-optimized c-Jun sequence of SEQ ID NO: 9, Construct 11 (FIG. 2D) and the control CD19t-R12-EGFRt construct at the single cell level.

[0238] These results demonstrated that inclusion of the codon-optimized c-Jun sequences of SEQ ID NO: 1 and 10 in the anti-ROR1-EGFRt constructs, constructs 3 and 12, could substantially increase c-Jun transgene expression in target cells. Overall transduction efficiency was similar to that of the R12-EGFRt construct (i.e., lacking c-Jun), and the codon-optimized c-Jun-anti-ROR1 CAR constructs were not associated with high vector copy numbers (data not shown).

[0239] In a separate experiment, the further codon-optimized c-Jun sequence described above was used to transduce T cells at various multiplicities of infection, and transgene expression levels for the various constructs were determined using flow cytometry. For each construct, an MOI that gave similar levels of transduction, as determined by co-expression of anti-ROR1 CAR and EGFRt, was selected for comparison of transgene expression by T cells.

[0240] A first experiment was performed (tier 1; FIG. 3A). In this experiment, construct 3 was shown to have the highest c-Jun expression while maintaining good expression of the anti-ROR1 CAR and EGFRt transgenes, with only a slight decrease in transduction efficiency compared to construct 5, which showed a slightly decreased but still good c-Jun expression, and the CD19t control. Construct 3 was selected and then compared to further codon-optimized c-Jun constructs in a tier 2 experiment shown in FIG. 3B. In the tier 2 comparison, constructs 3 and 8 showed high c-Jun expression while maintaining good expression of the anti-ROR1 CAR and EGFRt transgenes, with construct 3 having the highest c-Jun expression. Construct 7 also showed good c-Jun expression, but low transduction efficiency, as well as ROR1 CAR and EGFRt expression.

[0241] In summary, a number of codon-optimized c-Jun sequences were generated and screened for their effect on transduction efficiency and c-Jun, R12 and EGFRt transgene expression. These experiments showed that constructs 3, 8 and 12 (with codon-optimized c-Jun sequences shown in SEQ ID NOs: 1, 6 and 10, respectively) had high c-Jun expression with acceptable effects on transduction efficiency and transgene (R12 and EGFRt) expression. Construct 7 also showed good c-Jun and R12 expression and moderate reduction in transduction efficiency and EGFRt expression. These candidate codon-optimized c-Jun sequences can be used to reduce exhaustion of engineered immune cells, particularly for adoptive cellular immunotherapy.

Claims

1. a. a nucleotide sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1; b. a nucleotide sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2; c. a nucleotide sequence having at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:4; d. a nucleotide sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:5; e. a nucleotide sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6; f. a nucleotide sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:7; g. a nucleotide sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:8; h. a nucleotide sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:9; or i. a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

10. wherein said nucleotide sequence encodes a c-Jun protein.

2. 2. The polynucleotide of claim 1 , wherein the nucleotide sequence comprises: (a) the nucleic acid sequence set forth in SEQ ID NO:1; (b) the nucleic acid sequence set forth in SEQ ID NO:2; (c) the nucleic acid sequence set forth in SEQ ID NO:4; (d) the nucleic acid sequence set forth in SEQ ID NO:5; (e) the nucleic acid sequence set forth in SEQ ID NO:6; (f) the nucleic acid sequence set forth in SEQ ID NO:7; (g) the nucleic acid sequence set forth in SEQ ID NO:8; (h) the nucleic acid sequence set forth in SEQ ID NO:9; or (i) the nucleic acid sequence set forth in SEQ ID NO:

10.

3. The polynucleotide of claim 1 or 2, further comprising: (a) a nucleotide sequence encoding a ligand-binding protein; (b) a nucleotide sequence encoding a truncated EGFR (EGFRt); (c) a nucleotide sequence encoding a linker; or (d) any combination of (a) to (c).

4. 4. The polynucleotide of claim 3, wherein the ligand binding protein comprises a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric antibody-T cell receptor (caTCR), a chimeric signal transducing receptor (CSR), a T cell receptor mimetic (TCR mimetic), or a combination thereof.

5. The polynucleotide of claim 3 or 4, wherein the ligand binding protein comprises an antigen binding domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, or a combination thereof.

6. A vector comprising a polynucleotide according to any one of claims 1 to 5 and a regulatory element.

7. A composition comprising a polynucleotide according to any one of claims 1 to 5 or a vector according to claim 6.

8. A kit comprising a polynucleotide according to any one of claims 1 to 5, a vector according to claim 6, or a composition according to claim 7.

9. A polypeptide comprising a c-Jun protein encoded by the polynucleotide of any one of claims 1 to 5, the vector of claim 6, or the composition of claim 7.

10. A cell comprising a polynucleotide according to any one of claims 1 to 5, a vector according to claim 6, a composition according to claim 7, or a polypeptide according to claim 9.

11. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 5, a vector according to claim 6, a polypeptide according to claim 9, or a cell according to claim 10, and a pharma- ceutically acceptable carrier.

12. The pharmaceutical composition of claim 11 for treating a subject in need of treatment.

13. 12. The pharmaceutical composition of claim 11 for treating or preventing a disease or condition in a subject in need thereof.

14. The pharmaceutical composition of claim 13, wherein the disease or condition is cancer.

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