Cd19-targeted car t cell therapy

Abstract

A chimeric antigen receptor (CAR), for example a CD19-specific CAR, capable of targeting an antigen expressed on disease-associated cells, such as tumor cells. A polynucleotide encoding the CAR and, optionally: i) an miRNA silencer module capable of inhibiting the expression of an immune checkpoint protein (e.g., PD-1); ii) a cytokine (e.g., membrane-bound IL-15); and / or a cell tag (e.g., a HER-1 kill switch). A vector comprising the polynucleotide. A modified immune effector cell comprising the CAR, polynucleotide, or vector. Compositions and kits comprising the CAR, polynucleotide, vector, and / or modified immune effector cell. Use of the CAR, polynucleotide, vector, and / or modified immune effector cell in the manufacture of a medicament for the treatment of a disease or disorder. A method for treating a subject with a disease or disorder, comprising administering the CAR, polynucleotide, vector, and / or modified immune effector cell to a subject in need thereof.

Description

CD19-TARGETED CAR T CELL THERAPY CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority of U.S. Provisional Application No. 63 / 566,848, filed March 18, 2024, and U.S. Provisional Application No.63 / 516,565, filed July 31, 2023. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML copy, created on July 25, 2024, is named 75594-410122_SL.xml and is 784,341 bytes in size. FIELD OF THE INVENTION

[0003] The present invention relates to chimeric receptor therapy, particularly chimeric antigen receptor (CAR) T-cell therapies, such as those specific to CD19, for the treatment of diseases and disorders characterized by the overexpression of CD19. BACKGROUND OF THE INVENTION

[0004] Chimeric receptor therapies, including chimeric antigen receptor T (CAR-T) cells and T cell receptor (TCR) therapies, involve the use of cells engineered to express receptors that target specific antigens expressed on tumor cells or other disease-associated cells, such as cells involved in autoimmune diseases. In the context of cancer treatment, the engineered cells bind to tumor cells and initiate an immune response that results in the destruction of the tumor cell. Similarly, in the treatment of autoimmune disorders, the engineered cells can be directed towards specific immune cells responsible for the abnormal immune response. By targeting and depleting these aberrant immune cells, chimeric receptor therapies have the potential to modulate the immune system and alleviate the symptoms of autoimmune diseases. This approach offers a promising avenue for the treatment of both cancer and autoimmune conditions by harnessing the power of engineered immune cells to selectively eliminate harmful cells while sparing healthy tissues. 1

[0005] Antigens that are present on cancer cells, in particular those that may be present on normal cells but are overexpressed on cancer cells, are promising targets for chimeric receptor therapies. Several targets for such therapies have been identified to date, including but not limited to CD19, CD33, BCMA, CD44, α-Folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EGFR, Folate receptor α, Mucins such as MUC1, MUC4 or MUC16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123 or VEGF-R2. Among these, CD19, CD33, MUC1, MUC16, ROR1, and mesothelin have shown particular promise as targets for immunotherapy.

[0006] The expression of CD19 has been used as a diagnostic marker for certain hematological malignancies. For example, CD19 expression, often in combination with other markers, is used in the diagnosis of various B-cell malignancies, including relapsed and refractory B-cell lymphomas, acute lymphoblastic leukemia, mantle cell lymphoma, chronic lymphocytic leukemia, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, and B-cell precursor acute lymphoblastic leukemia. Long-term follow-up data indicates that treatments using CD19 directed CAR-T cells are effective for treating patients with these hematological malignancies, along with other cancers associated with the overexpression of CD19.

[0007] Ongoing research efforts aim to improve the durability of responses following CAR-T cell therapy. One of the challenges in CAR-T design is maintaining its long-lasting presence without exhaustion in the body. This aspect plays a crucial role in their efficacy and prevents relapse. Ensuring the sustained activity of CAR-T cells is vital for successful outcomes.

[0008] Immune checkpoint inhibition, which prevents the switching off of T-cells and promotes the activity of these cells, has shown promise. Immunotherapy utilizing blocking antibodies has been extensively evaluated in the clinic and has been shown to improve tumor regression across multiple malignancies, especially when administered in conjunction with CAR-T cells or cells expressing TCRs. Examples of checkpoint inhibitor targets include but are not limited to PD-1, PD-L1, CTLA-4, TIGIT, 4-1BB, PIK3IP1, CD27, CD28, CD40, CD70, CD122, CD137, OX40 (CD134), GITR, ICOS, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, or VISTA. Among these, CTLA4, PD-1, PD-L1, TIM3, TIGIT, LAG3, and / or PIK3IP1 have shown the most promise as targets. The PD-1 / programmed death ligand 1 (PD-L1) pathway 2  in particular plays a vital role in how tumor cells evade immune response and thus PD-1 and PD- L1 show particular promise as targets.

[0009] The addition of systemic checkpoint inhibition to traditional CAR-T therapy, however, further complicates the treatment as well as increases toxicity risk and cost. Further, checkpoint inhibitor blocking antibodies have not performed consistently across cancer types, may have limited access to the tumor microenvironment, require repeated administration, and may lose effectiveness over time. Genome editing is an alternate approach to checkpoint inhibition and has the advantage of restricting the checkpoint inhibitor to only the engineered CAR-T cells. However, gene editing adds complexity to the manufacturing process, which increases the turnaround time and cost of the cell therapy.

[0010] There is accordingly a continuing need in the art to obtain safer, more effective, less expensive therapies to antigen-associated diseases and conditions, including treatments that combine CAR-T and / or TCR therapy with systemic checkpoint inhibition.

[0011] There is also a need to devise ways of diversifying treatment regimens to provide a multi- pronged targeting of antigens in order to address complex in vivo biological issues such as loss of immunological surveillance, genetic alterations in tumor antigen composition and tumor heterogeneity (giving rise to cancer cell phenotypic differences).

[0012] In addition, the current state of traditional CAR-T manufacturing conducted in centralized GMP facilities is burdened by high costs and labor-intensive processes. The reliance on viral vectors and the requirement for ex vivo cell activation and extensive expansion to achieve an adequate cell count for treatment contribute to these inefficiencies. As a result, the prolonged production time not only leads to treatment delays but also increases the risk of CAR-T cell exhaustion. Given these challenges, there also exists a need in the field for more efficient and rapid manufacturing processes to streamline CAR-T therapy production and improve patient outcomes.

[0013] The present invention improves the efficacy and safety of CD19 CAR-T cell therapy and reduces burdensome costs by eliminating the need for gene editing or combination with checkpoint inhibitors. 3  SUMMARY OF THE INVENTION

[0014] The present invention relates in part to a non-naturally occurring polynucleotide encoding a CD19-specific chimeric receptor.

[0015] In certain embodiments, the chimeric receptor is a T-cell receptor.

[0016] In certain embodiments, the chimeric receptor is a chimeric antigen receptor.

[0017] In certain embodiments, the CD19-specific chimeric antigen receptor is encoded by a polynucleotide having at least 90% identity with SEQ ID NO: 939.

[0018] In certain embodiments, the polynucleotide further encodes a miRNA that inhibits the expression of an immune checkpoint protein.

[0019] In certain embodiments, the miRNA inhibits the expression of PD-1 (i.e., a “PD-1 silencing miRNA module” or a “PD-1 silencer”).

[0020] In certain embodiments, the PD-1 silencing miRNA module is encoded by two individual miRNAs.

[0021] In certain embodiments, one of the miRNAs in the PD-1 silencing miRNA module is encoded by a nucleic acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 348 or that is capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO: 348.

[0022] In certain embodiments, one of the miRNAs in the PD-1 silencing miRNA module is encoded by a nucleic acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 349 or that is capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO: 349.

[0023] In certain embodiments, the PD-1 silencing miRNA module is encoded by a nucleic acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 267 or that is capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO: 267. In other embodiments, the PD-1 silencing miRNA module is located within the 5’UTR. In certain such embodiments, the 5’UTR is encoded by a nucleic acid sequence having at least about 80% sequence identity with 4  SEQ ID NO: 944 or that is capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO: 944.

[0024] In certain embodiments, the polynucleotide further encodes a cytokine.

[0025] In certain embodiments, the cytokine is IL-15, or functional fragment or variant thereof. In certain such embodiments, the IL-15 is membrane bound.

[0026] In certain embodiments, the nucleic acid encodes a fusion protein comprising IL-15, or a functional fragment or variant thereof, and IL-15Rα, or a functional fragment or variant thereof.

[0027] In certain embodiments, the fusion protein comprises a polypeptide having at least 90% identity with SEQ ID NO: 523 or SEQ ID NO: 525.

[0028] In certain embodiments, the polynucleotide further encodes a cell tag.

[0029] In certain embodiments, the cell tag acts as a safety or kill switch.

[0030] In certain embodiments, the cell tag comprises a HER1 Domain III, or a functional fragment or variant thereof, and a truncated HER1 Domain IV, or a functional fragment or variant thereof.

[0031] In certain embodiments, the cell tag is encoded by a polynucleotide having at least 90% sequence identity with SEQ ID NO: 571.

[0032] In certain embodiments, the cell tag comprises a polypeptide having at least 90% identity with SEQ ID NO: 572.

[0033] In certain embodiments, the cell tag is encoded by a polynucleotide having at least 90% sequence identity with SEQ ID NO: 1035.

[0034] In certain embodiments, the polynucleotide further encodes a GM-CSFRa signal peptide, or a functional fragment or variant thereof.

[0035] In certain such embodiments, the GM-CSFRa signal peptide has the amino acid sequence of SEQ ID NO: 836, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than SEQ ID NO: 836 by at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% 5  sequence identity with SEQ ID NO: 836, and / or is a conservatively-substituted variant of SEQ ID NO: 836.

[0036] In certain embodiments, the polynucleotide encodes a CD19-specific VHdomain and a CD19-specific VL domain.

[0037] In certain embodiments, the polynucleotide encodes a CD19-specific scFv.

[0038] In some embodiments, the scFv comprises a polypeptide having at least 90% sequence identity with SEQ ID NO: 959.

[0039] In certain embodiments, the polynucleotide encodes a CD8 hinge and transmembrane domain. In certain embodiments, the CD8 hinge comprises a polypeptide having at least 90% sequence identity with SEQ ID NO: 816. In certain embodiments, the CD8 transmembrane domain comprises a polypeptide having at least 90% sequence identity with SEQ ID NO: 812.

[0040] In certain embodiments, the polynucleotide encodes a CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain comprises a polypeptide having at least 90% sequence identity with SEQ ID NO: 828.

[0041] In certain embodiments, the polynucleotide encodes a CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain comprises a polypeptide having at least 90% sequence identity with SEQ ID NO: 826.

[0042] In certain embodiments, the nucleic acid sequence encoding the chimeric receptor is framed by transposon repeat regions.

[0043] The present invention relates in part to a single vector comprising the expression cassette of the present invention.

[0044] In certain embodiments, the vector comprises a Tc1 / mariner transposon (e.g., a Sleeping Beauty transposon).

[0045] In certain embodiments, the Sleeping Beauty transposon comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 981 or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 6  99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 981 or a codon degenerate variant of SEQ ID NO: 981).

[0046] In certain embodiments, the Sleeping Beauty transposon comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 982 or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 982 or a codon degenerate variant of SEQ ID NO: 982).

[0047] The present invention relates in part to a modified immune effector cell comprising the polynucleotide of the present invention.

[0048] The present invention relates in part to a composition comprising the polynucleotide of the present invention or the modified immune effector cell of the present invention.

[0049] The present invention relates in part to a kit comprising the polynucleotide of the present invention or the cell of the present invention.

[0050] The present invention relates in part to a method of treating a disease or disorder, comprising administering to a subject in need thereof the modified immune effector cell of the present invention to a subject in need thereof.

[0051] In certain embodiments, the disease or disorder is a cancer.

[0052] In certain embodiments, the cancer is B cell lymphoma, acute lymphoblastic leukemia (ALL), mantle cell leukemia (MCL), breast cancer, cervical cancer, chronic lymphocytic leukemia (CLL), another hematological cancer, or any CD19+ malignancy.

[0053] In certain embodiments, the disease or disorder is automimmune disorder. In some embodiments, the autoimmune disease or disorder is rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), multiple sclerosis, myasthenia gravis (MG), type 1 diabetes, inflammatory bowel disease, psoriasis, and autoimmune thyroiditis, among others. In a particular embodiment, the autoimmune disease or disorder to be treated is SLE.

[0054] In certain embodiments, the modified immune effector cell does not undergo propagation or activation before administration. 7

[0055] The present invention relates in part to the use of a modified immune effector cell of the present invention in the manufacture of a medicament for the treatment of a disease or disorder.

[0056] In certain embodiments, the compositions described herein are administered as a combination therapy with an additional therapeutic agent.

[0057] In certain embodiments, the additional therapeutic agent is a vaccine, an interleukin (e.g., IL-12), an immunotherapeutic agent, an anti-cancer drug, a chemotherapeutic agent, or an immune checkpoint inhibitor.

[0058] In certain embodiments, the chemotherapeutic agent is a histone deacetylase inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG.1A is a schematic diagram of a single Sleeping Beauty transposon encoding CD19- specific CAR, mbIL15, and HER1t (alternatively referred to herein as “Vector 1” or “CD19 UltraCAR-T”).

[0060] FIG.1B is a schematic diagram of a single Sleeping Beauty transposon encoding CD19- specific CAR, mbIL15, HER1t, and two PD-1-silencing miRNAs (alternatively referred to herein as “Vector 2” or “NextGen CD19 UltraCAR-T”).

[0061] FIGs. 2A-2C show sensorgrams illustrating that CD19 binds to FMC63 scFv-Fc with binding affinity of 4.5 nM (FIG. 2A), that CD19 binds to positive control FMC63 IgG with 7.9 nM (FIG.2B), and CD19 does not bind to negative control scFv-Fc protein (FIG.2C).

[0062] FIGs.3A-3B show graphs illustrating a reduction in PD-1 expression in CD19 UltraCAR- T cells carrying a PD-1 silencer (produced using Vector 2) relative to UltraCAR-T cells without a PD-1 silencer (produced using Vector 1) via RT-qPCR (FIG. 3A) and RNAseq (FIG. 3B) data. The values represent fold-change in expression in UltraCAR-T cells with the PD-1 silencer compared to UltraCAR-T cells without the PD-1 silencer in samples generated from 10 healthy donors.

[0063] FIG.4A shows a bar graph reflecting fold-change in expression of miRNAs targeting PD- 1 in CD19 UltraCAR-T with PD-1 silencer (produced using Vector 2) over CD19 UltraCAR-T without PD-1 silencer (produced using Vector 1). FIG.4B shows an alignment of small RNA reads 8  to the PD-1 silencer module using IGV browser. The mapped miRNAs match the intended PD-1 targeting guide miRNAs, PD-1_1843 and PD-1_2061, with little to no visible passenger miRNAs. Data is representative of one donor Vector 2 sample. The X-axis indicates nucleotide position across the silencer module and the vertical gray bars indicate read depth.

[0064] FIGs.5A-5D show a series of bar graphs illustrating the specific cytotoxic ability of CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2) against Acute Lymphoblastic Leukemia cell line (NALM-6 on day 3 (FIG. 5A) and NLAM-6 / PD-L1 on Day 3 (FIG. 5B)) compared to CD19 UltraCAR-T cells without PD-1 silencer (produced using Vector 1). Various effector to target (E:T) ratios are shown. NALM-6 with CD19 knockout (FIG.5C) and MOLM- 13 (FIG.5D), a cell line that does not express CD19, are included as controls.

[0065] FIGs. 6A-6B show bar graphs of polyfunctionality (FIG. 6A) and polyfunctionality strength index (PSI) (FIG. 6B) in NALM-6 and NALM-6 / PDL1 target cell lines treated with Sleeping Beauty UltraCAR-T cells with PD-1 silencer (produced using Vector 2) and without PD- 1 silencer (produced using Vector 1).

[0066] FIGs. 7A-7B show bar graphs of polyfunctionality (FIG. 7A) and polyfunctionality strength index (PSI) (FIG.7B) in JEKO-1 / PDL1 target cell line treated with Sleeping Beauty CAR- T cells with PD-1 silencer (produced using Vector 2) and without PD-1 silencer (produced using Vector 1).

[0067] FIG.8 is a dot plot representing the cytotoxic function of CAR-T cells following multiple rounds of stimulation with CD19+ tumor cells. Results are presented as ratios of % target cell killing at each round of tumor cell addition relative to initial round killing capacity. The colors represent sustained cytotoxic potential of CD19 CAR-T cells with PD-1 silencer (produced using Vector 2; circles) in a highly stimulated setting in comparison to control CD19 UltraCAR-T cells without PD-1 silencer (produced using Vector 1; squares).

[0068] FIGs.9A-9G are a series of Western blots showing that CD19 UltraCAR-T cells with PD- 1 silencer (produced using Vector 2) and CD19 UltraCAR-T cells without PD-1 silencer (produced using Vector 1) express CD19-CAR (FIGs. 9A-9B), mbIL15 (FIGs. 9C-9D), HER1t (FIG. 9E), and that those UltraCAR-T cells with PD-1 silencer (Vector 2) have reduced PD-1 expression upon stimulation with CD3 / CD28 beads (FIGs.9F-9G). 9

[0069] FIG. 10A is a bar graph of results from an antibody-dependent (anti-HER1 antibody - cetuximab) cellular cytotoxicity assay involving CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2) and control UltraCAR-T cells without PD-1 silencer (produced using Vector 1), with the addition of either anti-HER1 antibody (cetuximab) or control anti-CD20 antibody (rituximab). FIG. 10B is flow cytometry data from the antibody-dependent cellular cytotoxicity assay.

[0070] FIG.11 is a dot plot showing results from a cytokine withdraw assay performed on CD19 UltraCAR-T cells with PD-1 silencer and with and without mbIL15 expression (cytokine) (produced using Vector 2) and without PD-1 silencer and with and without mbIL15 (cytokine) expression (IGE-2503).

[0071] FIG. 12A is a dot plot of IVIS (tumor burden) in NALM-6 tumor model utilizing NSG mice that were treated with CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2) and control UltraCAR-T cells without PD-1 silencer (produced using Vector 1). FIG.12B is in vivo luminescence imaging of CD19+ NALM-6 tumor bearing NSG mice that were treated.

[0072] FIG. 13A is a dot plot showing relative body weight in NSG mice treated with CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2) and control CD19 UltraCAR-T cells without PD-1 silencer module (produced using Vector 1) using NALM-6 tumor model. FIG. 13B is a dot plot showing survival in these same mice.

[0073] FIGs. 14A-14E are dot plots generated from characterizing, by flow cytometry, the peripheral blood from NSG mice (NALM-6 tumor model) treated with CD19 UltraCAR-T Cells with PD-1 silencer module (produced using Vector 2) and without PD-1 silencer module (produced using Vector 1). FIGs. 14A-C are dot plots of HER1t+ cell concentration following treatment; FIG.14D is a dot plot of PD-1 expression following treatment; and FIG.14E is a dot plot of the concentration of memory cells following treatment.

[0074] FIG.15A & FIG.16A are dot plots showing the tumor burden of NSG MHC Class I / II KO mice (NALM-6 tumor model) treated (Tx) with (i) CD19 CAR-T cells not expressing mbIL15 nor carrying a PD-1 silencer (produced using Vector 3); (ii) CD19 CAR-T cells with a PD1 silencer but not expressing mbIL15 (produced using Vector 4); (iii) CD19 UltraCAR-T cells expressing mbIL-15 but not carrying a PD-1 silencer (produced using Vector 1); and (iv) CD19 UltraCAR-T 10  cells expressing mbIL-15 and carrying a PD-1 silencer (produced using Vector 2). FIG. 15B & FIG. 16B are dot plots showing relative body weight of NSG MHC Class I / II KO mice treated (Tx) with the same.

[0075] FIGs. 17A-17E are a series of dot plots showing relative (%) change in body weight in NSG MHC Class I / II KO mice (NALM-6 tumor model) treated with (i) CD19 CAR-T cells not expressing mbIL15 nor carrying a PD-1 silencer (produced using Vector 3) (FIG.17B); (ii) CD19 CAR-T cells with PD-1 silencer but not expressing mbIL15; (produced using Vector 4) (FIG. 17C); (iii) CD19 UltraCAR-T cells expressing mbIL-15 but not carrying a PD-1 silencer (produced using Vector 1) (FIG. 17D); (iv) CD19 UltraCAR-T expressing mbIL-15 and carrying a PD-1 silencer (produced using Vector 2) (FIG.17E); and (v) saline (FIG.17A).

[0076] FIGs.18A-18B are a bar graphs (study day 21) showing levels of mbIL-15 (FIG.18A) and PD-1 expression (FIG. 18B) in NSG MHC Class I / II KO mice (NALM-6 tumor model) treated with (i) CD19 CAR-T cells not expressing mbIL15 nor carrying a PD-1 silencer (produced using Vector 3); (ii) CD19 CAR-T cells with PD-1 silencer but not expressing mbIL15 (produced using Vector 4); (iii) CD19 UltraCAR-T cells expressing mbIL-15 but not carrying a PD-1 silencer (produced using Vector 1); and (iv) CD19 UltraCAR-T cells expressing mbIL-15 and carrying a PD-1 silencer (produced using Vector 2). FIGs. 18C-18F are dot plots showing HER1t concentration in blood in similarly treated mice after treatment.

[0077] FIG.19 is a dot plot of memory cells count on study day 21, in NSG MHC Class I / II KO mice (NALM-6 tumor model) treated with (i) CD19 CAR-T cells not expressing mbIL15 nor carrying a PD-1 silencer (produced using Vector 3); (ii) CD19 CAR-T cells carrying a PD-1 silencer but not expressing mbIL15 (produced using Vector 4); (iii) CD19 UltraCAR-T cells expressing mbIL-15 but not carrying a PD-1 silencer (produced using Vector 1); and (iv) CD19 UltraCAR-T cells expressing mbIL-15 and carrying a PD-1 silencer (produced using Vector 2).

[0078] FIG. 20A-B and FIG. 21A-B are dot plots of tumor burden (dorsal and ventral IVIS) in NSG mice (Jeko-1 / PD-L1 tumor model) following treatment with 1x106(FIGs.20A-B) and 5x106(FIGs.21A-B) of NextGen CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2) and control CD19 UltraCAR-T cells without PD-1 silencer (produced using Vector 1). FIG. 20C and FIG.21C are in vivo luminescence imaging (tumor burden) of the CD19+ Jeko-1 / PD-L1 11  tumor bearing NSG mice that were treated.

[0079] FIGs.22A-22B are dot plots of the relative body weight (%) in NSG mice (Jeko-1 / PD-L1 tumor model) following treatment with 1x106(FIG.22A) and 5x106(FIG.22B) NextGen CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2) and control CD19 UltraCAR-T cells without PD-1 silencer (produced using Vector 1).

[0080] FIGs.23A and 23Bare dot plots showing HER1t concentration in NSG mice (Jeko-1 / PD- L1 tumor model) following treatment with 1x106(FIG.23A) and 5x106(FIG.23B) NextGen CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2) and control CD19 UltraCAR-T cells without PD-1 silencer (produced using Vector 1). FIGs.23C and 23D are a dot plots showing PD-1 levels in NSG mice (Jeko-1 / PD-L1 tumor model) following treatment with 1x106(FIG.23C) and 5x106(FIG. 23D) NextGen CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2) and control CD19 UltraCAR-T cells without PD-1 silencer (produced using Vector 1). FIGs.23E and 23Fare dot plots showing memory cell concentration in NSG mice (Jeko-1 / PD-L1 tumor model) following treatment with 1x106(FIG. 23E) and 5x106(FIG. 23F) NextGen CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2) and control CD19 UltraCAR-T cells without PD-1 silencer (produced using Vector 1).

[0081] FIGs.24A-24B are dot plots of tumor burden (FIG.24A) and HER1t concentration (FIG. 24B) in Raji tumor model utilizing NSG mice treated with saline (HBSS), CD19 CAR-T cells expressing HER1t but not mbIL-15, and not carrying a PD-1 silencer (produced using Vector 5), control CD19 UltraCAR-T cells without PD-1 silencer (produced using Vector 1), or NextGen CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2).

[0082] FIGs.25A-25C are dot plots of HER1t concentration in the blood (FIG.25A), spleen (FIG. 25B), and bone marrow (BM, FIG. 25C) of humanized mice models following treatment with saline (HBSS) or NextGen CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2).

[0083] FIGs. 26A-26C are dot plots of CD19+ B-cell concentration in the blood (FIG. 26A), spleen (FIG. 26B), and bone marrow (BM, FIG. 26C) of humanized mice models following treatment with saline (HBSS) or NextGen CD19 UltraCAR-T cells with PD-1 silencer (produced using Vector 2). 12

[0084] FIGs.27A-27H show the results of NSG MHC Class I / II KO mice (NALM-6 tumor model) in a primary challenge and a rechallenge study. The mice were treated with: (i) CD19 CAR-T cells not expressing mbIL15 nor carrying a PD-1 silencer (produced using Vector 3) (FIG. 27A and 27C) or (ii) CD19 UltraCAR-T cells expressing mbIL-15 and carrying a PD-1 silencer (produced using Vector 2) (FIG.27B and 27D). FIG.27A-27D show dot plots of tumor burden as measused by total flux. FIG.27E is an schematic of an exemplary study protocol. FIG.27F-27G are dot plots of tumor burden of naïve mice (FIG. 27F) or tumor free mice previously treated with Vector 2 (FIG.27G) following rechallenge with NALM-6 tumor. FIG.27H shows a survival curve of naïve mice and tumor free mice following tumor rechallenge.

[0085] FIGs.28A-28I show manufacturing of NextGen CD19 UltraCAR-T cells (produced using Vector 2) from an SLE / LN donor and a healthy donor. FIG.28A is a bar graph of the transfection efficiency of T cells. FIG.28B-28E is a series of graphs showing that NextGen CD19 CAR-T cells generated from an SLE / LN donor and a healthy donor exhibited similar T cell phenotypes as measured by the percentage of cells having a (FIG. 28B) CD4+phenotype, (FIG. 28C) CD8+phenotype, (FIG. 28D) ratio of CD4+and CD8+, and (FIG. 28E) memory cell phenotype (using CD45RA and CD62L markers). FIG.28F-28I is a series of bar graphs and dot plots showing that Nextgen CD19 CAR-T cells (produced using Vector 2) generated from an SLE / LN donor and a healthy donor elicited a complete autologous B-Cell depletion in vitro. FIG.28F depicts the result of a co-culture experiment of Target cells (B-cells) and effector cells (CAR-T) cells in different ratios, with the bar graph showing the percentage of CD19+B-cells killed by NextGen CD19 UltraCAR-T cells after electroporation and overnight resting. FIG. 28G is a dot plot showing successful expansion of CAR-T cells by measuring CAR-T cell counts over time post transfection. FIGs.28H-28I are bar graphs showing the percentage of CD19+B-cells killed by NextGen CD19 UltraCAR-T cells after 3 weeks of expansion and 24 hours (FIG.28H) or 48 hours (FIG.29I) of exposure of B-cells to the CAR-T cells.

[0086] FIGs. 29A-29M show that Nextgen CD19 CAR-T cells (Vector 2) generated from an SLE / LN donor and a healthy donor elicited a complete autologous B-Cell depletion in a humanized MHC I / II double knock out (DKO) mice. FIG.29A is an exemplary schematic of the study design. FIGs. 29B-29C are dot plots showing the B-cell count in blood after 7 and 14 days without and with autologous CAR-T treatment prepared either from the healthy donor (HD) (FIG.29B) or the 13  SLE / LN donor (FIG. 29C). FIG. 29D is a bar graph showing the frequency of CD19+viable B- cells in blood, which was utilized to calculate the percentage killing in vivo. FIG.29E is a dot plot showing the expansion of HER1t+ CAR-T cells in blood after 7 days (study day 14) and 14 days (study day 21) following administration of the NextGen CD19 UltraCAR-T cells. FIGs.29F-29G are dot plots showing the B-cell count in bone marrow after 7 and 14 days without and with autologous CAR-T treatment prepared either from a healthy donor (HD) (FIG.29F) or an SLE / LN donor (FIG.29G). FIG.29H is a bar graph showing the frequency of CD19+viable B-cells in bone marrow, which was utilized to calculate the percentage killing in vivo. FIG. 29I is a dot plot showing the expansion of HER1t+ CAR-T cells in bone marrow (BM) after 7 days (study day 14) and 14 days (study day 21) following administration of the NextGen CD19 UltraCAR-T cells. FIGs.29J-29K are dot plots showing the B-cell count in spleen tissue after 7 and 14 days without and with autologous CAR-T treatment prepared either from healthy donor (HD) (FIG. 29J) or SLE / LN donor (FIG. 29K). FIG. 29L is a bar graph showing the frequency of CD19+viable B- cells in spleen tissue, which was utilized to calculate the percentage killing in vivo. FIG.29M is a dot plot showing the expansion of HER1t+ CAR-T cells in spleen tissue after 7 days (study day 14) and 14 days (study day 21) following administration of the NextGen CD19 UltraCAR-T cells. DETAILED DESCRIPTION OF THE INVENTION

[0087] The following description and examples illustrate embodiments of the present disclosure in detail.

[0088] It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Those of skill in the art will recognize that there are variations and modifications of the present disclosure, which are encompassed within its scope.

[0089] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0090] The section headings used herein are for organizational purposes only and are not to be 14  construed as limiting the subject matter described.

[0091] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. Definitions

[0092] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0093] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0094] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0095] Use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting; i.e., “including” does not mean “limited to.”

[0096] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. 15

[0097] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional possible components, elements, or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0098] The term “about” signifies a range of values that deviate slightly from a specific numerical value. This deviation typically encompasses the inherent experimental variability associated with biological assays and measurements commonly used in the biotechnological arts. The acceptable range for “about” can vary depending on the specific context and the parameter being measured. However, it is generally understood to be within ±5-10% of the stated value. For example, the phrase “about 10 mg” could be interpreted as a range from 9.5 mg to 10.5 mg, acknowledging potential variations due to factors like weighing errors or instrument limitations. When the term “about” precedes a listing of numerical values or percentages, the term “about” indicates that the value can be about any of the recited numerical values or percentages. For example, the phrase “about 80%, 85%, 90%, 98%, or 99%,” means “about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%.”

[0099] The term “approximately” is similar to “about” but can imply a slightly larger range of deviation from the stated value. It suggests a range within ±10-15% of the specified number. This term is often used when the precise value is less critical or when a wider range of acceptable values is expected due to inherent biological variability. For example, “approximately 2 hours” for an incubation step might be acceptable even if the actual incubation time varies slightly between experiments due to factors like temperature fluctuations. When the term “approximately” precedes a listing of numerical values or percentages, the term “approximately” indicates that the value can be approximately any of the recited numerical values or percentages. For example, the phrase “approximately 10, 15, 20, or 25,” means “approximately 10, approximately 15, approximately 20, or approximately 25.” 16

[0100] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0101] The term “isolated” and its grammatical equivalents as used herein refer to the removal of a nucleic acid, protein, polypeptide, cell, or other material from its natural environment. The term “purified” and its grammatical equivalents as used herein refer to a molecule or composition, whether removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions, that has been increased in purity, wherein “purity” is a relative term, not “absolute purity.” It is to be understood, however, that nucleic acids and proteins can be formulated with diluents or adjuvants and still for practical purposes be isolated. For example, nucleic acids typically are mixed with an acceptable carrier or diluent when used for introduction into cells. The term “substantially purified” and its grammatical equivalents as used herein refer to a nucleic acid sequence, polypeptide, protein or other compound which is essentially free, i.e., is more than about 50% free of, more than about 70% free of, more than about 90% free of, the polynucleotides, proteins, polypeptides and other molecules that the nucleic acid, polypeptide, protein or other compound is naturally associated with.

[0102] “Nucleic acid,” “nucleic acid molecule,” “polynucleotide,” “polynucleotide construct,” “oligonucleotide,” and their grammatical equivalents as used herein refer to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, triplex DNA, as well as double and single stranded RNA It also includes modified, for example, by methylation and / or by capping, and unmodified forms of the polynucleotide. The term is also meant to include molecules that include non-naturally occurring, synthetic, and semi-synthetic nucleotides and polynucleotides as well as nucleotide analogs. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving 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 polynucleotide” is a polynucleotide that has undergone a 17  molecular biological manipulation. The polynucleotide sequences and vectors disclosed or contemplated herein can be introduced into a cell by, for example, transfection, transformation, or transduction.

[0103] The term “fragment,” as applied to a polynucleotide or nucleic acid sequence, refers to a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least 6, 8, 9, 10, 12, 15, 18, 20, 21 , 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51 , 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000, or more consecutive nucleotides of a nucleic acid according to the invention.

[0104] As used herein, an “isolated polynucleotide” or “isolated nucleic acid fragment” refers to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non- natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

[0105] The term “gene” and its grammatical equivalents refers to a polynucleotide comprising nucleotides that encode a functional molecule, including functional molecules produced by transcription only (e.g., a bioactive RNA species) or by transcription and translation (e.g., a polypeptide). The term “gene” encompasses cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expresses a specific RNA, protein or polypeptide, including regulatory sequences preceding (5’ non-coding sequences) and following (3’ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and / or coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A chimeric gene may comprise coding sequences derived from different sources and / or regulatory sequences derived from different sources. 18  “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene or “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0106] The term “genome” includes chromosomal as well as mitochondrial, chloroplast and viral DNA or RNA. The term “probe” refers to a single-stranded nucleic acid molecule that can base pair with a complementary single stranded target nucleic acid to form a double-stranded molecule.

[0107] “Heterologous DNA” refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. The heterologous DNA may include an exogenous gene. “Exogenous gene” means a gene foreign to the subject, that is, a gene which is introduced into the subject through a transformation process, an unmutated version of an endogenous mutated gene or a mutated version of an endogenous unmutated gene. Exogenous genes can be either natural or synthetic genes which are introduced into the subject in the form of DNA or RNA which may function through a DNA intermediate such as by reverse transcriptase. Such genes can be introduced into target cells, directly introduced into the subject, or indirectly introduced by the transfer of transformed cells into the subject.

[0108] A “primer” refers to an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction or for DNA sequencing.

[0109] A DNA “coding sequence” or “coding region” refers to a double-stranded DNA sequence that encodes a polypeptide and can be transcribed and translated into a polypeptide in a cell, ex vivo, in vitro or in vivo when placed under the control of suitable regulatory sequences. “Suitable regulatory sequences” refers to nucleotide sequences located upstream (5’ non-coding sequences), within, or downstream (3’ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, 19  polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem- loop structures. The boundaries of the coding sequence are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is intended for expression in an eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3’ to the coding sequence.

[0110] “Open reading frame” is abbreviated ORF and refers to a length of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

[0111] The term “downstream” refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0112] The term “upstream” refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0113] The term “response element” refers to one or more cis-acting DNA elements which confer responsiveness on a promoter mediated through interaction with the DNA- binding domains of a transcription factor. This DNA element may be either palindromic (perfect or imperfect) in its sequence or composed of sequence motifs or half sites separated by a variable number of nucleotides. The half sites can be similar or identical and arranged as either direct or inverted repeats or as a single half site or multimers of adjacent half sites in tandem. The response element may comprise a minimal promoter isolated from different organisms depending upon the nature of the cell or organism into which the response element is incorporated. The DNA binding domain of the transcription factor binds, in the presence or absence of a ligand, to the DNA sequence of a 20  response element to initiate or suppress transcription of downstream gene(s) under the regulation of this response element.

[0114] The term “operably linked” as used herein refers to refers to the physical and / or functional linkage of a DNA segment to another DNA segment in such a way as to allow the segments to function in their intended manners. A DNA sequence encoding a gene product is operably linked to a regulatory sequence when it is linked to the regulatory sequence, such as, for example, promoters, enhancers and / or silencers, in a manner which allows modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter, in the correct reading frame with respect to the transcription initiation site and allows transcription elongation to proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence coding for a gene product when it is ligated to the DNA sequence in such a manner as to increase or decrease, respectively, the transcription of the DNA sequence. Enhancers and silencers can be located upstream, downstream or embedded within the coding regions of the DNA sequence. A DNA for a signal sequence is operably linked to DNA coding for a polypeptide if the signal sequence is expressed as a pre-protein that participates in the secretion of the polypeptide. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or via adapters or linkers inserted in the sequence using restriction endonucleases known to one of skill in the art.

[0115] As used herein, the term “codon degenerate variant” refers to a modified nucleic acid sequence that encodes the same amino acid sequence as the original sequence but differs in the specific nucleotides comprising the codons. The genetic code is degenerate, meaning that multiple codons can code for the same amino acid. For example, the amino acid leucine can be encoded by six different codons: CTG, CTT, CTC, CTA, TTG, and TTA. A codon degeneracy table, also known as a genetic code table or codon table, is a chart that provides information about the relationship between codons (sequences of three nucleotides) and the corresponding amino acids they encode. The table lists the 64 possible codons and indicates which amino acid each codon represents. Table 1 is an example of a codon degeneracy table: Table 1: Codon Degeneracy Table 21  Nucleotide Position in Codon – Amino Acid Encoded First Second nucleotide Third tideNA; both of which form complementary base-pairs with “A” (Adenine); “*” indicates stop codons.)

[0116] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0117] Furthermore, publicly available software resources are readily available for computer- generated “reverse-translation,” also known as, “back translation” of polypeptide sequences, i.e., converting polypeptide sequences into nucleotide sequences encoding same). See, e.g., Madeira, F., et al., Nucleic Acids Res, 47(Wl), W636-W641 (2019); Madeira, F., et al., Curr Protoc in Bioinformatics, 66(1):e74 (2019); Chojnacki, S, et al., Nucleic Acids Res. 2017 Jul 3;45(Wl):W550-W553 (2017); Athey, J., et al., BMC Bioinformatics 18:391 (2017).

[0118] As used herein, a codon degenerate variant may be utilized to optimize gene expression or 22  enhance protein production. By modifying the codons within a nucleic acid sequence, it is possible to utilize codons that are more frequently used or preferred by the host organism’s translational machinery. This can lead to increased efficiency in protein expression or improved compatibility with a specific host organism.

[0119] The term “expression” as used herein refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.

[0120] The terms “cassette,” “expression cassette” and “gene expression cassette” refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination. The segment of DNA comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation. “Transformation cassette” refers to a specific vector comprising a polynucleotide that encodes a polypeptide of interest and having elements in addition to the polynucleotide that facilitate transformation of a particular host cell. Cassettes, expression cassettes, gene expression cassettes and transformation cassettes of the invention may also comprise elements that allow for enhanced expression of a polynucleotide encoding a polypeptide of interest in a host cell. These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like. The expression cassettes described herein may comprise a total length of between about 500 to about 10,000 bp, about 1,000 to about 5,000 bp, about 1,500 to about 4,500 bp, about 1,800 to about 4,400 bp, about 2,000 to about 4,500 bp, about 2,100 to about 4,400 bp, about 2,200 to about 4,300 bp, about 2,300 to about 4,200 bp, about 2,400 to about 4,100 bp, about 2,500 to about 4,000 bp, about 2,600 to about 3,900 bp, about 2700 to about 3800 bp, about 2,800 to about 3,800 bp, about 2,900 to about 3,700 bp, about 3,000 to about 3,600 bp, about 3,100 to about 3,500 bp, about 3,150 to about 3,450 bp, about 3,200 to about 3,400 bp, about 3,250 to about 3,350 bp, or about 3300 bp. Alternatively, the expression cassette may comprise any number of base pairs falling within these ranges. For instance, the expression cassette may comprise about 500 bp, about 750 bp, about 1,000 bp, about 1,250 bp, about 1,500 bp, about 1,750 bp, about 2,000 bp, about 2,250 bp, about 2,500 bp, about 23  2,550 bp, about 2,600 bp, about 2,650 bp, about 2,700 bp, about 2,750 bp, about 2,800 bp, about 2,850 bp, about 2,900 bp, about 2,950 bp, about 3000 bp, about 3,050 bp, about 3,100 bp, about 3,150 bp, about 3,200 bp, about 3,250 bp, about 3,300 bp, about 3,350 bp, about 3,,400 bp, about 3450 bp, about 3,500 bp, about 3,550 bp, about 3,600 bp, about 3,650 bp, about 3,700 bp, about 3,750 bp, about 3,800 bp, about 3,850 bp, about 3,900 bp, about 3,950 bp, about 4,000 bp, about 4,050 bp, about 4,100 bp, about 4,150 bp, about 4,200 bp, about 4,250 bp, about 4,300 bp, about 4,350 bp, about 4,400 bp, about 4,450 bp, or about 4,500 bp. In one aspect, the expression cassette comprises about 2,800 bp. In another aspect, the expression comprises 2,825 bp.

[0121] As used herein, the term “vector” refers to any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, eosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both, viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. Possible vectors include, for example, plasmids or modified viruses including, for example bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives, or the Bluescript vector. Another example of vectors that are useful in the invention is the ULTRAVECTOR®Production System (Intrexon Corp., Blacksburg, VA) as described in WO 2007 / 038276. For example, the insertion of the DN fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate DNA fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the DNA molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) into the DNA termini. Such vectors may be engineered to contain selectable marker genes that provide for the selection of cells that have incorporated the marker into the cellular genome. Such markers allow identification and / or selection of host cells that incorporate and express the proteins encoded by the marker.

[0122] As used herein, the term “plasmid” refers to an extra-chromosomal element often carrying 24  a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0123] As used herein, the terms “cloning vector” and “replicon” refer to a unit length of a nucleic acid, preferably DNA, that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. Cloning vectors may be capable of replication in one cell type and expression in another (“shuttle vector”). Cloning vectors may comprise one or more sequences that can be used for selection of cells comprising the vector and / or one or more multiple cloning sites for insertion of sequences of interest.

[0124] As used herein, the term “viral vector” as used herein refers to a virus, viral particle, or derivative thereof, capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. Viral vectors, and particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. In addition to a nucleic acid, a vector may also comprise one or more regulatory regions, and / or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).

[0125] As used herein, the term “adenovirus” and “adenoviral vector” as used herein, refers to an adenovirus that retains the ability to participate in the adenovirus life cycle and / or which has been physically inactivated by, for example, disruption (e.g., sonication), denaturing (e.g., using heat or solvents), or cross-linkage (e.g., via formalin cross-linking). The “adenovirus life cycle” includes 25  (1) virus binding and entry into cells, (2) transcription of the adenoviral genome and translation of adenovirus proteins, (3) replication of the adenoviral genome, and (4) viral particle assembly (see, e.g., Fields Virology, 5thed., Knipe et al. (eds.), Lippincott Williams & Wilkins, Philadelphia, PA (2006)). Adenoviruses, as used and described herein may also be rendered replication deficient (i.e., do not retain ability to participate in the adenovirus life cycle) by deletion of one or more parts of the naturally occurring viral genome. “Adenoviruses” and “Adenoviral vector,” as used and described herein, may include an adenovirus in which the adenoviral genome has been manipulated to accommodate a nucleic acid sequence that is non-native with respect to the adenoviral genome. Typically, an adenoviral vector is generated by introducing one or more mutations (e.g., a deletion, insertion, or substitution) into the adenoviral genome of the adenovirus so as to accommodate the insertion of a non-native nucleic acid sequence, for example, for gene transfer, into the adenovirus.

[0126] As used herein, the terms “MOI” or “Multiplicity of Infection” refer to the average number of virus particles that infect a single cell in a specific experiment (e.g., recombinant virus or control virus).

[0127] As used herein, the terms “transfection,” “transduction,” and “nucleofection” refer to the uptake of exogenous or heterologous RNA or DNA by a cell. A cell has been “transfected” by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced inside the cell. A cell has been “transformed” by exogenous or heterologous RNA or DNA when the transfected RNA or DNA effects a phenotypic change. The transforming RNA or DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

[0128] As used herein, the term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

[0129] As used herein, the term terms “induce,” “induction” and their grammatical equivalents as used herein refer to an increase in nucleic acid sequence transcription, promoter activity and / or expression brought about by a transcriptional regulator, relative to some basal level of 26  transcription.

[0130] As used herein, the terms “promoter” and “promoter sequence” are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3’ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the 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. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters.” Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters.” Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0131] The promoter sequence is typically bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is found a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0132] The source of the promoter that is inserted into the gene switch can be natural or synthetic, and the source of the promoter should not limit the scope of the invention described herein. In other words, the promoter may be directly cloned from cells, or the promoter may have been previously cloned from a different source, or the promoter may have been synthesized. 27

[0133] As used herein, the term “transcriptional regulator” refers to a biochemical element that acts to prevent or inhibit the transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein), or to permit or stimulate the transcription of the promoter-driven DNA sequence under certain environmental conditions (e.g., an inducer or an enhancer).

[0134] As used herein, the term “enhancer” refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly- used CMV promoter) also comprise enhancer sequences. Enhancers can be located upstream, within, or downstream of coding sequences. The term “Ig enhancers” refers to enhancer elements derived from enhancer regions mapped within the immunoglobulin (Ig) locus (such enhancers include for example, the heavy chain (mu) 5’ enhancers, light chain (kappa) 5’ enhancers, kappa and mu intronic enhancers, and 3’ enhancers (see generally Paul W. E. (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363; and U.S. Pat. No.5,885,827).

[0135] A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.

[0136] “Transcriptional and translational control sequences” refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences. Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor 1 (EF 1) enhancer, yeast enhancers, viral gene enhancers, and the like. 28

[0137] The terms “3’ non-coding sequences” and “3’ untranslated region (UTR)” refer to DNA sequences located downstream (3’) of a coding sequence and may comprise polyadenylation [poly(A)] recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3’ end of the mRNA precursor.

[0138] As used herein, the term “regulatory region” refers to a nucleic acid sequence that regulates the expression of a second nucleic acid sequence. A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin that are responsible for expressing different proteins or even synthetic proteins (a heterologous region). In particular, the sequences can be sequences of prokaryotic, eukaryotic, or viral genes or derived sequences that stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, promoters, enhancers, transcriptional termination sequences, and signal sequences which direct the polypeptide into the secretory pathways of the target cell.

[0139] As used herein, the term “modulate” means to induce, reduce or inhibit nucleic acid or gene expression, resulting in the respective induction, reduction or inhibition of protein or polypeptide production.

[0140] As used herein, the term “CAP” or “cap” refers to a modified nucleotide, generally a 7- methyl guanosine, linked 3’ to 5’ (7meG-ppp-G), to the 5’ end of a eukaryotic mRNA, that serves as a required element in the normal translation initiation pathway during expression of protein from that mRNA.

[0141] As used herein, the term “Sleeping Beauty (SB) Transposon System” refers to a synthetic Tc1 / mariner transposon system for introducing DNA sequences into a cell or into the chromosomes of vertebrates. Some exemplary embodiments of the system are described, for example, in U.S. Pat. Nos.6,489,458, 8,227,432, 9,228,180 and WO / 2016 / 145146. The Sleeping Beauty transposon system is composed of a Tc1 / mariner transposase called a Sleeping Beauty (SB) 29  transposase and a SB transposon. In embodiments, the Sleeping Beauty transposon system can include the SB11 transposon system, the SB100X transposon system, or the SB110 transposon system.

[0142] As used herein, the term “transposon” or “transposable element” (TE) refers to a vector DNA sequence that can change its position within the genome, sometimes creating or reversing mutations and altering the cell’s genome size. Transposition often results in duplication of the TE. Class I TEs are copied in two stages: first they are transcribed from DNA to RNA, and the RNA produced is then reverse transcribed to DNA This copied DNA is then inserted at a new position into the genome. The reverse transcription step is catalyzed by a reverse transcriptase, which can be encoded by the TE itself The characteristics of retrotransposons are similar to retroviruses, such as HIV. The cut-and-paste transposition mechanism of class II TEs does not involve an RNA intermediate. The transpositions are catalyzed by several transposase enzymes. Some transposases non-specifically bind to any target site in DNA, whereas others bind to specific DNA sequence targets. The transposase makes a staggered cut at the target site resulting in single-strand 5’ or 3’ DNA overhangs (sticky ends). This step cuts out the DNA transposon, which is then ligated into a new target site; this process involves activity of a DNA polymerase that fills in gaps and of a DNA ligase that closes the sugar-phosphate backbone. This results in duplication of the target site. The insertion sites of DNA transposons can be identified by short direct repeats which can be created by the staggered cut in the target DNA and filling in by DNA polymerase, followed by a series of inverted repeats important for the TE excision by transposase. Cut-and-paste TEs can be duplicated if their transposition takes place during S phase of the cell cycle when a donor site has already been replicated, but a target site has not yet been replicated. Transposition can be classified as either autonomous or non-autonomous in both Class I and Class II TEs. Autonomous TEs can move by themselves while non-autonomous TEs require the presence of another TE to move. This is often because non-autonomous TEs lack transposase (for class II) or reverse transcriptase (for class I).

[0143] As used herein, the term “transposase” refers to an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. 30

[0144] As used herein, the terms “polypeptide,” “peptide,” “polypeptide construct,” and “peptide construct” and their grammatical equivalents, refer to a polymeric compound comprised of covalently linked amino acid residues. A “mature protein” is a protein which is full-length and which, optionally, includes glycosylation or other modifications typical for the protein in a given cellular environment. As disclosed herein, embodiments of the invention include HPV antigens / antigenic polypeptides, peptides, and mature proteins described herein and also polynucleotides (DNA or RNA) that encode the same. Polypeptides and proteins disclosed herein (including functional fragments and functional variants thereof) can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, α- amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4- hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4- carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α- naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N’-benzyl-N’-methyl-lysine, N’,N’-dibenzyl-lysine, 6-hydroxylysine, omithine, α- aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbomane)-carboxylic acid, α,γ-diaminobutyric acid, α,β- diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0145] As used herein, the term “polypeptide fragment” refers to a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence. Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part. Such fragments of a polypeptide according to the invention may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300 or more amino acids.

[0146] As used herein, the terms “isolated polypeptide,” “isolated peptide,” or “isolated protein” refer to a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is not meant to exclude artificial or synthetic mixtures with other 31  compounds, or the presence of impurities which do not interfere with biological activity, and which may be, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.

[0147] As used herein, the term “identical” or “sequence identity” in the context of two nucleic acid sequences or amino acid sequences of polypeptides refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A “comparison window,” as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J Mal. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad Sci US.A., 85:2444 (1988); by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC / Gene program by Intelligentics, Mountain View Calif, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is well described by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is also often performed by inspection and manual alignment.

[0148] In one class of embodiments, the polypeptides herein are at least about 80%, 85%, 90%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to a reference polypeptide, or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to a reference nucleic acid or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) 32  using default parameters. When one molecule is said to have certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, said percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned.

[0149] As used herein, the term “percent identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described above or in, e.g., Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using sequence analysis software such as the MegAlign (or more recently MegAlign Pro) program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences may be performed using a Clustal method of alignment (Higgins et al., CABIOS.5:151 1989) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using a Clustal method may be selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0150] As used herein, the term “substantially similar” and its grammatical equivalents as applied to nucleic acid or amino acid sequences mean that a nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, such as at least 95%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and at least 99.99% sequence identity to a reference sequence using the comparison programs described above, e.g., BLAST, using standard 33  parameters. The term “substantially identical” and its grammatical equivalents as applied to nucleic acid or amino acid sequences mean that a nucleic acid or amino acid sequence comprises a sequence that has at least 99%, such as at least at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and at least 99.99% sequence identity to a reference sequence using the comparison programs described above, e.g., BLAST, using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. In embodiments, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, over a region of at least about 100 residues, and in embodiments, the sequences are substantially identical over at least about 150 residues. In embodiments, the sequences are substantially identical over the entire length of the coding regions.

[0151] Also contemplated and included herein are nucleic acid molecules that hybridize to the disclosed sequences. Hybridization conditions may be mild, moderate, or stringent, as is warranted. Appropriate stringency conditions which promote DNA hybridization, for example, 6× sodium chloride / sodium citrate (SSC) at about 45° C, followed by a wash of 2×SSC at 50° C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. As used herein, the phrase “stringent hybridization conditions” refers to those conditions that include a salt concentration of about 1.0 M NaCl in 50% formamide, at a temperature of about 37° C for about 4 to 12 hours, followed by a wash in 0.1×SSC at about 60- 34  65° C.

[0152] As used herein, the term “functional fragment,” or its grammatical equivalents, is used herein to mean a portion, fragment, or segment of a biological molecule that retains the essential functional characteristics or activities of the original biological molecule.

[0153] The term “functional variant,” or its grammatical equivalents, is used herein to mean a modified form of a biological molecule that retains the essential functional characteristics or activities of the original molecule while exhibiting some degree of variation. It includes a biological molecule that has been altered, such as through genetic engineering or mutagenesis techniques, to introduce specific changes while preserving the biological molecule’s overall functionality. A functional variant may have one or more amino acid substitutions, insertions, or deletions compared to the original molecule, while still maintaining the desired biological activity or function. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art. In one embodiment, a variant biological comprises at least about 14 monomers (e.g., nucleotides or amino acids). In certain embodiments, the functional variant of a referenced amino acid sequence has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the referenced amino acid sequence and / or is a conservatively-substituted variant of the referenced sequence. When used with reference to a nucleic acid, the phrase “functional variant” refers to a nucleic acid that differs from the referenced nucleic acid but encodes a polypeptide having the same primary function as the polypeptide encoded by the referenced nucleic acid. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the referenced nucleic acid sequence, hybridizes under stringent hybridization conditions with the complement of the referenced nucleic acid sequence, or is a codon degenerate variant of the nucleic acid sequence.

[0154] As used herein, the term “homology” in all its grammatical forms and spelling variations refers to the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known to the art. For example, homology can be determined by a direct comparison of the 35  sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s) and size determination of the digested fragments.

[0155] The term “substitution,” when used in the context of an amino acid sequence refers to a variation wherein one amino acid in the amino acid sequence is replaced by another. The nomenclature used to denote amino acid substitutions follows a standardized format. Taking “L50G” as an example, “L” represents the amino acid leucine (abbreviated as “L”) at the original position, “50” signifies the position of the amino acid in the amino acid sequence in relation to the N-terminus thereof (in this case, the amino acid is the 50thamino acid from the N-terminus of the sequence), and “G” indicates the substituted amino acid, in this instance, glycine (abbreviated as “G”). Therefore, an “L50G” denotes a substitution where leucine at position 50 of the amino acid sequence (relative to the N-terminus thereof) has been replaced by glycine.

[0156] As used herein, the term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (see Schulz, G. E. and Schirmer, R.H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, for example, lysine for arginine and vice versa such that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; serine for threonine such that a free -OH can be maintained; and glutamine for asparagine such that a free -NH2 can be maintained. Exemplary conservative amino acid substitutions are shown in the following chart: 36  Table 2. Exemplary conservative amino acid substitutions

[0157] An amino acid sequence that differs from a reference amino acid sequence by only conservative amino acid substitutions will be referred to herein as a “conservatively-substituted variant” of the reference sequence. Given the established knowledge and well-known techniques in protein science, it is well within the skill of a person of ordinary skill in the art to determine the functional impact of a “conservatively-substituted variant” as compared to the reference amino acid sequence.

[0158] In some embodiments, the functional variant may be a conservatively-substituted variant of the reference sequence. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by 100 or fewer conservative amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by 90 or fewer amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by 80 or fewer amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by 70 or fewer conservative amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by 60 or fewer conservative amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by 50 or fewer conservative amino acid substitutions. In some embodiments, the 37  conservatively-substituted variant may differ from the amino acid sequence of the reference protein by 40 or fewer conservative amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by 30 or fewer conservative amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by 20 or fewer conservative amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by 10 or fewer conservative amino acid substitutions. In some embodiments, the conservatively-substitute variant may differ from the reference sequence by 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conservative amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by at least 100 and 150 conservative amino acid substitutions. In some embodiments, the conservatively-substituted variant may differ from the amino acid sequence of the reference sequence by at least 150 conservative amino acid substitutions.

[0159] An amino acid sequence that differs from a reference amino acid sequence by at least one non-conservative amino acid substitution will be referred to herein as a “non-conservatively- substituted variant” of the reference sequence. As used herein, the term “non-conservative amino acid substitution” refers to an amino acid substitution between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non- conservative amino acid substitution to not interfere with, or inhibit the biological activity of, the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the homologous parent protein. Amino acid substitutability is discussed in more detail, for example, in L. Y. Yampolsky and A. Stoltzfus, “The Exchangeability of Amino acids in Proteins,” Genetics 2005 Aug.; 170(4):1459-1472. Given the established knowledge and well-known techniques in protein science, it is well within the skill of a person of ordinary skill in the art to determine the functional impact of a non-conservative amino acid substitution in a functional variant as compared to the reference amino acid sequence. 38

[0160] In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by at least one non-conservative amino acid substitution. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by between ten and 20 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by between 21 and 30 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by between 31 and 40 non- conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by between 41 and 50 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by between 51 and 60 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by between 61 and 70 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by between 71 and 80 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by between 81 and 90 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by between 91 and 100 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by at least 100 non-conservative amino acid substitutions.

[0161] As used herein, the term “antibody” refers to monoclonal or polyclonal antibodies. The term “monoclonal antibodies,” as used herein, refers to antibodies that are produced by a single clone of B-cells and bind to the same epitope. In contrast, “polyclonal antibodies” refer to a population of antibodies that are produced by different B-cells and bind to different epitopes of the same antigen. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each 39  of the heavy chains contains one N-terminal variable domain (VH domain, also referred to as a VH region) and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain contains one N-terminal variable domain (VLdomain, also referred to as a VLregion) and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH domain and VL domain have a similar general structure, with each region comprising four framework regions, whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDRI, CDR2, and CDR3, form the “hypervariable region” of an antibody, which is responsible for antigen binding.

[0162] As used herein, the term “functional antibody fragment” and “functional fragment of an antibody,” or their grammatical equivalents, are used interchangeably to mean a portion, fragment, or segment of the antibody that retains the essential functional characteristics or activities of the original antibody. In one embodiment, that activity is the ability to specifically bind to an antigen. (See, generally, Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). The functional antibody fragment may comprise, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Non-limiting examples of functional antibody fragments include: (i) an antigen-binding fragment (Fab), which is a monovalent fragment consisting of the VL domain, VH dp,aom, CL domain, and CH1 domain; (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the stalk region; (iii) a variable fragment (“Fv”) consisting of the VL and VH domains of a single arm of an antibody; (iv) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VLand VHdomains) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778 (1998)) and (v) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VHdomain connected to a VL domain by a peptide linker that is too short to allow pairing between the VH domain and VL domain on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH-VLdomain polypeptide chains to generate a dimeric molecule having two functional antigen binding sites. Functional antibody fragments are known 40  in the art and are described in more detail in, e.g., U.S. Patent 8,603,950.

[0163] As used herein, the term “antibody-like molecules” can be for example proteins that are members of the Ig-superfamily which are able to selectively bind a partner. MHC molecules and T cell receptors are such molecules. In one embodiment, the antibody-like molecule is a TCR. In one embodiment, the TCR has been modified to increase its MHC binding affinity.

[0164] As used herein, the term “antigen recognition moiety” or “antigen recognition domain” refers to a molecule or portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, antibody-like molecule or fragment thereof and the antigen is a tumor antigen.

[0165] As used herein, the term “immune cells” includes dendritic cells, macrophages, neutrophils, mast cells, eosinophils, basophils, natural killer cells and lymphocytes (e.g., B and T cells).

[0166] As used herein, the terms “T cell” or “T lymphocyte” refer to a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T- cell receptor (TCR) on the cell surface. “TCRs” are protein molecules found on the surface of T cells, which are a type of white blood cell involved in the adaptive immune response. A TCR’s variable domain contains the highly polymorphic loops referred to as complementarity determining regions (CDRs), which are responsible for binding to the peptide-presenting MHC. There are two major forms of TCRs: αβ TCR and γδ TCR. Both forms consist of two protein chains, known as alpha (α) and beta (β) chains for αβ TCRs and gamma (γ) and delta (δ) chains for γδ TCRs. These chains come together to form a heterodimeric structure. The majority of T cells in the human immune system express αβ TCRs. The α chain and β chain of αβ TCRs are encoded by separate gene segments, which undergo recombination during T cell development to generate diverse TCR specificities. The α and β chains each contain variable (V), diversity (D), and joining (J) gene segments, similar to the antibody gene rearrangement process. The combination of V, D, and J gene segments contributes to the unique antigen-binding specificity of the αβ TCR. The αβ TCR recognizes antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of 41  antigen-presenting cells. In contrast to αβ TCRs, γδ TCRs are less prevalent in the immune system but still play important roles. The γ and δ chains of γδ TCRs are also encoded by separate gene segments and undergo recombination during T cell development. The γδ TCR gene rearrangement process is distinct from that of αβ TCRs. γδ T cells often exhibit a tissue-specific distribution and are found in epithelial tissues, such as the skin and gut. γδ TCRs can recognize a variety of antigens, including certain peptides and non-peptide molecules, independently of MHC presentation. Both αβ TCRs and γδ TCRs participate in immune surveillance and response, but they have different functions and specificities. The αβ TCRs are predominantly involved in recognizing peptides presented by major histocompatibility complex (MHC) molecules, while γδ TCRs can have more diverse antigen recognition capabilities.

[0167] TCRs, and constructs encoding TCRs, that recognize MHC–antigen complexes, can be generated and introduced into T cells (known as TCR T cells), and the ensuing TCR–peptide-MHC interaction can be exploited to trigger an immune response. Greenbaum et al., Cancer Immunol Res 1 November 2021; 9 (11): 1252–1261. There is an interest in using TCRs with higher than normal range of affinity for peptide-MHC antigens (type I), referred to as high affinity TCRs, to: 1) driving the activity of CD4 helper T cells (which do not have a CD8 co-receptor), or 2) developing soluble TCRs that can be used to directly target cells by attaching “effector” molecules (e.g., antibody Fc regions, toxic drugs, or antibody scFvs such as anti-CD 3 antibodies to form bispecific proteins) (Ashfield and Jakobsen, IDrugs,9,554-9 (2006); Foote and Eisen Proc Natl Acad Sci U S A, 97:10679-81 (2000); Holler et al., Proc Natl Acad Sci U S A, 97:5387-92 (2000); Molloy et al., Curr Opin Pharmacol, 5:438-43 (2005); Richman and Kranz, Biomol Eng, 24:361- 73 (2007)). This approach may also overcome the problem faced by some cancer patients whereby their T cells do not express TCRs with sufficient specificity and binding affinity for the underlying tumor antigen. For example, over 300 MHC restricted T cell defined tumor antigens (Cheever et al., Clin Cancer Res.2009;15(17):5323-5337) have been identified. These tumor antigens include mutated peptides, differentiation antigens, and over-expressed antigens, all of which serve as targets for therapy. Since most cancer antigens described to date are derived from intracellular proteins that can only be targeted at the cell surface in the context of MHC molecules, TCRs are ideal candidates for therapy as they have evolved to recognize this class of antigens. Similarly, TCRs can detect peptides derived from viral proteins that have been naturally processed in infected 42  cells and displayed on the cell surface by MHC molecules. However, patients with these diseases may not have an optimized TCR that binds and destroys infected cells. Finally, in methods with high specificity, TCRs may be used as receptor antagonists for autoimmune targets, or as delivery agents to immunosuppress local immune cell responses, thereby avoiding general immunosuppression.

[0168] As used herein, the term “T helper cells” (TH or Th cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surfaces. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including THI, TH2, TH3, TH9, THI 7, TH22 or TFH (T follicular helper cells), which secrete different cytokines to facilitate different types of immune responses. Signaling from the APCs directs T cells into particular subtypes.

[0169] As used herein, the term “cytotoxic T cells” (TC cells, or CTLs) or “cytotoxic T lymphocytes” destroy virus-infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surfaces. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0170] As used herein, the term “memory T cells” refers to a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with memory against past infections. Memory T cells comprise three subtypes: central memory T cells (TcM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface proteins CD45RO, CD45RA and / or CCR7. 43

[0171] As used herein, the term “regulatory T cells” (Treg cells), formerly known as suppressor T cells, refer to T cells that play a role in the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress autoreactive T cells that escaped the process of negative selection in the thymus.

[0172] As used herein, the term “Natural killer T cells” (NKT cells - not to be confused with natural killer cells of the innate immune system) refer to those cells that bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD Id. Once activated, these cells can perform functions ascribed to both T helper (TH) and cytotoxic T (TC) cells (i.e., cytokine production and release of cytolytic / cell killing molecules). They are also able to recognize and eliminate some tumor cells and cells infected with herpes viruses.

[0173] As used herein, the term “proliferative disease” refers to a unifying concept in which excessive proliferation of cells and / or turnover of cellular matrix contributes significantly to the pathogenesis of the disease, including cancer.

[0174] “Patient” or “subject” as used herein refers to a mammalian subject diagnosed with or suspected of having or developing a disease or disorder such as cancer. In some embodiments, the term “patient” refers to a mammalian subject with a higher than average likelihood of developing a proliferative disorder such as cancer. Exemplary patients can be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents and other mammalians that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female. “Patient in need thereof” or “subject in need thereof” is referred to herein as a patient diagnosed with or suspected of having a disease or disorder, for instance, but not restricted to human papilloma virus (HPV) infection.

[0175] “Administering” is referred to herein as providing one or more compositions described herein to a patient or a subject. By way of example and not limitation, composition administration, e.g., injection, can be performed by intravenous injection, subcutaneous injection, intradermal injection, intraperitoneal injection, or intramuscular injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual 44  perfusion over time. Alternatively, or concurrently, administration can be by the oral route. Additionally, administration can also be by surgical deposition, or positioning of a medical device. A pharmaceutical composition can comprise a composition of the invention as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0176] As used herein, the term “therapeutic product” refers to a therapeutic polypeptide or therapeutic polynucleotide which imparts a beneficial function to the host cell in which such product is expressed. Therapeutic polypeptides may include, without limitation, peptides as small as three amino acids in length, single- or multiple-chain proteins, and fusion proteins. Therapeutic polynucleotides may include, without limitation, antisense oligonucleotides, small interfering RNAs, ribozymes, and RNA external guide sequences. The therapeutic product may comprise a naturally occurring sequence, a synthetic sequence or a combination of natural and synthetic sequences.

[0177] As used herein, the term “treatment,” “treating,” or its grammatical equivalents refers to obtaining a desired pharmacologic and / or physiologic effect. In embodiments, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptom or pathological manifestation attributable to the disease. To this end, the inventive method comprises administering a therapeutically effective amount of a composition of the invention expressing the inventive nucleic acid sequence, or a vector comprising the inventive nucleic acid sequences.

[0178] As used herein, a “treatment interval” refers to a treatment cycle, for example, a course of administration of a therapeutic agent that may be repeated, e.g., on a regular schedule. In some embodiments, a dosage regimen may have one or more periods of no administration of the therapeutic agent in between treatment intervals.

[0179] As used herein, a “dosage regimen” or “dosing regimen” includes a treatment regimen based on a determined set of doses. The terms “dose” and “dosing” as used herein refers to the 45  administration of a substance to achieve a therapeutic objective (e.g., the treatment of a tumor).

[0180] The terms “administered in combination,” “co-administration,” or “co-administering,” or “co-providing” as used herein means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with a disease or disorder, for example, the two or more treatments are delivered after the subject has been diagnosed with the disease or disorder and before the disease or disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments may be partially additive, wholly additive, or greater than additive. The delivery may be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0181] In some embodiments of the present invention, a first treatment and a second treatment may be administered simultaneously (e.g., at the same time), in the same or in separate compositions, or sequentially. Sequential administration refers to administration of one treatment before (e.g., immediately before; less than 5, 10, 15, 30, 45, or 60 minutes before; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 or more hours before; 4, 5, 6, 7, 8, 9 or more days before; or 1, 2, 3, 4, 5, 6, 7, 8 or more weeks before) administration of an additional (e.g., secondary) treatment. The order of administration of the first and secondary treatment may also be reversed.

[0182] The term “therapeutically effective amount,” “therapeutic amount,” “immunologically effective amount,” “anti-tumor effective amount,” “tumor-inhibiting effective amount” or its grammatical equivalents refers to an amount effective, at dosages and for periods of time 46  necessary, to achieve a desired therapeutic result. The therapeutically effective amount can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a composition described herein to elicit a desired response in one or more subjects.

[0183] Alternatively, the pharmacologic and / or physiologic effect of administration of one or more compositions described herein to a patient or a subject of can be “prophylactic,” i.e., the effect completely or partially prevents a disease or symptom thereof. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease or prevention of manifestation of a target pathology). miRNA(s)

[0184] As used herein, the terms “miR,” “mir” and “miRNA” are used to refer to microRNA, a class of small non-coding RNA molecules that are capable of affecting the expression of a gene (the “target gene”) by modulating the translation of messenger RNA transcribed therefrom (either increasing or decreasing the gene’s expression) and / or destabilizing such messenger RNA.

[0185] The term “primary miRNA,” abbreviated “pri-miRNA,” refers to an miRNA containing at least one RNA hairpin. The RNA hairpin(s) are cleaved from the pri-miRNA in the cell nucleus to form one or more precursor miRNAs (“pre-miRNAs”). This pre-miRNA is exported into the cytoplasm where the stem loop structure is cleaved to produce a double-stranded miRNA comprising a miRNA-5p strand from the former 5’ arm of the hairpin loop and a miRNA-3p strand from the former 3’ arm of the hairpin loop. The Argonaute protein then binds the double-stranded miRNA and one of the strands (either the miRNA-5p sequence or the miRNA-3p sequence) is released. The remaining bound strand becomes the “guide strand” whereas the released strand is known as the “passenger strand” and preferably degrades. The guide strand then goes on to interact with the messenger RNA derived from the target gene, thus affecting its translation.

[0186] Both the miRNA-5p and miRNA-3p strand sequences will be referred to herein as “mature miRNA” sequences. The remaining portions of a pri-miRNA or pre-miRNA (the portion thereof 5’ to the miRNA-5p sequence, the portion thereof 3’ to the miRNA-3p sequence, and the stem loop sequence in between the miRNA-5p and miRNA-3p sequences) will be collectively referred 47  to as miRNA backbone sequences. The term “5’ backbone sequence” will be used herein to refer to the backbone sequence that, in a pri- or pre-miRNA, is 5’ of the miRNA-5p sequence. The term “3’ backbone sequence” will be used herein to refer to the backbone sequence that, in a pri- or pre- miRNA, is 3’ of the miRNA-3p sequence. The term “loop sequence” refers to the backbone sequence that, in a pri- or pre-miRNA, is between the miRNA-5p and miRNA-3p sequences.

[0187] The term “miRNA,” unless otherwise indicated, refers generically to the mature, primary, and precursor forms of a particular microRNA and functional fragments and variants thereof.

[0188] The miRNAs can be non-naturally occurring. The terms “non-naturally occurring,” “non- natural,” “synthetic,” and “artificial,” as used to describe miRNA(s) herein, are used interchangeably and refer to an miRNA having a sequence that does not occur in nature.

[0189] The present invention relates in part to a ribonucleic acid comprising two non-natural pre- miRNA sequences, wherein each pre-miRNA sequence comprises a guide miRNA that inhibits the expression of an immune checkpoint protein. In certain embodiments, the RNA comprises more than two such non-natural pre-miRNA sequences, for example three, four, five, six, seven, eight, nine, ten, or more such sequences. It is understood that each guide miRNA may target the same or different gene. In embodiments wherein two or more guide miRNAs target the same gene, such guide miRNAs may target the same or different regions of such gene.

[0190] In certain embodiments, each non-natural pre-miRNA sequence in the ribonucleic acid forms a stem-loop secondary structure that is distinct and non-complementary from that formed by a different non-natural pre-miRNA sequence in the ribonucleic acid. In certain embodiments, the non-natural pre-miRNA sequences have less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 55%, or less than about 50% sequence identity with each other.

[0191] In certain embodiments, the secondary structure of each non-natural pre-miRNA is sufficiently similar to that of a naturally-occurring pre-miRNA sequence so as to reduce or prevent cellular RNAi-based anti-pathogen toxicity. In certain such embodiments, the nucleic acid sequence of a non-natural pre-miRNA has at least about 50%, at least about 55%, at least about 48  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% sequence identity, at least about 99.1% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or at least about 99.99% sequence identity with that of a naturally-occurring pre-miRNA and / or can hybridize under stringent hybridization conditions with a naturally-occurring pre-miRNA.

[0192] In certain embodiments, the secondary structure of each pri-miRNA containing a non- natural pre-miRNA (hereinafter, a “non-natural pri-miRNA”) is sufficiently similar to that of a naturally-occurring pri-miRNA sequence so as to reduce or prevent cellular RNAi-based anti- pathogen toxicity. In certain such embodiments, the nucleic acid sequence of a non-natural pri- miRNA 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%, at least about 99% sequence identity, at least about 99.1% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or at least about 99.99% sequence identity with that of a naturally- occurring pri-miRNA and / or can hybridize under stringent hybridization conditions with a naturally-occurring pri-miRNA.

[0193] The non-natural pre-miRNA of the present invention may be produced from a naturally- occurring pre-miRNA by removing the native mature miRNA sequences and replacing them with non-native mature miRNA sequences wherein one of the sequences is capable of serving as a guide miRNA targeting a gene of interest.

[0194] In certain embodiments, each non-natural pre-miRNA comprises backbone sequences derived from a naturally-occurring pre-miRNA, for example from that present in mouse, rat, or human. In certain embodiments, the backbone sequences (the 3’ backbone sequence, the 5’ backbone sequence, and the loop sequence) of the non-natural pre-miRNA have 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%, at least about 99% sequence identity, at least about 99.1% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence 49  identity, or at least about 99.99% sequence identity with the corresponding backbone sequences of a naturally-occurring pre-miRNA and / or can hybridize under stringent hybridization conditions with such corresponding backbone segments. In certain embodiments, the backbone segments of the non-natural pre-miRNA sequences are identical to the corresponding backbone segments of a naturally-occurring pre-miRNA. In certain embodiments, the naturally-occurring pre-miRNA is miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915. In certain embodiments, the naturally-occurring pre-miRNA is miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In certain embodiments, the naturally- occurring pre-miRNA is miR16, miR21, miR22, miR204, or miR206. In certain embodiments, the naturally-occurring pre-miRNA is miR204 or miR206.

[0195] In certain embodiments, each non-natural pri-miRNA comprises backbone sequences derived from a naturally-occurring pri-miRNA, for example from that present in mouse, rat, or human. In certain embodiments, the backbone sequences (the 3’ backbone sequence, the 5’ backbone sequence, and the loop sequence) of the non-natural pri-miRNA have 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%, at least about 99% sequence identity, at least about 99.1% sequence identity, at least about 99.5% sequence identity, at least about 99.9% sequence identity, or at least about 99.99% sequence identity with the corresponding backbone sequences of a naturally-occurring pri-miRNA and / or can hybridize under stringent hybridization conditions with such corresponding backbone segments. In certain embodiments, the backbone segments of the non-natural pri-miRNA sequences are identical to the corresponding backbone segments of a naturally-occurring pri-miRNA. In certain embodiments, the naturally-occurring pri-miRNA is miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915. In certain embodiments, the naturally-occurring pre-miRNA is miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In certain embodiments, the naturally- occurring pre-miRNA is miR16, miR21, miR22, miR204, or miR206. In certain embodiments, the naturally-occurring pre-miRNA is miR204 or miR206. 50

[0196] While the miRNA-5p and miRNA-3p sequences hybridize with each other, they are not necessarily exactly complementary. In the design of a non-naturally occurring miRNA, compensatory mutations can be made in the miRNA-5p and / or miRNA-3p sequences so as to maintain the RNA folding and free energy of the native miRNA. In certain embodiments, the sequence encoding the miRNA-3p sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the complement to the sequence encoding the miRNA-5p sequence or is capable of hybridizing under stringent hybridization conditions with the sequence encoding the miRNA-5p sequence.

[0197] In certain embodiments, the two non-natural pre-miRNA sequences are separated from each other by at least about 1, at least about 2, at last about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, 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 60, at least about 70, at least about 80, at least about 90, 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, or at least about 250 nucleotides. In certain embodiments, the two non-natural pre-miRNA sequences are separated from each other by about 5 to 250 nucleotides, about 10 to 250 nucleotides, about 10 to 200 nucleotides, about 10 to 150 nucleotides, about 10 to 100 nucleotides, about 10 to 50 nucleotides, about 10 to 40 nucleotides, about 10 to 30 nucleotides, about 10 to 20 nucleotides, about 16 to 250 nucleotides, about 16 to 200 nucleotides, about 16 to 150 nucleotides, about 16 to 100 nucleotides, about 16 to 50 nucleotides, about 16 to 40 nucleotides, about 16 to 30 nucleotides, about 16 to 20 nucleotides, about 20 to 200 nucleotides, about 20 to 150 nucleotides, about 20 to 100 nucleotides, about 20 to 50 nucleotides, about 20 to 45 nucleotides, about 20 to 40 nucleotides, about 20 to 35 nucleotides, about 20 to 30 nucleotides, about 20 to 25 nucleotides, about 30 to 200 nucleotides, about 30 to 150 nucleotides, about 30 to 100 nucleotides, about 30 to 50 nucleotides, or about 30 to 40 nucleotides. In certain embodiments, the two non-natural pre-miRNA sequences are separated from each other by at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 51  75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 2010, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides.

[0198] In certain embodiments, two non-natural pri-miRNA sequences adjoin each other with the 3’ nucleotide of one pri-miRNA being directly bonded to the 5’ nucleotide of another pri-miRNA. In such embodiments, the nucleotides separating the respective non-natural pre-miRNAs contained in each pri-miRNA form part of the pri-miRNA sequences.

[0199] In certain embodiments, the non-natural pre-miRNA comprises a mature miRNA sequence that is capable of binding to an mRNA and thereby interfering with the translation thereof and / or prompting its degradation. The mRNA may be produced from the expression of a target gene.

[0200] In certain embodiments, the target gene encodes an immune checkpoint protein. Thus, the pre-miRNA sequences inhibit the expression of an immune checkpoint protein by targeting the gene expressing the same. In certain such embodiments, the immune checkpoint protein is PD-1, PD-L1, CTLA4, TIGIT, 4-1BB, PIK3IP1, CD27, CD28, CD40, CD70, CD122, CD137, OX40 (CD134), GITR, ICOS, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM3, or VISTA. In certain such embodiments, the immune checkpoint protein is CTLA4, CD70, PD-1, PD-L1, TIGIT, TIM3, LAG3, GITR, or PIK3IP1. In certain embodiments, immune checkpoint protein is CTLA4, CD70, PD-1, TIGIT, TIM3, LAG3, GITR, or PIK3IP1. In certain embodiments, immune checkpoint protein is CD70, PD-1, or TIGIT. In certain embodiments, immune checkpoint protein is PD-1.

[0201] In certain embodiments, each non-natural pre-miRNA targets a different gene. In certain different regions of the same gene. 52

[0202] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; and (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1.

[0203] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; and (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1.

[0204] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets PD-1; and (b) a pre-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets PD-1.

[0205] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets PD-1; and (b) a pri-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets PD-1.

[0206] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets TIGIT.

[0207] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets TIGIT.

[0208] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR21 and a guide miRNA that targets TIGIT.

[0209] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR21 and a guide miRNA that targets TIGIT.

[0210] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA 53  comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT.

[0211] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT.

[0212] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT.

[0213] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT.

[0214] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR142 and a guide miRNA that targets TIGIT.

[0215] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR142 and a guide miRNA that targets TIGIT.

[0216] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets TIGIT.

[0217] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets TIGIT.

[0218] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR21 and a guide miRNA that targets TIGIT. 54

[0219] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR21 and a guide miRNA that targets TIGIT.

[0220] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR21 and a guide miRNA that targets TIGIT.

[0221] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR21 and a guide miRNA that targets TIGIT.

[0222] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR142 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets TIGIT.

[0223] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR142 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets TIGIT.

[0224] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR142 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT.

[0225] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR142 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT.

[0226] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR142 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR21 and a guide miRNA that targets TIGIT.

[0227] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR142 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA 55  comprising backbone sequences from miR21 and a guide miRNA that targets TIGIT.

[0228] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets TIGIT.

[0229] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets TIGIT.

[0230] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT.

[0231] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets TIGIT.

[0232] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets CD70; and (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets CD70.

[0233] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets CD70; and (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets CD70.

[0234] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets CD70; and (b) a pre-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets CD70.

[0235] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets CD70; and (b) a pri-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets CD70. 56

[0236] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR26a1 and a guide miRNA that targets CD70; and (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets CD70.

[0237] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR26a1 and a guide miRNA that targets CD70; and (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets CD70.

[0238] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR26a1 and a guide miRNA that targets CD70; and (b) a pre-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets CD70.

[0239] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR26a1 and a guide miRNA that targets CD70; and (b) a pri-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets CD70.

[0240] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets CD70; and (b) a pre-miRNA comprising backbone sequences from miR26a1 and a guide miRNA that targets CD70.

[0241] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets CD70; and (b) a pri-miRNA comprising backbone sequences from miR26a1 and a guide miRNA that targets CD70.

[0242] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets CD70; and (b) a pre-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets CD70.

[0243] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets CD70; and (b) a pri-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets CD70.

[0244] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR150 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA 57  comprising backbone sequences from miR206 and a guide miRNA that targets PD-1.

[0245] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR150 and a guide miRNA that targets TIGIT; and (b) a pie-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1.

[0246] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pre-miRNA comprising backbone sequences from miR17 and a guide miRNA that targets TIGIT.

[0247] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pri- miRNA comprising backbone sequences from miR17 and a guide miRNA that targets TIGIT.

[0248] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR17 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1.

[0249] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR17 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1.

[0250] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pre-miRNA comprising backbone sequences from miR150 and a guide miRNA that targets TIGIT.

[0251] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pri- miRNA comprising backbone sequences from miR150 and a guide miRNA that targets TIGIT. 58

[0252] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pre-miRNA comprising backbone sequences from miR16 and a guide miRNA that targets CD70.

[0253] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pri- miRNA comprising backbone sequences from miR16 and a guide miRNA that targets CD70.

[0254] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pre-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets CD70.

[0255] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pri- miRNA comprising backbone sequences from miR22 and a guide miRNA that targets CD70.

[0256] In certain embodiments, the ribonucleic acid comprises: (a) a pre-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pre-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pre-miRNA comprising backbone sequences from miR22 and a guide miRNA that targets CD70.

[0257] In certain embodiments, the ribonucleic acid comprises: (a) a pri-miRNA comprising backbone sequences from miR204 and a guide miRNA that targets PD-1; (b) a pri-miRNA comprising backbone sequences from miR206 and a guide miRNA that targets PD-1; and (c) a pri- miRNA comprising backbone sequences from miR22 and a guide miRNA that targets CD70.

[0258] The present invention also relates in part to a deoxyribonucleic acid encoding any of the aforementioned ribonucleic acids.

[0259] Examples of deoxyribonucleic acid sequences that encode backbone sequences that may 59  be used in the practice of the present invention include, but are not limited, to those listed in Table 3 below. The symbols of “X” and “Y” in Table 3 indicate nucleic acid sequences encoding, respectively, the guide miRNA (which may be either miRNA-5p or miRNA-3p) and the passenger miRNA (which may be either miRNA-5p or miRNA-3p), whereas the symbol of “n” indicates the number of nucleotides in such sequences, for example 16–30, preferably 18–25. In some embodiments, n can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides. In certain embodiments, the deoxyribonucleic acids encoding backbone sequences are those that hybridize under stringent hybridization conditions with the complement of any one of the sequences listed in Table 3. Table 3: Deoxyribonucleic acid sequences encoding miRNA backbone sequences Pri-miRNA from Nucleic acid sequences encoding which backbone 5’ Backbone Sequence – Xn-Stem Loop Sequence –Yn-3’ Backbone Sequence60  miR26a1 SEQ ID NO: 43-Xn-SEQ ID NO: 44-Yn-SEQ ID NO: 45

[0260] In any of the foregoing embodiments, the sequence encoding the pre-miRNA comprises: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively; SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively; SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively; SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively; 61  SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively; SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, respectively; SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively; SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively; SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively; SEQ ID NO: 338, SEQ ID NO: 339, and SEQ ID NO: 340, respectively; SEQ ID NO: 341, SEQ ID NO: 342, and SEQ ID NO: 343, respectively; or SEQ ID NO: 344, SEQ ID NO: 345, and SEQ ID NO: 346, respectively; or sequences having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any of the foregoing sequences, or that are capable of hybridizing under stringent hybridization conditions to the complements of such sequences.

[0261] Non-limiting examples of nucleic acid sequences encoding the guide miRNA targeting genes encoding such checkpoint inhibitors are listed in Table 4. Table 4 also lists the sequences encoding the passenger strand. As previously discussed, the guide and passenger strand are not necessarily complementary. It is contemplated that the passenger strand may also serve to target the messenger RNA associated with the target gene. It is also contemplated that sequences that hybridize under stringent hybridization conditions with the complements of the sequences listed in Table 4 may also be used. The mature miRNA sequences used may be combined with a specific pri-miRNA backbone. Table 4 also lists backbones that can be combined with the mature guide and passenger miRNAs listed therein. Table 4: Deoxyribonucleic acid sequences encoding mature miRNA sequences miRNA Backbone DNA encoding guide miRNA DNA encoding passenger62  PD-1 miR-204(SEQ ID NO: 705)PD-1 miR-204(SEQ ID NO: 706)TIGITmiR-22(SEQ ID NO: 120)(SEQ ID NO: 121)TIGITmiR-142(SEQ ID NO: 122)(SEQ ID NO: 123)CD70 miR204(SEQ ID NO: 305)(SEQ ID NO: 306)CD70 miR19(SEQ ID NO: 307)(SEQ ID NO: 308)acid comprising a nucleic acid sequence having at least about 80% sequence identity with any one of SEQ ID NOs: 64–83, 85, 87–171, 293–322, and 704–713 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 64–83, 85, 87– 171, 293–322, and 704–713. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 704, 705, 709, and 710 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 704, 705, 709, and 710.

[0263] In certain embodiments, the sequence encoding the guide miRNA sequence has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 293, 295, 65  297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 704, 705, 709, and 710; or is capable of hybridizing under stringent hybridization conditions to the complement of any one of such sequences.

[0264] In certain embodiments, the sequence encoding the passenger miRNA sequence has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity to any one of SEQ ID NOs: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 706–708, and 711–713; or is capable of hybridizing under stringent hybridization conditions to the complement of any one of such sequences.

[0265] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets CTLA. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 65–71 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 65–71. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 64, 66, 68, and 70 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 64, 66, 68, and 70.

[0266] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets PD-1. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 72–83, 85, 87, and 704–713 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 72–83, 85, 87, and 704–713. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% 66  sequence identity with any one of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 704, 705, 709, and 710 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 704, 705, 709, and 710.

[0267] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets TIGIT. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 88–145 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 88–145. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, and 138 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 64, 66, 68, and 70 SEQ ID NOs: 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, and 138.

[0268] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets TIM3. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 146–157 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 146–157. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 146, 148, 150, 152, 154, and 156 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 146, 148, 150, 152, 154, and 156.

[0269] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets LAG3. In certain such embodiments, the present invention relates to a polynucleotide comprising 67  a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 158–161 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 158–161. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 158 and 160 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 158 and 160.

[0270] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets GITR. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 162–165 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 162–165. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 162 and 164 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 162 and 164.

[0271] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets PIK3IP1. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 166–171 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 166–171. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 166, 168, and 170 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 166, 168, and 170.

[0272] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets 68  CD70. In certain such embodiments, the present invention relates to a polynucleotide comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 293–322 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 293–322. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, and 321 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, and 321.

[0273] In certain embodiments, the present invention relates to a deoxyribonucleic acid wherein each sequence encoding a pre-miRNA comprises: a) a sequence encoding a 5’ miRNA backbone sequence; b) a sequence encoding a guide miRNA sequence; c) a sequence encoding a stem loop sequence; d) a sequence encoding a passenger miRNA sequence; and e) a sequence encoding a 3’ backbone sequence.

[0274] In certain embodiments, the sequence encoding the pre-miRNA comprises: a) a guide miRNA sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 704, 705, 709, and 710, or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of such sequences; and 69  b) a passenger sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity to, respectively, any one of SEQ ID NOs: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 706–708, and 711–713, or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of such sequences.

[0275] Deoxyribonucleic acids encoding exemplary non-natural pre-miRNA sequences targeting specific checkpoint inhibitors are described in Table 5. In certain embodiments, the deoxyribonucleic acid may comprise a sequence that is capable of hybridizing under stringent hybridization conditions with the complement of any one of the sequences listed in Table 5. Table 5: Deoxyribonucleic acid sequences encoding non-natural miRNA sequences miRNA Backbone DNA Sequence DNA Sequence Target sequences derived Encoding pri-miRNA Encoding pre- ce )))))))))))))))))) miRNA Backbone DNA Sequence DNA Sequence Target sequences derived Encoding pri-miRNA Encoding pre- nce )))))))))))))))))))))))))))))))))))))) miRNA Backbone DNA Sequence DNA Sequence Target sequences derived Encoding pri-miRNA Encoding pre- nce )))))))))))))))))))))))))))))))))))))) miRNA Backbone DNA Sequence DNA Sequence Target sequences derived Encoding pri-miRNA Encoding pre- nce )))))))

[0276] In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 347–447 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 347–447.

[0277] In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 178–263 and 323–337 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 178–263 and 323–337.

[0278] In certain embodiments, a miRNA targets CTLA4. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 347, 419, 420, and 421 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 347, 419, 420, and 421. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 178 and 250–252 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 178 and 250–252. 73

[0279] In certain embodiments, a pre-miRNA targets PD-1. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 348, 349, and 410–418 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 348, 349, and 410–418. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 179, 180, and 241–249 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 179, 180, and 241–249. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NO: 348 or 349 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 348 or 349. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NO: 179 or 180 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 179 or 180.

[0280] In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 179 and a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 180 or that is capable of hybridizing under stringent hybridization conditions to the complement of a nucleic acid comprising SEQ ID NO: 179 and SEQ ID NO: 180.

[0281] In certain embodiments, a pre-miRNA targets TIGIT. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 350–377 and 404–409 or that is capable of hybridizing under 74  stringent hybridization conditions to the complement of any one of SEQ ID NOs: 350–377 and 404–409. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 181–208 and 235–240 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 181–208 and 235–240.

[0282] In certain embodiments, a pre-miRNA targets TIM3. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 378–389 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 378–389. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 209–220 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 209–220.

[0283] In certain embodiments, a pre-miRNA targets LAG3. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 390–396 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 390–396. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 221–227 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 221–227.

[0284] In certain embodiments, a pre-miRNA targets GITR. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 397–403 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 397–403. In certain such 75  embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 228–234 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 228–234.

[0285] In certain embodiments, a pre-miRNA targets PIK3IP1. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 422–424 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 422–424. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 253–255 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 253–255.

[0286] In certain embodiments, a pre-miRNA targets CD70. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 433–447 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 433–447. In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 323–337 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 323–337.

[0287] In embodiments of the present invention, the two or more pre-miRNAs encoded by the deoxyribonucleic acid may each contain guide miRNA sequences that target the same target gene or the various guide miRNAs may target different genes. In addition, each pre-miRNA design of that of the pri-miRNA containing them may be based on a different native miRNA backbone to reduce the likelihood of misfolding of one miRNA with another. Table 6 provides examples of deoxyribonucleic acid sequences encoding two or more pri-miRNAs. 76  Table 6: Deoxyribonucleic acid sequences comprising two or more pri-miRNAs miRNA Target Backbone sequences derived DNA Sequence from miRNA Target Backbone sequences derived DNA Sequence from

[0288] In certain such embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 267–290, 448–460, and SEQ ID NO: 944 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 267–290, 448–460, and SEQ ID NO: 944.

[0289] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs that target PD-1. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 267, 282, and 944, or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 267, 282, and 944. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 267, or that is capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO: 267.

[0290] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets PD-1 and a pre-miRNA that targets TIGIT. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 269–274, 287, 288, and 290 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 269–274, 287, 288, and 290. 78

[0291] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs that target PD-1 and a pre-miRNA that targets TIGIT. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 275–280 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 275–280.

[0292] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets PD-1 and a pre-miRNA that targets CTLA4. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 281, 283, and 284 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 281, 283, and 284.

[0293] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets TIGIT and a pre-miRNA that targets CTLA4. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 285, 286, and 289 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 285, 286, and 289.

[0294] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs that target PD-1 and a pre-miRNA that targets CD70. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 448 or 451 or that is capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO: 448 or 451.

[0295] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNAs that targets PD-1 and two pre-miRNAs that target CD70. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 449 79  or that is capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO: 449.

[0296] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNAs that targets PD-1 and a pre-miRNA that target CD70. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 450 or that is capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID NO: 450.

[0297] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs that each CD70. In certain embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 452–460 or that is capable of hybridizing under stringent hybridization conditions to the complement of any one of SEQ ID NOs: 452–460. Chimeric Antigen Receptors (CARs)

[0298] A polynucleotide of the present disclosure can further encode a chimeric receptor, such as a chimeric antigen receptor (CAR). Thus, in some embodiments, a polynucleotide of the present disclosure can encode miRNA(s) and a CAR as described herein. In any of the embodiments of the present disclosure, a modified immune effector cell can comprise a chimeric antigen receptor as described herein.

[0299] A CAR is an engineered receptor that grafts an exogenous specificity onto an immune effector cell. In some instances, a CAR comprises an extracellular domain (ectodomain) that comprises an antigen-binding domain, a transmembrane domain, and an intracellular (endodomain) domain. The intracellular domain comprises an intracellular signaling domain. In certain embodiments, the extracellular domain further comprises a spacer between the antigen- binding domain and the transmembrane domain. A. Antigen-Binding Domain 80

[0300] The extracellular domain of a CAR comprises an antigen-binding domain that is capable of recognizing and binding to an epitope on a target antigen.

[0301] An antigen-binding domain can comprise complementary determining regions (CDRs) that bind to an epitope on a target antigen, for example the CDRs of a monoclonal antibody that binds to the antigen. A complementarity determining region (CDR) is a short amino acid sequence found in the variable domains of antigen receptor (e.g., immunoglobulin and T-cell receptor) proteins that bind an antigen and therefore provides the receptor with its specificity for that particular antigen. Each polypeptide chain of an antigen receptor can contain three CDRs (CDR1, CDR2, and CDR3).

[0302] In certain embodiments, the antigen-binding domain comprises a Fv, Fab, Fab2, Fab’, F(ab’)2, or F(ab’)3 fragment of an antibody that binds to an antigen.

[0303] In certain embodiments, the antigen binding domain comprises the variable domain of the heavy chain of an antibody (VHdomain) and / or the variable domain of the light chain of an antibody (VL domain) that binds the antigen, or functional fragments or variants thereof. In certain embodiments, the functional fragments or variants comprise the CDRs that bind to the antigen. For example, the functional fragment or variant of the VHdomain may comprise CDR1, CDR2, and CDR3 of the VH domain, and / or the functional fragment or variant of the VL domain may comprise the CDR1, CDR2, and CDR3 of the VL domain.

[0304] In certain embodiments, the antigen-binding domain comprises a scFv, sc(Fv)2, a dsFv, a diabody, a minibody, a nanobody, or binding fragments thereof. In certain embodiments, the antigen-binding domain further comprises an Fc fragment of an antibody, for example it may comprise an scFv linked with an Fc fragment.

[0305] In some embodiments, the CAR targets an antigen that is elevated in cancer cells or in autoimmune cells. Autoimmune diseases can include graft-versus-host disease, rheumatoid arthritis, lupus, lupus nephritis (LN), myasthenia gravis (MG), celiac disease, Crohn’s disease, Sjogren Syndrome, polymyalgia rheumatic, multiple sclerosis, neuromyelitis optica, ankylosing spondylitis, Type 1 diabetes, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris, and autoimmune uveitis. 81

[0306] In some embodiments, a CAR described herein comprises an antigen-binding domain that binds to an epitope on B7H4, BCMA, BTLA, CAIX, CA125, CCR4, CD3, CD4, CD5, CD7, CD16, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / v8, CD47, CD52, CD56, CD70, CD79b, CD80, CD81, CD86, CD123, CD133, CD137, CD138, CD151, CD171, CD174, CD276, CEA, CEACAM6, CLL-1, c-MET, CS1, CSPG4, CTLA-4, DLL3, EDB-F, EGFR, EGFR2, EGFRvIII, EGP-2, EGP-40, EphA2, FAP, FLT1, FLT4, Folate-binding Protein, Folate Receptor, Folate receptor α, α-Folate receptor, Frizzled, GD2, GD3, GHR, GHRHR, GITR, GPC3, Gp100, gp130, HBV antigens, HER1, HER2, HER3, HER4, HER1 / HER3, h5T4, HPV antigens, HVEM, IGF1R, IgKAppa, IL-1-RAP, IL-2R, IL6R, IL-11Rα, IL-13R-a2, KDR, KRASG12V, LewisA, LewisY, L1-CAM, LIFRP, LRP5, LTPR, MAGE-A, MAGE-A1, MAGE-A10, MAGE-A3, MAGEA3 / A6, MAGE-A4, MAGE-A6, MART-1, MCAM, mesothelin, PSCA, Mucins such as MUC1, MUC-4 or MUC16, NGFR, NKG2D, Notch-1-4, NY- ESO-1, O-acetylGD2, O-acetylGD3, OX40, P53, PD-1, PDE10A, PD-L1, PD-L2, PMSA, PRAME, PSCA, PSMA, PTCH1, RANK, Robol, ROR1, ROR1R, ROR-2, TACI, TAG-72, TCRa, TCRp, TGF, TGFBeta, TGFBeta-II, TGFBR1, TGFBR2, Titin, TLR7, TLR9, TNFR1, TNFR2, TNFRSF4, TRBC1, TWEAK-R, VEGF, VEGF-R2, or WT-1.

[0307] In some embodiments, a CAR described herein comprises an antigen-binding domain that binds to an epitope on CD19.

[0308] Antigen binding can be assessed by flow cytometry or a cell-based assay or any other equivalent assay. Cell based assays may utilize a cell type expressing antigen of interest on the surface to assess antigen-binding. An antigen or a fragment thereof expressed as a soluble protein can be utilized to assess antigen-binding using flow cytometry or similar assay. Improvements in antigen-binding may be indirectly assessed by functional measurement of antigen-binding domain or a chimeric receptor. For example, improved antigen-binding of a chimeric receptor or a CAR, as described herein, can be measured by increased specific cytotoxicity against target cells expressing the antigen.

[0309] Cell surface expression level of a polypeptide of the present disclosure can be assessed, for example, using a flow cytometry based assay. Improved expression of an antigen-binding polypeptide can be measured as percentage of analyzed cells expressing said antigen-binding 82  polypeptide or alternatively as average density of said antigen-binding polypeptide on the surface of a cell. Additional suitable methods that can be used for assessing cell surface expression of the antigen-binding polypeptides described herein include western blotting or any other equivalent assay.

[0310] CD19 is a cell surface glycoprotein of the immunoglobulin superfamily. In some instances, CD19 has been detected in solid tumors such as pancreatic cancer, liver cancer, and prostate cancer. As used herein, the term ‘CD19’ refers to the Cluster of Differentiation 19 protein, which is an antigenic determinant commonly expressed on B cells and various B-cell malignancies. CD19 is a transmembrane protein that plays a crucial role in B cell development, activation, and differentiation. The amino acid and nucleic acid sequences of CD19 from various species can be found in public databases, such as GenBank, UniProt, and Swiss-Prot. These sequences include, but are not limited to, the human CD19 protein sequence (e.g., UniProt / Swiss- Prot Accession No. P15391) and its corresponding nucleotide sequence (e.g., GenBank Accession No. NM_001178098), as well as their counterparts in other species. The term “CD19” also includes any: (a) naturally occurring allelic variants and isoforms of CD19 found in humans, mice, or other organisms; (b) species homologs of CD19; (c) fragments of CD19 that retain characteristic CD19 function or antigenic properties; (d) mutants or derivatives of CD19, whether naturally occurring or artificially created, that retain characteristic CD19 function or antigenic properties; (e) fusion proteins comprising CD19 or a portion thereof; and (f) any CD19 sequence with conservative or non-conservative amino acid substitutions, deletions, or insertions.

[0311] In some embodiments, the antigen binding domain of a CAR described herein, is specific to CD19. A CD19-specific CAR, when expressed on the cell surface, may redirect the specificity of T cells to human CD19.

[0312] In some embodiments, the antigen binding domain comprises CDRs that bind CD19. In certain embodiments, the antigen binding domain comprises the CDR1, CDR2, and CDR3 of the VLdomain. In certain embodiments, the antigen domain comprises the CDR1, CDR2, and CDR3 of the VH domain. In certain embodiments, the antigen binding domain comprises the CDR1, CDR2, and CDR3 of the VL domain and the CDR1, CDR2, and CDR3 of the VH domain. 83

[0313] In some embodiments, the antigen binding a VL domain that binds CD19, for example a variant domain light chain from a CD19-specific monoclonal anti-CD19 antibody, or a functional fragment or variant of a VLdomain. In certain embodiments, the functional fragment or variant of the VL domain comprises the CDR1, CDR2, and CDR3 of the VL domain.

[0314] In some embodiments, the antigen binding domain comprises a VH domain that binds CD19, for example a variant domain heavy chain from a CD19-specific monoclonal anti-CD19 antibody, or a functional fragment or variant of a VH domain. In certain embodiments, the functional fragment or variant of the VH domain comprises the CDR1, CDR2, and CDR3 of the VH domain.

[0315] In some embodiments, the antigen binding domain comprises a single chain antibody fragment (scFv) comprising a variable domain light chain (VL domain) and variable domain heavy chain (VHdomain) that binds CD19, for example VLand VHdomains from a CD19-specific antibody, or functional fragments or variants of such VLand VHdomains. The VHand VLdomains may be joined by a linker, for example a flexible linker, such as a glycine-serine linker or a Whitlow linker. In embodiments, the scFv is SJ25Cl and / or FMC63. In embodiments, the scFv is humanized. In some embodiments, the antigen binding moiety may comprise VHand VLdomains that are directionally linked, for example, from N to C terminus, VH domain-linker-VL domain or VL domain-linker-VH domain.

[0316] In certain embodiments, the linker comprises: (a) the amino acid sequence of SEQ ID NO: 535 ((G4S)3) or a conservatively-substituted variant thereof; or (b) an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 535. In certain such embodiments, the linker is encoded by SEQ ID NO: 536, hybridizes under stringent conditions to the complement of SEQ ID NO: 536, or is a codon degenerate version of SEQ ID NO: 536.

[0317] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 527, or an amino acid sequence comprising 1, 2, 3, 4 or 5 amino acid modifications to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 1023, 1024, 1025, or 1026. In some embodiments, the linker is encoded 84  by a polynucleotide sequence at least 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide of SEQ ID NO: 528. In some embodiments, the linker is encoded by the polynucleotide of SEQ ID NO: 528. In some embodiments, the linker is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90% 95%, 96%, 97%, %, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide of SEQ ID NO: 536. In some embodiments, the linker is encoded by the polynucleotide of SEQ ID NO: 536.

[0318] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics).

[0319] In some embodiments, a CD19-specific CAR-T cell described herein comprises an anti- CD19 antibody described in US20160152723.

[0320] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19 (Kite Pharma, Inc.). In some embodiments, described herein include a CD19-specific CAR-T cell, in which the antigen binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19.

[0321] In some embodiments, a CD19-specific CAR-T cell described herein comprises an anti- CD antibody described in WO2015187528 or fragment or derivative thereof.

[0322] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTL019 (Novartis). In some embodiments, described herein include a CD19- specific CAR-T cell, in which the antigen binding domain recognizes an epitope on CD19 that is also recognized by CTL019.

[0323] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART19 (Cellectis). In some embodiments, described herein include a CD19-specific CAR-T cell, in which the antigen binding domain recognizes an epitope on CD19 that is also recognized by UCART19.

[0324] In some embodiments, the antigen binding domain recognizes an epitope on CD19 that is also recognized by BPX-401 (Bellicum). In some embodiments, described herein include a 85  CD19-specific CAR-T cell, in which the antigen binding domain recognizes an epitope on CD19 that is also recognized by BPX-401.

[0325] In some cases, the antigen binding domain recognizes an epitope on CD19 that is also recognized by blinatumomab (Amgen), coltuximabravtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), denintuzumabmafodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomabpaptox (National Cancer Institute), XmAb 5871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex) or AFM11 (Affimed).

[0326] In addition to the above, exemplary CD19-specific CARs, including antigen-binding domains comprising VHand VLdomains described in the art as capable of targeting CD19, or functional fragments or variants thereof, may be used. Examples of such CD19-specific CARs, antigen-binding domains, and VH and VL domains are described in each of US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985, the full contents of each of which is incorporated by reference herein.

[0327] In certain embodiments, the antigen-binding domain comprises a VL domain comprising the amino acid sequence of any one of the sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, 86  WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than any one of the aforementioned sequences by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of the sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No. 18 / 259,985, and / or is a conservatively-substituted variant thereof.

[0328] In certain embodiments, the antigen-binding domain comprises a VLdomain encoded by a polynucleotide comprising any one of the nucleic acid sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, %, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of the sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, 87  US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985; hybridizes under stringent hybridization conditions with the complement of any one of the sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985; or is a codon degenerate variant of any one of the sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985.

[0329] In certain embodiments, the antigen-binding domain comprises a VHdomain comprising any one of the amino acid sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, 88  US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than any one of the aforementioned sequences by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, %, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of the sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No. 18 / 259,985, and / or is a conservatively-substituted variant thereof..

[0330] In certain embodiments, the antigen-binding domain comprises a VH domain encoded by a polynucleotide comprising any one of the nucleic acid sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985, or a functional fragment or variant thereof. In certain embodiments, 89  the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, %, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of the sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985; hybridizes under stringent hybridization conditions with the complement of any one of the sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985; or is a codon degenerate variant of any one of the sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985.

[0331] In certain embodiments, the antigen-binding domain comprises: (i) a VL domain 90  comprising any one of the amino acid sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985, or a functional fragment or variant thereof, for example a functional fragment that is shorter than any one of the aforementioned sequences by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus, or a function variant having at least about 80%, 85%, 90%, 95%, 96%, 97%, %, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of the aforementioned sequences, and / or is a conservatively-substituted variant thereof; and (ii) a VH domain comprising any one of the amino acid sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985, or a functional fragment or variant thereof, for example a functional fragment that is shorter than any one of the aforementioned sequences by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus, or a function variant having at least about 80%, 85%, 90%, 95%, 96%, 97%, %, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any of the aforementioned sequences, and / or is a conservatively- substituted variant thereof..

[0332] In certain embodiments, the antigen-binding domain comprises: (i) a VL domain encoded 91  by a polynucleotide comprising any one of the nucleic acid sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985, or a functional fragment or variant thereof, for example a functional variant having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any of the aforementioned sequences, hybridizes under stringent hybridization conditions with the complement of any of the aforementioned sequences, or is a codon degenerate variant of any one of the aforementioned sequences; and (ii) a VH domain encoded by a polynucleotide comprising any one of the nucleic acid sequences disclosed in US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, WO2019246546, and U.S. Application No.18 / 259,985, or a functional fragment or variant thereof, for example a functional variant having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any of the aforementioned sequences, hybridizes under stringent hybridization conditions with the complement of any of the aforementioned sequences, or is a codon degenerate variant of any of the aforementioned sequences.

[0333] In some embodiments, the VHdomain comprises three complementarity determining regions (CDRs): VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the VH domain 92  comprises the VH CDR1, VH CDR2, and VH CDR3 set forth in SEQ ID NO: 947, or a functional fragment or variant thereof. In some embodiments, the amino acid sequence of VHCDR1 comprises the amino acid sequence of SEQ ID NO: 954, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 954); the amino acid sequence of VH CDR2 comprises the amino acid sequence of SEQ ID NO: 955, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 955); the amino acid sequence of VH CDR3 comprises the amino acid sequence of SEQ ID NO: 956, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 956). In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of SEQ ID NO: 954; the amino acid sequence of VHCDR2 comprises the amino acid sequence of SEQ ID NO: 955; and the amino acid sequence of VHCDR3 comprises the amino acid sequence of SEQ ID NO: 956.

[0334] In some embodiments, the VL domain comprises three CDRs: VL CDR1, VL CDR2, and VLCDR3. In some embodiments, the VLcomprises the VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NO: 946, or a functional fragment or variant thereof. In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of SEQ ID NO: 948, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 948); the amino acid sequence of VL CDR2 comprises the amino acid sequence of SEQ ID NO: 949, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 949); the amino acid sequence of VLCDR3 comprises the amino acid sequence of SEQ ID NO: 950, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 950). In some embodiments, the amino acid sequence of VL CDR1 comprises the amino acid sequence of SEQ ID NO: 948; the amino acid sequence of VL CDR2 comprises the amino acid sequence of SEQ ID NO: 949; and the amino acid sequence of VLCDR3 comprises the amino acid sequence of SEQ ID NO: 950. 93

[0335] In some embodiments, the VH domain comprises an amino acid sequence that is a functional fragment or variant thereof of SEQ ID NO: 947. In certain such embodiments, the VHdomain is a functional variant of SEQ ID NO: 947 comprising CDRs having the amino acid sequences of: (a) SEQ ID NO: 954, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 954); (b) SEQ ID NO: 955, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 955); and (c) SEQ ID NO: 956, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 956). In some embodiments, the VHdomain has an amino acid sequence of any one of SEQ ID NOs: 947, 1031, 1032, 1033, or 1034.

[0336] In some embodiments, the VLdomain comprises an amino acid sequence that is a functional fragment or variant thereof of SEQ ID NO: 946. In certain such embodiments, the VLdomain is a functional variant of SEQ ID NO: 946 comprising CDRs having the amino acid sequences of: (a) SEQ ID NO: 948, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 948); (b) SEQ ID NO: 949, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 949); and (c) SEQ ID NO: 950, or a functional fragment or variant thereof (e.g., an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO: 950). In some embodiments, the VL domain has an amino acid sequence of SEQ ID NO: 946, 1027, 1028, 1029, or 1030.

[0337] In some embodiments, the amino acid sequence of VHCDR1 comprises the amino acid sequence of SEQ ID NO: 954; the amino acid sequence of VH CDR2 comprises the amino acid sequence of SEQ ID NO: 955; and the amino acid sequence of VHCDR3 comprises the amino acid sequence of SEQ ID NO: 956; and the amino acid sequence of VLCDR1 comprises the amino acid sequence of SEQ ID NO: 948; the amino acid sequence of VL CDR2 comprises the amino acid sequence of SEQ ID NO: 949; and the amino acid sequence of VL CDR3 comprises the amino acid sequence of SEQ ID NO: 950. 94

[0338] In some embodiments, the amino acid sequence of VH CDR1 consists of the amino acid sequence of SEQ ID NO: 954; the amino acid sequence of VHCDR2 consists of the amino acid sequence of SEQ ID NO: 955; and the amino acid sequence of VHCDR3 consists of the amino acid sequence of SEQ ID NO: 956; and the amino acid sequence of VL CDR1 consists of the amino acid sequence of SEQ ID NO: 948; the amino acid sequence of VL CDR2 consists of the amino acid sequence of SEQ ID NO: 949; and the amino acid sequence of VLCDR3 consists of the amino acid sequence of SEQ ID NO: 950.

[0339] In some embodiments, the VH domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 947. In some embodiments, the VHdomain comprises the amino acid sequence of SEQ ID NO: 947. In some embodiments, the amino acid sequence of the VHdomain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 947. In some embodiments, the amino acid sequence of the VH domain consists of the amino acid sequence of SEQ ID NO: 947.

[0340] In some embodiments, the VLdomain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 946. In some embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 946. In some embodiments, the amino acid sequence of the VLdomain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 946. In some embodiments, the amino acid sequence of the VLdomain consists of the amino acid sequence of SEQ ID NO: 946.

[0341] In some embodiments, the VHdomain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 947; and the VLdomain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 946. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 947; and the VL domain comprises the amino acid sequence of SEQ ID NO: 946. In some embodiments, the amino acid sequence of the VHdomain consists of a sequence 95  at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 947; and the amino acid sequence of the VLdomain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 946. In some embodiments, the amino acid sequence of the VH domain consists of the amino acid sequence of SEQ ID NO: 947; and the amino acid sequence of the VLdomain consists of the amino acid sequence of SEQ ID NO: 946.

[0342] In some embodiments, the VL and VH domains are linked by a linker, for example a Whitlow linker, or a functional fragment or variant thereof. In some embodiments, the linker comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 527. In some embodiments, the amino acid sequence of the linker consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the amino acid sequence of the linker consists of the amino acid sequence of SEQ ID NO: 527. In certain embodiments, the VL domain is N terminal to the linker whereas the VHdomain is C terminal to the linker.

[0343] In some embodiments, the CD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 958. In some embodiments, the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 958. In some embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 958. In some embodiments, the amino acid sequence of the CD19 binding domain consists the amino acid sequence of SEQ ID NO: 958. In some embodiments, the amino acid sequence of the CD19 binding domains comprises 1, 2, 3, 4, or 5 conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 958. In some such embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 958.

[0344] In some embodiments, the CD19 binding domain comprises an amino acid sequence at 96  least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 959. In some embodiments, the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 959. In some embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 959. In some embodiments, the amino acid sequence of the CD19 binding domain consists the amino acid sequence of SEQ ID NO: 959. In some embodiments, the amino acid sequence of the CD19 binding domains comprises 1, 2, 3, 4, or 5 conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 959. In some such embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 959.

[0345] In some embodiments, the CD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 960. In some embodiments, the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 960. In some embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 960. In some embodiments, the amino acid sequence of the CD19 binding domain consists the amino acid sequence of SEQ ID NO: 960. In some embodiments, the amino acid sequence of the CD19 binding domains comprises 1, 2, 3, 4, or 5 conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 960. In some such embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 960.

[0346] In some embodiments, the CD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 961. In some embodiments, the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 961. In some embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 961. In some embodiments, the amino acid 97  sequence of the CD19 binding domain consists the amino acid sequence of SEQ ID NO: 961. In some embodiments, the amino acid sequence of the CD19 binding domains comprises 1, 2, 3, 4, or 5 conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 961. In some such embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 961.

[0347] In some embodiments, the CD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 962. In some embodiments, the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 962. In some embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 962. In some embodiments, the amino acid sequence of the CD19 binding domain consists the amino acid sequence of SEQ ID NO: 962. In some embodiments, the amino acid sequence of the CD19 binding domains comprises 1, 2, 3, 4, or 5 conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 962. In some such embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 962.

[0348] In some embodiments, the CD19 binding domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 963. In some embodiments, the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 963. In some embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 963. In some embodiments, the amino acid sequence of the CD19 binding domain consists the amino acid sequence of SEQ ID NO: 963. In some embodiments, the amino acid sequence of the CD19 binding domains comprises 1, 2, 3, 4, or 5 conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 963. In some such embodiments, the amino acid sequence of the CD19 binding domain consists of a sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 98  99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 963.

[0349] In some embodiments, the VHdomain comprises: a VHCDR1 encoded by the polynucleotide sequence of SEQ ID NO: 970, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 970; a VH CDR2 encoded by the polynucleotide sequence of SEQ ID NO: 971, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 971; a VH CDR3 encoded by the polynucleotide sequence of SEQ ID NO: 972, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 972. In some embodiments, the VHdomain comprises a VH CDR1 encoded by the polynucleotide sequence of SEQ ID NO: 970; a VH CDR2 encoded by the polynucleotide sequence of SEQ ID NO: 971; and a VHCDR3 encoded by the polynucleotide sequence of SEQ ID NO: 972.

[0350] In some embodiments, the VL domain comprises: a VL CDR1 encoded by the polynucleotide sequence of SEQ ID NO: 967, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 967; a VL CDR2 encoded by the polynucleotide sequence of SEQ ID NO: 968, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 968; a VLCDR3 encoded by the polynucleotide sequence of SEQ ID NO: 969, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 969. In some embodiments, the VLdomain comprises: a VLCDR1 encoded by the polynucleotide sequence of SEQ ID NO: 967; a VLCDR2 encoded by the polynucleotide sequence of SEQ ID NO: 968; and a VL CDR3 encoded by the polynucleotide sequence of SEQ ID NO: 969.

[0351] In some embodiments, the VHdomain comprises: a VHCDR1 encoded by the polynucleotide sequence of SEQ ID NO: 970, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 970; a VHCDR2 encoded by the polynucleotide sequence of SEQ ID 99  NO: 971, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 971; a VHCDR3 encoded by the polynucleotide sequence of SEQ ID NO: 972, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 972; and the VL domain comprises: VL CDR1 encoded by the polynucleotide sequence of SEQ ID NO: 967, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 967; a VL CDR2 encoded by the polynucleotide sequence of SEQ ID NO: 968, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 968; a VL CDR3 encoded by the polynucleotide sequence of SEQ ID NO: 969, or a polynucleotide sequence comprising up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modifications to the polynucleotide acid sequence of SEQ ID NO: 969.

[0352] In some embodiments, the VH domain comprises a VH CDR1 encoded by the polynucleotide sequence of SEQ ID NO: 970; a VH CDR2 encoded by the polynucleotide sequence of SEQ ID NO: 971; and a VHCDR3 encoded by the polynucleotide sequence of SEQ ID NO: 972; and the VL domain comprises a VL CDR1 encoded by the polynucleotide sequence of SEQ ID NO: 967; a VL CDR2 encoded by the polynucleotide sequence of SEQ ID NO: 968; and a VL CDR3 encoded by the polynucleotide sequence of SEQ ID NO: 969.

[0353] In some embodiments, the VH domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 966. In some embodiments, the VHdomain is encoded by the polynucleotide sequence of SEQ ID NO: 966.

[0354] In some embodiments, the VL domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 965. In some embodiments, the VLdomain is encoded by the polynucleotide sequence of SEQ ID NO: 965.

[0355] In some embodiments, the VH domain is encoded by a polynucleotide sequence at least 100  75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 966; and the VLdomain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 965. In some embodiments, the VH domain is encoded by the polynucleotide sequence of SEQ ID NO: 966; and the VLdomain that is encoded by the polynucleotide sequence of SEQ ID NO: 965.

[0356] In some embodiments, the CD19 binding domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 974. In some embodiments, the CD19 binding domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 975. In some embodiments, the CD19 binding domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence of SEQ ID NO: 976. In some embodiments, the CD19 binding domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 977. In some embodiments, the CD19 binding domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 978. In some embodiments, the CD19 binding domain is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 979. B. Transmembrane Domain

[0357] The transmembrane domain of the CAR is responsible for its location on the cell surface of the engineered T cell.

[0358] The transmembrane domain can be derived from either a natural or a synthetic source. Where the source is natural, the domain can, for example, be derived from any membrane-bound or transmembrane protein. Suitable transmembrane domains include transmembrane domains 101  from a TCR-alpha chain, a TCR-beta chain, a TCR-γ1 chain, a TCR-δ chain, a TCR-zeta chain, CD28, CD3 epsilon, CD3ζ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, GITR, CD152 (CTLA-4), or CD154, or a functional fragment or variant thereof. Alternatively, the transmembrane domain can be synthetic, and can comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine is found at one or both termini of a synthetic transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain, a CD152 (CTLA-4), TCRγ1, TCRδ or a CD3ζ transmembrane domain.

[0359] Optionally, a short oligonucleotide or polypeptide linker, in some embodiments between 2 and 10 amino acids in length, may link the transmembrane domain with the intracellular signaling domain of a CAR. In some embodiments, the linker is a glycine-serine linker.

[0360] In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain or a CD3ζ transmembrane domain, or a functional fragments or variants thereof.

[0361] In certain embodiments, the transmembrane domain comprises a CD8α transmembrane domain, or a functional fragment or variant thereof. In certain such embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 812 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 812 by at most about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 812, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 812.

[0362] In certain embodiments, the CD8α transmembrane domain, or functional fragment or variant thereof, is encoded by SEQ ID NO: 813 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 813, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 813, or is a codon degenerate variant of SEQ ID NO: 813. 102

[0363] In certain embodiments, the transmembrane domain comprises a CD28 transmembrane domain, or a functional fragment or variant thereof. In certain such embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 814 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 814 by at most about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 814, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 814.

[0364] In certain embodiments, the CD28 transmembrane domain, or functional fragment or variant thereof, is encoded by SEQ ID NO: 815 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 815, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 815, or is a codon degenerate variant of SEQ ID NO: 815. C. Spacer

[0365] In some embodiments, a CAR of the present disclosure comprises a spacer that links the antigen-binding domain to the transmembrane domain. In some embodiments, the spacer is flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition.

[0366] In certain embodiments, a CAR comprising a spacer has improved functional activity compared to an otherwise identical CAR lacking the spacer. In certain embodiments, a CAR comprising a spacer has increased expression on a cell surface compared to an otherwise identical CAR lacking the spacer. In an embodiment, a CAR comprising a spacer is a polypeptide that, were it not for the spacer, would not express on the cell membrane surface and / or would not be able to bind its target due to lack of proximity or steric hindrance.

[0367] In certain embodiments, the spacer comprises a stalk region, for example a hinge region from an antibody. In some embodiments, the stalk region can be from about 20 to about 300 amino 103  acids in length. In some cases, the stalk region can be about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or greater amino acids in length. In other cases, the stalk region can be about: 100, 125, 150, 175, 200, 225, 250, 275 or 300 amino acids in length. In some cases a stalk region can be less than 20 amino acids in length.

[0368] In some instances, the stalk region comprises the hinge region from an IgG, for example IgG1. In alternative instances, the stalk region comprises the CH2CH3 region of immunoglobulin and, optionally, portions of CD3.

[0369] In some embodiments, the stalk region comprises: a CD8α hinge domain, for example one comprising the sequence of SEQ ID NO: 816; an IgG4-Fc 12 amino acid hinge region, for example one comprising the sequence of SEQ ID NO: 818; a CD28 hinge domain; a CTLA-4 hinge domain; or a functional fragment or variant thereof.

[0370] In certain embodiments, the stalk region comprises a CD8α hinge region, or a functional fragment or variant thereof. In certain such embodiments, the spacer comprises the amino acid sequence of SEQ ID NO: 816, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 816 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In one such embodiment, the functional fragment or variant consists of an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1013. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 816, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 816. In some embodiments, the amino acid sequence of the spacer comprises 1, 2, or 3 conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 816. In some such embodiments, the spacer may comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identity with the amino acid sequence of SEQ ID NO: 1014.

[0371] In certain embodiments, the CD8α hinge region, or functional fragment or variant thereof, is encoded by SEQ ID NO: 817, or a functional fragment or variant thereof. In certain 104  embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 817, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 817, or is a codon degenerate variant of SEQ ID NO: 817.

[0372] In certain embodiments, the stalk region comprises a IgG4-Fc 12 amino acid hinge region, or a functional fragment or variant thereof. In certain such embodiments, the spacer comprises the amino acid sequence of SEQ ID NO: 818, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 818 by at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C- terminus. In one such embodiment, the functional fragment or variant consists of an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1015. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 818, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 818. In some embodiments, the amino acid sequence of the spacer comprises 1, 2, or 3 conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 818. In some such embodiments, the spacer comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identity with the amino acid sequence of SEQ ID NO: 1016.

[0373] In some embodiments, the stalk region can be capable of dimerizing with a homologous stalk region of a second CAR.

[0374] In certain embodiments, in addition to a stalk region, the spacer may comprise one or more stalk extension region(s). In certain embodiments, the stalk extension region is a polypeptide that is homologous to the stalk region. For example, it may comprise at least one amino acid residue substitution as compared with the stalk region. In some embodiments, the stalk extension region comprises a sequence with at least about 70%, 75%, 80%, 85%, 90%, 95% , 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% identity to the stalk region to which it is attached, for example a CD8α hinge domain, a CD28 hinge domain, or a CTLA-4 hinge domain. 105

[0375] In some embodiments, the spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 stalk extension regions.

[0376] In certain embodiments, the stalk region can be linked to the stalk extension region by way of a linker.

[0377] In certain embodiments, the stalk extension region can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the length of the stalk region as measured by number of amino acids.

[0378] In some embodiments, the stalk region comprises at least one dimerization site. In certain embodiments, the stalk region may comprise one or more dimerization sites to form homo- or hetero-dimerized chimeric polypeptides. In other embodiments, the stalk region or one or more stalk extension regions may contain mutations that eliminate dimerization sites altogether.

[0379] In certain embodiments, the stalk extension region has at least one fewer dimerization site as compared to a stalk region. For example, if a stalk region comprises two dimerization sites, a stalk extension region can comprise one or zero dimerization sites. As another example, if a stalk region comprises one dimerization site, a stalk extension region can comprise zero dimerization sites. In some examples, a stalk extension region lacks a dimerization site. In some cases, one or more dimerization site(s) in the spacer can be membrane proximal (e.g., the spacer comprises a stalk region containing a dimerization site wherein the stalk region is proximal to the membrane and stalk extension region(s) that do not contain a dimerization site that are distal to the membrane). In other cases, one or more dimerization site(s) can be membrane distal (e.g., the spacer comprises a stalk region containing a dimerization site wherein the stalk region is distal to the membrane and stalk extension region(s) that do not contain a dimerization site that are proximal to the membrane).

[0380] In certain embodiments, the dimerization site is a cysteine residue capable of forming a disulfide bond. In certain embodiments, the stalk extension region is capable of forming fewer disulfide bond(s) as compared to a stalk region. For example, if a stalk region is capable of forming two disulfide bonds, a stalk extension region may be capable of forming one or no disulfide bonds. As another example, if a stalk region is capable of forming one disulfide bond, a stalk extension region may be capable of forming no such bonds. 106

[0381] Each of the stalk extension regions can be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or greater amino acids in length.

[0382] In certain embodiments, the stalk extension region is homologous to the CD8α hinge region. In certain such embodiments, the stalk extension region comprises the amino acid sequence of SEQ ID NO: 820 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 820 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In one such embodiment, the functional fragment or variant consists of an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1017. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 820, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 820. In some embodiments, the amino acid sequence of the spacer comprises 1, 2, or 3 conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 820. In some such embodiments, the spacer comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identity with the amino acid sequence of SEQ ID NO: 1018.

[0383] In certain embodiments, the stalk extension region is encoded by any one of SEQ ID NOs: 821–823, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of SEQ ID NOs: 821–823, hybridizes under stringent hybridization conditions with the complement of any one of SEQ ID NOs: 821–823, or is a codon degenerate variant of any one of SEQ ID NOs: 821–823.

[0384] In certain embodiments, the spacer comprises a stalk region and 1 to 3 stalk extension regions. In certain such embodiments, the spacer comprises a stalk region and 2 stalk extension regions, for example a CD8α hinge region and 2 stalk extension regions wherein each stalk extension region is homologous to the CD8α hinge region.

[0385] In some embodiments, each of the stalk region and stalk extension region(s) can be derived from at least one of a CD8α hinge domain, a CD28 hinge domain, a CTLA-4 hinge domain, 107  a LNGFR extracellular domain, IgG1 hinge, IgG4 hinge and CH2-CH3 domain. The stalk and stalk extension region(s) can be separately derived from any combination of CD8α hinge domain, CD28 hinge domain, CTLA-4 hinge domain, LNGFR extracellular domain, IgG1 hinge, IgG4 hinge or CH2-CH3 domain. As an example, the stalk region can be derived from CD8α hinge domain and at least one stalk extension region can be derived from CD28 hinge domain thus creating a hybrid spacer. As another example, the stalk region can be derived from an IgG1 hinge or IgG4 hinge and at least one stalk extension region can be derived from a CH2-CH3 domain of IgG.

[0386] In certain such embodiments, the spacer comprises the amino acid sequence of SEQ ID NO: 824, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 824, and / or is a conservatively- substituted variant of the amino acid sequence of SEQ ID NO: 824.

[0387] In certain embodiments, the spacer is encoded by SEQ ID NO: 825, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 825, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 825, or is a codon degenerate variant of SEQ ID NO: 825. D. Intracellular Signaling Domain

[0388] The intracellular signaling domain of the CAR may be responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, can be cytolytic activity or helper activity including the secretion of cytokines. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. In some embodiments, the intracellular domain further comprises a signaling domain for T-cell activation. 108

[0389] In some embodiments, the intracellular cell signaling domain interacts with a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell.

[0390] The intracellular domain can comprise an amino acid sequence derived from FCER1G, CD19, CD40, KIR3DL1, KIR3DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, SIRPA, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, FCGR1A, FCGR2A, FCGR2B, FCGR3A, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, PILRB, NCR1, NCR2, NCR3, NKG2A, NKG2C, NKG2D, DAP12, FCER1G, DAP10, CD84, CD19, KIR3DL1, KIR3DL2, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL2, KIR3DL3, SIRPA, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, CD4, CD8A, CD8B, LAT, FCGR1A, FCGR2A, FCGR2B, FCGR3A, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, NCR1, NCR2, NCR3, LY9, NKG2C, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3ζ, CD5, CD22, CD79a, CD79b or CD66d, or a functional fragment or variant thereof. In some cases, the signaling domain for T-cell activation comprises a domain derived from CD3ζ, or a functional fragment or variant thereof.

[0391] In certain embodiments, the intracellular signaling domain comprises a CD3ζ intracellular signaling domain, or a functional fragment or variant thereof. In certain such embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 826, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than SEQ ID NO: 826 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 826, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 826.

[0392] In certain embodiments, the CD3ζ intracellular signaling domain, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 827 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 827, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 827, or is a codon degenerate variant of SEQ ID NO: 827. 109

[0393] The intracellular signaling domain can further comprise one or more co-stimulatory domains. Exemplary co-stimulatory domains include, but are not limited to, CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), and CD3-zeta co-stimulatory domains, and functional fragments or variants thereof. In some instances, a CAR described herein comprises one or more, or two or more of co-stimulatory domains selected from CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, and OX40 (CD134) co-stimulatory domains, and functional fragments or variants thereof. In some instances, a CAR described herein comprises one or more, or two or more of co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, and OX40 (CD134) co-stimulatory domains, and functional fragments or variants thereof. In some instances, a CAR described herein comprises one or more, or two or more of co-stimulatory domains selected from CD8, CD28, 4-1BB (CD137), DAP10, and DAP12 co-stimulatory domains, and functional fragments or variants thereof. In some instances, a CAR described herein comprises one or more, or two or more co-stimulatory domains selected from CD28 and 4-1BB (CD137) co-stimulatory domains, and functional fragments or variants thereof. In some instances, a CAR described herein comprises CD28 and 4-1BB (CD137) co-stimulatory domains, or their respective functional fragments or variants. In some instances, a CAR described herein comprises CD28 and OX40 (CD134) co-stimulatory domains, or their respective functional fragments and variants. In some instances, a CAR described herein comprises CD8 and CD28 co-stimulatory domains, or their respective functional fragments and variants. In some instances, a CAR described herein comprises a CD28 co-stimulatory domain, or a functional fragment or variant thereof. In some instances, a CAR described herein comprises a 4-1BB (CD137) co-stimulatory domain, or a functional fragment or variant thereof. In some instances, a CAR described herein comprises an OX40 (CD134) co-stimulatory domain, or a functional fragment or variant thereof. In some instances, a CAR described herein comprises a CD8 co-stimulatory domain, or a functional fragment or variant thereof. In some instances, the CAR described herein comprises a DAP10 co- stimulatory domain or a functional fragment or variant thereof. In some instances, the CAR described herein comprises a DAP12 co-stimulatory domain, or a functional fragment or variant thereof.

[0394] In certain embodiments, the intracellular signaling domain comprises a CD28 co- stimulatory domain, or a functional fragment or variant thereof. In certain such embodiments, the 110  intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 828, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 828 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 828, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 828.

[0395] In certain embodiments, the CD28 co-stimulatory domain, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 829 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 829, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 829, or is a codon degenerate variant of SEQ ID NO: 829.

[0396] In certain embodiments, the intracellular signaling domain comprises a 4-1BB co- stimulatory domain, or a functional fragment or variant thereof. In certain such embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 830, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 830 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 830, and / or is a conservatively-substituted variant of SEQ ID NO: 830.

[0397] In certain embodiments, the 4-1BB co-stimulatory domain, or functional fragment or variant thereof, is encoded by SEQ ID NO: 831 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 831, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 831, or is a codon degenerate variant of SEQ ID NO: 831. 111

[0398] In certain embodiments, the intracellular signaling domain comprises a DAP10 co- stimulatory domain, or a functional fragment or variant thereof. In certain embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 832, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 832 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 832, and / or is a conservatively-substituted variant of SEQ ID NO: 832.

[0399] In certain embodiments, the DAP10 co-stimulatory domain, or functional fragment or variant thereof, is encoded by the sequence of SEQ ID NO: 833, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 833, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 833, or is a codon degenerate variant of SEQ ID NO: 833.

[0400] In certain embodiments, the intracellular signaling domain comprises a DAP12 co- stimulatory domain, or a functional fragment or variant thereof. In certain embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 834, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 834 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 834, and / or is a conservatively-substituted variant of SEQ ID NO: 834.

[0401] In certain embodiments, the DAP12 co-stimulatory domain, or functional fragment or variant thereof, is encoded by the sequence of SEQ ID NO: 835, or functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 835, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 835, or 112  is a codon degenerate variant of SEQ ID NO: 835.

[0402] In certain embodiments, the intracellular signaling domain comprises both a CD28 co- stimulatory domain and a 4-1BB co-stimulatory domain, or respective functional fragments or variants thereof, as described above.

[0403] In certain embodiments, the intracellular signaling domain comprises a CD3ζ intracellular signaling domain, for example one comprising the sequence of SEQ ID NO: 826, or a functional fragment or variant thereof, and a CD28 co-stimulatory domain, for example one comprising the sequence of SEQ ID NO: 828, or a functional fragment or variant thereof. E. Signal Peptide

[0404] In an embodiment, a signal peptide directs the nascent CAR protein into the endoplasmic reticulum. This is, for example, if the receptor is to be glycosylated and anchored in the cell membrane. Any eukaryotic signal peptide sequence is expected to be functional. Generally, the signal peptide natively attached to the protein or, in the case of a fusion protein, the component closest to the N-terminus is used (e.g., in a scFv with the VL domain at closest to the N-terminus, the native signal of the light chain is used). In some embodiments, the signal peptide is native for GM-CSFRa (SEQ ID NO: 836) or Ig Kappa (IgK) (SEQ ID NO: 838), Immuno-globulin E (IgE) (SEQ ID NO: 834), or a functional fragment or variant thereof. Other signal peptides that can be used include those native to CD8α (SEQ ID NO: 842) and CD28. In some embodiments, the signal peptide is that native to Mouse Ig VHregion 3 (SEQ ID NO: 844), β2M signal peptide (SEQ ID NO: 846), Azurocidin (SEQ ID NO: 848), Human Serum Albumin signal peptide (SEQ ID NO: 850), A2M receptor associated protein signal peptide (SEQ ID NO: 852), IGHV3-23 (SEQ ID NO: 854), IGKV1-D33 (HuL1) (SEQ ID NO: 856), IGKV3-D33 (L14F) (HuH7) (SEQ ID NO: 858), a TVB2 (T21A) signal peptide (SEQ ID NO: 860), a CD52 signal peptide (SEQ ID NO: 862), a low-affinity nerve growth factor receptor (LNGFR, TNFRSF16) signal peptide (SEQ ID NO: 864), or a functional fragment or variant thereof.

[0405] In certain embodiments, the CAR is linked to a GM-CSFRa signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the GM-CSFRa signal peptide has the amino acid sequence of SEQ ID NO: 836, or a functional fragment or variant thereof. In certain 113  embodiments, the functional fragment is shorter than SEQ ID NO: 836 by at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 836, and / or is a conservatively-substituted variant of SEQ ID NO: 836.

[0406] In certain embodiments, the GM-CSFRa signal peptide, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 837, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 837, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 837, or is a codon degenerate variant of SEQ ID NO: 837. F. Exemplary CD19 CAR Constructs

[0407] By way of example only, but not limitation, the CD19 CAR can comprise: (a) a signal peptide; (b) a VH domain and / or VL domain; (b) a transmembrane domain; and (c) an intracellular signaling domain. In certain embodiments, the CD19 CAR further comprises a spacer domain.

[0408] In certain embodiments, the signal peptide is a GM-CSFRa signal peptide, or a functional fragment or variant thereof. In certain such embodiments, the GM-CSFRa signal peptide has the amino acid sequence of SEQ ID NO: 836, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than SEQ ID NO: 836 by at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 836, and / or is a conservatively-substituted variant of SEQ ID NO: 836.

[0409] In certain embodiments, the CD19-specific CAR construct comprises a VH domain and / or a VLdomain. In some embodiments, the CAR construct comprises both a VHdomain and a VLdomain. In further embodiments, the CAR construct comprises a VHdomain, linker, and an VL domain scFv structure. In some embodiments, the scFv comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 959, or a functional fragment or variant 114  thereof. In certain embodiments, the functional fragment is shorter than SEQ ID NO: 959 by at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 959, and / or is a conservatively-substituted variant of SEQ ID NO: 959.

[0410] In certain embodiments, the CD19-specific CAR comprises a CD8α transmembrane domain, or a functional fragment or variant thereof. In some embodiments, the CD8α transmembrane domain, or functional fragment or variant thereof, comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 812 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than SEQ ID NO: 812 by at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 812, and / or is a conservatively-substituted variant of SEQ ID NO: 812.

[0411] In certain embodiments, the spacer comprises a CD8α hinge domain, or afunctional fragment or variant thereof. In some embodiments, the CD8α hinge domain, or functional fragment or variant thereof, comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 816, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than SEQ ID NO: 816 by at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 816, and / or is a conservatively-substituted variant of SEQ ID NO: 816. In certain embodiments, the CD19-specific CAR construct comprises an intracellular signaling domain. In certain embodiments, the intracellular signaling domain comprises a CD3ζ signaling domain, or a functional fragment or variant thereof. In some embodiments, the CD3ζ signaling domain, or functional fragment or variant thereof, comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 826, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than SEQ ID NO: 826 by at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain 115  embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 826, and / or is a conservatively-substituted variant of SEQ ID NO: 826.

[0412] In certain embodiments, the intracellular signaling domain further comprises a CD28 costimulatory signaling domain, or a functional fragment or variant thereof In some embodiments, the CD28 costimulatory signaling domain, or functional fragment or variant thereof, comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 828, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than SEQ ID NO: 828 by at most 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C- terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 828, and / or is a conservatively-substituted variant of SEQ ID NO: 828.

[0413] In certain embodiments, the CD19-specific CAR construct is encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO: 939 or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 939 or a codon degenerate variant of SEQ ID NO: 939).

[0414] In some embodiments, the CD19-specific CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 983, 984, 985, 986, 987, 988, 989, 990, 991 or 992. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 983. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 984. In some embodiments, the CAR comprises an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 985. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 986. In some embodiments, 116  the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 987. In some embodiments, the CAR comprises an amino acid sequence at 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 988. In some embodiments, the CAR comprises an amino acid sequence at 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 989. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 990. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 991. In some embodiments, the CAR comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of SEQ ID NO: 992.

[0415] In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, or 1004. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 993. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 994. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 995. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 996. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 117  997. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 998. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 999. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 1000. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 1001. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 1002. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 1003. In some embodiments, the CAR is encoded by a polynucleotide sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 1004.

[0416] In some embodiments, the CAR comprises the amino acid sequence of CAR CTL019. In some embodiments, the CAR is CAR CTL019. In some embodiments, the CAR comprises the amino acid sequence of the CAR expressed by the CAR T-cell tisagenlecleucel. In some embodiments, the CAR is the CAR expressed by the CAR T-cell tisagenlecleucel. In some embodiments, the CAR comprises the amino acid sequence of the CAR expressed by the CAR T- cell KYMRIAH®. In some embodiments, the CAR is the CAR expressed by the CAR T-cell KYMRIAH®. In some embodiments, the CAR comprises the amino acid sequence of CAR KTE- C19. In some embodiments, the CAR is CAR KTE-C19. In some embodiments, the CAR comprises the amino acid sequence of the CAR expressed by the CAR T-cell axicabtagene ciloleucel. In some embodiments, the CAR is the CAR expressed by the CAR T-cell axicabtagene 118  ciloleucel. In some embodiments, the CAR comprises the amino acid sequence of the CAR expressed by the CAR T-cell YESCARTA®. In some embodiments, the CAR is the CAR expressed by the CAR T-cell YESCARTA®.

[0417] CARs and CAR construction as well as compositions are also described, for example, in: ^ Chimeric Antigen Receptor (CAR) T-Cell Therapies for Cancer: A Practical Guide, Edited by: Daniel W. Lee and Nirali N. Shah, 2020 (ISBN 978-0-323-66181-2; DOI doi.org / 10.1016 / C2017-0-04066-1); ^ Second Generation Cell and Gene-based Therapies, Biological Advances, Clinical Outcomes and Strategies for Capitalisation, Editors-in-Chief: Alain A. Vertès, Devyn M. Smith, Nathan J. Dowden, 2020 (ISBN 978-0-12-812034-7; DOI doi.org / 10.1016 / C2016-0-02070-3); ^ Basics of Chimeric Antigen Receptor (CAR) Immunotherapy, Author: Mumtaz Yaseen Balkhi, 2020 (ISBN 978-0-12-819573-4, DOI doi.org / 10.1016 / C2018-0-05356-6); ^ Engineering and Design of Chimeric Antigen Receptors, Authors: Sonia Guedan, Hugo Calderon, Avery D. Posey, Jr., and Marcela V. Maus, Molecular Therapy: Methods & Clinical Development, Vol. 12, March (2019) (cell.com / molecular-therapy-family / methods / pdf / S2329- 0501(18)30133-5.pdf); ^ Chimeric Antigen Receptor T Cell Therapy Pipeline at a Glance: A Retrospective and Systematic Analysis from Clinicaltrials.Gov, Authors: Eider F Moreno Cortes, Caleb K Stein, Paula A Lengerke Diaz, Cesar A Ramirez-Segura, Januario E. Castro, MD, Blood (2019) 134 (Supplement_1): 5629 (doi.org / 10.1182 / blood-2019-132273); ^ WO2020209934 (PCT / US2020 / 017794) - Novel chimeric antigen receptors and libraries (MIT); ^ WO2020037142 (PCT / US2019 / 046691) - Compositions and methods for high- throughput activation screening to boost t cell effector function (Yale); ^ WO2015123642 (PCT / US2015 / 016057) - Chimeric antigen receptors and methods of 119  making (Univ. TX); ^ WO2019079486 (PCT / US2018 / 056334) - Polypeptide compositions comprising spacers (Precigen) ^ WO2017214333 (PCT / US2017 / 036440) - Cd33 specific chimeric antigen receptors (Precigen) ^ WO2016126608 (PCT / US2016 / 015978) - Car-expressing cells against multiple tumor antigens and uses thereof ^ US 2020 / 0377589 - Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy ^ US 10,800,840 - Compositions and methods for generating a persisting population of T cells useful for the treatment of cancer ^ CN 109400713 – Use of novel chimeric antigen receptor modified T cells for the treatment of cancer ^ U.S. Application No. 18 / 259,985 - Recombinant Vectors Comprising Polycistronic Expression Cassettes and Methods of Use Thereof

[0418] Each of the above, along with US89006682, WO2019213282, US20200268860, WO2020227177, US10457730, WO2019159193, US10287350, US10221245, US20190125799, WO2018201794, US20170368098, US20160145337, US9701758, WO2014153270, WO2012079000, WO2019160956, WO2019161796, WO2020222176, WO2020219848, US20190135894, US10774388, WO2020180882, US10765701, WO2020172641, WO2020172440, WO2016149578, WO2020124021, WO2020108646, WO2020108643, WO2020113188, WO2020108644, WO2020108645, WO2020108642, US10669549, WO2020102770, US10501539, WO2020069409, US10603380, US10533055, WO2020010235, and WO2019246546, is hereby incorporated by reference. Accordingly, any one of the CARs described in each of the above, especially any one of the CD19-specific CARs disclosed therein, may comprise the expression cassette described herein. 120  Cytokine

[0419] In some embodiments, the modified immune effector cell of the present invention can comprise a cytokine. The cytokine may, for example, be encoded by the polynucleotide of the present disclosure. For example, the polynucleotide may encode: the miRNA(s), CAR and cytokine; the miRNA(s) and cytokine; or the CAR and the cytokine.

[0420] In some cases, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variant or combination thereof. In certain embodiments, the cytokine is an interferon, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, hGH, and / or a ligand of human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, IFN-alpha, IFN-beta, or IFN-gamma.

[0421] In certain embodiments, the cytokine is an interleukin. In some cases the interleukin is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL- 16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL- 30, IL-31, IL-32, IL-33, IL-35, or a functional variant or fragment thereof.

[0422] In certain embodiments, the cytokine may be IL-12, or a functional fragment or variant thereof. In some embodiments, the IL-12 is a single chain IL-12 (scIL-12), protease sensitive IL- 12, destabilized IL-12, membrane bound IL-12, intercalated IL-12. In some instances, the IL-12 variants are as described in WO2015 / 095249, WO2016 / 048903, WO2017 / 062953.

[0423] In certain embodiments, the cytokine may be IL-15, or a functional fragment or variant thereof. In certain embodiments, the IL-15, or functional fragment or variant thereof, is membrane- bound. Such may occur when IL-15, or a functional fragment or variant thereof, is bound to membrane-bound IL-15Rα, or a functional fragment or variant thereof. Thus, certain embodiments of the present invention may involve a fusion protein comprising IL-15 and IL-15Rα, or their respective functional fragments or variants (such a fusion protein is referred to herein as “mbIL15”).

[0424] In certain embodiments, the IL-15, or functional fragment or variant thereof, comprises the amino acid sequence of SEQ ID NO: 519, or a functional fragment or variant thereof. In certain 121  embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 519 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C- terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 519, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 519.

[0425] In certain embodiments, the IL-15, or functional fragment or variant thereof, is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 520, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 520, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 520, or is a codon degenerate variant of SEQ ID NO: 520.

[0426] In certain embodiments, the IL-15Rα, or functional fragment or variant thereof, comprises the amino acid sequence of SEQ ID NO: 521 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 521 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 521, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 521.

[0427] In certain embodiments, the IL-15Rα, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 522, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 522, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 522, or is a codon degenerate variant of SEQ ID NO: 522.

[0428] In certain embodiments, the IL-15, or functional fragment or variant thereof, is linked to the IL-15Rα, or functional fragment thereof by way of a linker. 122

[0429] In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 529 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than SEQ ID NO: 529 by at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 529, and / or is a conservatively-substituted variant of SEQ ID NO: 529.

[0430] In certain embodiments, the linker is encoded by a nucleic acid comprising SEQ ID NO: 530 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 530, hybridizes under stringent hybridization conditions with SEQ ID NO: 530, or is a codon degenerate variant of SEQ ID NO: 530.

[0431] In certain embodiments, the fusion protein comprising IL-15 and IL-15Rα comprises the amino acid sequence of SEQ ID NO: 523 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 523 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C- terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 523, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 523.

[0432] In certain embodiments, the fusion protein comprising IL-15 and IL-15Rα is encoded by a nucleic acid comprising SEQ ID NO: 524 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 524, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 524, or is a codon degenerate variant of SEQ ID NO: 524.

[0433] In certain embodiments, the cytokine is linked to a signal peptide. Any signal for use in eukaryotic cells, including those described above for use with the CARs may be linked to the cytokine. In certain embodiments, the cytokine is linked to an IgE signal peptide, for example one 123  comprising the sequence of SEQ ID NO: 840, or a functional fragment or variant thereof.

[0434] In certain embodiments, the fusion protein comprising IL-15 and IL-15Rα comprises the amino acid sequence of SEQ ID NO: 525 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 525 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C- terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 525, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 525.

[0435] In certain embodiments, the fusion protein comprising IL-15 and IL-15Rα is encoded by a nucleic acid comprising SEQ ID NO: 526 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 526, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 526, or is a codon degenerate variant of SEQ ID NO: 526. Cell Tag

[0436] In some embodiments, the modified immune effector cell of the present invention can comprise a cell tag. The cell tag may, for example, be encoded by the polynucleotide of the present disclosure. For example, the polynucleotide may encode the miRNA(s), CAR and / or cytokine described herein as well as a cell tag. In some aspects, the cell tag is used as a kill switch, selection marker, a biomarker, or a combination thereof.

[0437] In certain embodiments, the cell tag is capable of being bound by a predetermined binding partner. In certain such embodiments, the cell tag is non-immunogenic. In certain such embodiments, the cell tag comprises a polypeptide that is truncated so that it is non-immunogenic.

[0438] In certain embodiments, the administration of the predetermined binding partner allows for depletion of infused CAR-T cells. For example, the administration of cetuximab or any antibody that recognizes HER1 allows for the elimination of cells expressing a cell tag comprising 124  truncated non-immunogenic HER1. The truncation of the HER1 sequence eliminates the potential for EGF ligand binding, homo- and hetero-dimerization of EGFR, and / or EGFR-mediated signaling while keeping cetuximab-binding ability intact (Ferguson, K., 2008. A structure-based view of Epidermal Growth Factor Receptor regulation. Annu Rev Biophys, Volume 37, pp.353- 373).

[0439] In certain embodiments, the cell tag comprises at least one of a truncated non- immunogenic HER1 polypeptide, a truncated non-immunogenic LNGFR polypeptide, a truncated non-immunogenic CD20 polypeptide, or a truncated non-immunogenic CD52 polypeptide, or a functional fragment or variant thereof.

[0440] In certain embodiments, the cell tag comprises a truncated non-immunogenic HER1 polypeptide comprising a HER1 Domain III and a truncated HER1 Domain IV. Such domains and the nucleic acid sequences encoding the same include those described in WO 2018 / 226897. Such a cell tag is referred to herein as a “HER1t kill switch” and the truncated non-immunogenic HER1 is referred to as “HER1t”.

[0441] In certain embodiments, the HER1 Domain III comprises the amino acid sequence of SEQ ID NO: 565 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 565 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 565, and / or is a conservatively-substituted variant the amino acid sequence of sequence of SEQ ID NO: 565.

[0442] In certain embodiments, the HER1 Domain III, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 566 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 566, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 566, or is a codon degenerate variant of SEQ ID NO: 566. 125

[0443] In certain embodiments, the truncated HER1 Domain IV comprises the amino acid sequence of SEQ ID NO: 567 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 567 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 567, and / or is a conservatively-substituted variant the amino acid sequence of sequence of SEQ ID NO: 567.

[0444] In certain embodiments, the truncated HER1 Domain IV, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 568 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 568, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 568, or is a codon degenerate variant of SEQ ID NO: 568.

[0445] In certain such embodiments, the truncated non-immunogenic HER1 comprises the amino acid sequence of SEQ ID NO: 569 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 569 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C- terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 569, and / or is a conservatively-substituted variant the amino acid sequence of sequence of SEQ ID NO: 569.

[0446] In certain embodiments, the truncated non-immunogenic HER1, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 570 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 570, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 570, or is a codon degenerate variant of SEQ ID NO: 570. 126

[0447] In certain embodiments, the cell tag comprises a truncated non-immunogenic CD20, or CD20t-1, or a functional fragment or variant thereof. In certain such embodiments, the cell tag comprises the amino acid sequence of SEQ ID NO: 573, SEQ ID NO: 575, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequences of SEQ ID NO: 573 or SEQ ID NO: 575 by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 573 or SEQ ID NO: 575, and / or is a conservatively- substituted variant of the amino acid sequence of SEQ ID NO: 573 or SEQ ID NO: 575.

[0448] In certain embodiments, the truncated non-immunogenic CD20, or CD20t-1, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 574 or SEQ ID NO: 576 or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 574 or SEQ ID NO: 576, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 574 or SEQ ID NO: 576, or is a codon degenerate variant of SEQ ID NO: 574 or SEQ ID NO: 576.

[0449] In certain embodiments, the cell tag further comprises a transmembrane domain. The transmembrane domain can be derived from either a natural or a synthetic source. Where the source is natural, the domain can, for example, be derived from any membrane-bound or transmembrane protein. Suitable transmembrane domains can include the transmembrane domain(s) of alpha, beta or zeta chain of the T-cell receptor; or a transmembrane domain from CD28, CD3 epsilon, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154, or a functional fragment or variant thereof. Alternatively, the transmembrane domain can be synthetic, and can comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine is found at one or both termini of a synthetic transmembrane domain.

[0450] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, or a functional fragment or variant thereof. In certain such embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 828 or a functional 127  fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 828 by at most about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 828, and / or is a conservatively- substituted variant of the amino acid sequence of SEQ ID NO: 828.

[0451] In certain embodiments, the CD28 transmembrane domain, or functional fragment or variant thereof, is encoded by a nucleic acid comprising SEQ ID NO: 829, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 829, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 829, or is a codon degenerate variant of SEQ ID NO: 829.

[0452] In certain embodiments, the cell tag comprises a truncated HER1, or functional fragment or variant thereof, and a transmembrane domain, or a functional fragment or variant thereof. In some embodiments, the cell comprises the amino acid sequence of SEQ ID NO: 1035 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 1035 by at most about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 1035, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 1035.

[0453] In other embodiments, the cell tag comprises the amino acid sequence of SEQ ID NO: 569, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 569 by at most about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 569, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 569. 128

[0454] In other embodiments, the cell tag comprises the amino acid sequence of SEQ ID NO: 571, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 571 by at most about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 571, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 571.

[0455] In certain embodiments, the cell tag is encoded by a nucleic acid comprising SEQ ID NO: 572, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 572, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 572, or is a codon degenerate variant of SEQ ID NO: 572.

[0456] In other embodiments, the cell tag comprises the amino acid sequence of SEQ ID NO: 569 or a functional fragment or variant thereof. In certain embodiments, the functional fragment is shorter than the amino acid sequence of SEQ ID NO: 569 by at most about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the amino acid sequence of SEQ ID NO: 569, and / or is a conservatively-substituted variant of the amino acid sequence of SEQ ID NO: 569.

[0457] In certain embodiments, the cell tag is encoded by a nucleic acid comprising SEQ ID NO: 570, or a functional fragment or variant thereof. In certain embodiments, the functional variant has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 570, hybridizes under stringent hybridization conditions with the complement of SEQ ID NO: 570, or is a codon degenerate variant of SEQ ID NO: 570.

[0458] In certain embodiments, the cell tag is linked with a signal peptide. The signal peptide can be any signal peptide suitable for use in a eukaryotic cell including those described with respect to CARs herein. In certain embodiments, the signal peptide is a Igκ signal peptide comprising the amino acid sequence of SEQ ID NO: 491, or a functional fragment or variant thereof 129

[0459] As mentioned above, in some aspects, the cell tag is used as a kill switch. In this approach, one or more “suicide" genes that initiate apoptotic pathways are incorporated into the CAR construct (Budde et al. PLoS1, 2013 doi:10.1371 / journal.pone.0082742). Activation of these suicide genes may be initiated by the addition of AP1903 (also known as rimiducid), a lipid- permeable tachrolimus analog that initiates homodimerization of the human protein FKBP12 (Fv), to which the apoptosis-inducing proteins are translationally fused. Examples of drug-inducible suicide genes (i.e., kill switches) include inducible caspase 9 and human simplex virus thymidine kinase, where the addition of small molecule inducers trigger the suicide genes and kill the engineered T cells that express them. These features allow clinicians to kill off the CAR T cells if CRS develops and becomes life-threatening. Linkers

[0460] In certain embodiments, the polypeptides of the present invention (e.g., the CAR, the cytokine, and the cell tag) are linked by linker polypeptide(s). The linkers may also be used to link domains of a polypeptide (e.g., the VH and VL domains of a CAR, the truncated HER1 and transmembrane domains of the cell tag, and the IL-15 and IL-15Rα domains).

[0461] Linkers suitable in the present invention include flexible linkers, rigid linkers, and in vivo cleavable linkers. In some cases, the linker acts to link functional domains together (as in flexible and rigid linkers) or to release a free functional domain in vivo as in in vivo cleavable linkers.

[0462] As noted, in some cases, the linker sequence may include a flexible linker. Flexible linkers can be applied when a joined domain requires a certain degree of movement or interaction. Flexible linkers can be composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. A flexible linker can have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). An example of a flexible linker can have the sequence of (Gly-Gly-Gly- Gly-Ser)n (SEQ ID NO: 1037). By adjusting the copy number “n”, the length of this exemplary GS linker can be optimized to achieve appropriate separation of functional domains, or to maintain necessary inter-domain interactions. For example, (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 535), wherein n is 3 is a (G4S)3 linker. Besides GS linkers, other flexible linkers can be utilized for recombinant fusion proteins. In some cases, flexible linkers can contain additional amino acids 130  such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu can be used to improve solubility.

[0463] Flexible linkers can be suitable choices when certain movements or interactions are desired for fusion protein domains. In addition, although flexible linkers do not have rigid structures, in some cases they can serve as a passive linker to keep a distance between functional domains. The length of a flexible linker may be adjusted to allow for proper folding or to achieve optimal biological activity of the fusion proteins.

[0464] A rigid linker can be utilized to maintain a fixed distance between domains of a polypeptide. Examples of rigid linkers include Alpha helix-forming linkers, Pro-rich sequence, (XP)n, X-Pro backbone, A(EAAAK)nA (n = 2-5) (SEQ ID NO: 563) and functional fragments and variants thereof, to name a few. Rigid linkers can exhibit relatively stiff structures by adopting α-helical structures or by containing multiple Pro residues in some cases.

[0465] A linker useful in the present invention can be cleavable in some cases. In other cases, the linker is not cleavable. Linkers that are not cleavable can covalently join functional domains together to act as one molecule throughout an in vivo processes or an ex vivo process. A linker can also be cleavable in vivo. A cleavable linker can be introduced to release free functional domains in vivo.

[0466] A cleavable linker can be cleaved by the presence of reducing reagents, proteases, to name a few. For example, a reduction of a disulfide bond can be utilized to produce a cleavable linker. In the case of a disulfide linker, a cleavage event through disulfide exchange with a thiol, such as glutathione, could produce a cleavage. In some cases, a cleavable linker can allow for targeted cleavage. For example, an in vivo cleavage of a linker in a recombinant fusion protein can also be carried out by proteases that can be expressed in vivo under pathological conditions (e.g., cancer or inflammation), in specific cells or tissues, or constrained within certain cellular compartments. A cleavable linker can comprise a hydrazone, peptides, a disulfide, or a thioester. For example, a hydrazone can confer serum stability. In other cases, a hydrazone can allow for cleavage in ...

Claims

CLAIMS 1. A polynucleotide encoding: (a) a first miRNA that inhibits the expression of an immune checkpoint protein; and (b) a CD19-specific chimeric antigen receptor (CAR).

2. The polynucleotide of claim 1, wherein the immune checkpoint protein is PD-1, PD-L1, CTLA-4, TIGIT, 4-1BB, PIK3IP1, CD27, CD28, CD40, CD70, CD122, CD137, OX40 (CD134), GITR, ICOS, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, or VISTA.

3. The polynucleotide of claim 1, wherein the immune checkpoint protein is PD-1.

4. The polynucleotide of claim 1, further encoding a second miRNA that inhibits the expression of an immune checkpoint protein.

5. The polynucleotide of claim 4, wherein both the first and second miRNA inhibit the expression of PD-1.

6. The polynucleotide of claim 4, wherein the first miRNA is encoded by a nucleic acid comprising SEQ ID NO:

72.

7. The polynucleotide of claim 4, wherein the first miRNA is encoded by a nucleic acid having at least 90% identity with SEQ ID NO:

348.

8. The polynucleotide of claim 4, wherein the first miRNA is encoded by a nucleic acid having at least 90% identity with SEQ ID NO:

179.

9. The polynucleotide of claim 4, wherein the first miRNA is encoded by a nucleic acid comprising SEQ ID NO:

74.

10. The polynucleotide of claim 4, wherein the first miRNA is encoded by a nucleic acid having at least 90% identity with SEQ ID NO:

349.

11. The polynucleotide of claim 4, wherein the first miRNA is encoded by a nucleic acid having at least 90% identity with SEQ ID NO:

180. 364  12. The polynucleotide of claim 4, wherein the first and second miRNAs are encoded by a nucleic acid having at least 90% identity with SEQ ID NO:

267.

13. The polynucleotide of claim 1, wherein the CD19-specific CAR comprises: (a) an antigen-binding domain comprising: (i) a first polypeptide comprising: the amino acid sequence of SEQ ID NO: 948 or a functional variant thereof, the amino acid sequence of SEQ ID NO: 949 or a functional variant thereof, and the amino acid sequence of SEQ ID NO: 950 or a functional variant thereof; and (ii) a second polypeptide comprising: the amino acid sequence of SEQ ID NO: 954 or a functional variant thereof, the amino acid sequence of SEQ ID NO: 955 or a functional variant thereof, and the amino acid sequence of SEQ ID NO: 956 or a functional variant thereof; (b) a transmembrane domain; and (c) an intracellular signaling domain.

14. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises: the amino acid sequence of SEQ ID NO: 948 or a sequence that differs therefrom by up to 5 conservative amino acid substitutions, the amino acid sequence of SEQ ID NO: 949 or a sequence that differs therefrom by up to 5 conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 950 or a sequence that differs therefrom by up to 5 conservative amino acid substitutions; and (ii) the second polypeptide comprises: the amino acid sequence of SEQ ID NO: 954 or a sequence that differs therefrom by up to 5 conservative amino acid substitutions, the amino acid sequence of SEQ ID NO: 955 or a sequence that differs therefrom by up to 5 conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 956 or a sequence that differs therefrom by up to 5 conservative amino acid substitutions.

15. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises: the amino acid sequence of SEQ ID NO: 948 or a sequence that differs therefrom by up to 2 conservative amino acid substitutions, the amino acid sequence of SEQ ID NO: 949 or a sequence that differs therefrom by up to 2 conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 950 or a sequence that differs therefrom by up to 2 conservative amino acid substitutions; 365  and (ii) the second polypeptide comprises: the amino acid sequence of SEQ ID NO: 954 or a sequence that differs therefrom by up to 2 conservative amino acid substitutions, the amino acid sequence of SEQ ID NO: 955 or a sequence that differs therefrom by up to 2 conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 956 or a sequence that differs therefrom by up to 2 conservative amino acid substitutions.

16. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises: the amino acid sequence of SEQ ID NO: 948 or a sequence that differs therefrom by a single amino acid substitution, the amino acid sequence of SEQ ID NO: 949 or a sequence that differs therefrom by a single amino acid substitution, and the amino acid sequence of SEQ ID NO: 950 or a sequence that differs therefrom by a single amino acid substitution; and (ii) the second polypeptide comprises: the amino acid sequence of SEQ ID NO: 954 or a sequence that differs therefrom by a single amino acid substitution, the amino acid sequence of SEQ ID NO: 955 or a sequence that differs therefrom by a single amino acid substitution, and the amino acid sequence of SEQ ID NO: 956 or a sequence that differs therefrom by a single amino acid substitution.

17. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises: the amino acid sequence of SEQ ID NO: 948, the amino acid sequence of SEQ ID NO: 949, and the amino acid sequence of SEQ ID NO: 950; and (ii) the second polypeptide comprises: the amino acid sequence of SEQ ID NO: 954, the amino acid sequence of SEQ ID NO: 955, and the amino acid sequence of SEQ ID NO:

956.

18. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 946; and (ii) the second polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO:

947.

19. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 946; and (ii) the second polypeptide comprises an amino acid sequence having at least 97% identity with SEQ ID NO:

947.

20. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 946; and (ii) the second polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO:

947. 366  21. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 946; and (ii) the second polypeptide comprises an amino acid sequence having at least 99% identity with SEQ ID NO:

947.

23. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 946 or differs therefrom by up to 5 conservative amino acid substitutions; and (ii) the second polypeptide comprises the amino acid sequence of SEQ ID NO: 947 or differs therefrom by up to 5 conservative amino acid substitutions.

24. The polynucleotide of claim 1, wherein: (i) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 946; and (ii) the second polypeptide comprises the amino acid sequence of SEQ ID NO:

947.

25. The polynucleotide of claim 1, wherein the antigen-binding domain comprises an amino acid sequence having at least 95% identity with SEQ ID NO:

959.

26. The polynucleotide of claim 1, wherein the antigen-binding domain comprises an amino acid sequence having at least 97% identity with SEQ ID NO:

959.

27. The polynucleotide of claim 1, wherein the antigen-binding domain comprises an amino acid sequence having at least 98% identity with SEQ ID NO:

959.

28. The polynucleotide of claim 1, wherein the antigen-binding domain comprises an amino acid sequence having at least 99% identity with SEQ ID NO:

959.

29. The polynucleotide of claim 1, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 959 or differs therefrom by up to 5 conservative amino acid substitutions.

30. The polynucleotide of claim 1, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 959 or differs therefrom by up to 2 conservative amino acid substitutions. 367  31. The polynucleotide of claim 1, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 959 or differs therefrom by a single amino acid substitution.

32. The polynucleotide of claim 1, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

959.

33. The polynucleotide of claim 1, wherein the antigen-binding domain comprises an amino acid sequence having at least 95% identity with SEQ ID NO:

961.

34. The polynucleotide of claim 1, wherein the transmembrane domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

812.

35. The polynucleotide of claim 1, wherein the intracellular signaling domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

826.

36. The polynucleotide of claim 1, wherein the intracellular signaling domain comprises a co- stimulatory domain.

37. The polynucleotide of claim 1, wherein the co-stimulatory domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

828.

38. The polynucleotide of claim 1, wherein the CAR further comprises a stalk region comprising an amino acid sequence having at least 90% identity with SEQ ID NO:

816.

39. The polynucleotide of claim 1, wherein the CAR comprises: (a) an antigen-binding domain comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 959; (b) a transmembrane domain comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 812; (c) an intracellular signaling domain comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 826 and an amino acid sequence having at least 95% identity with SEQ ID NO: 828; and (d) a stalk region comprising an amino acid sequence having at least 95% identity with SEQ ID NO:

816.

40. The polynucleotide of claim 1, wherein the CAR comprises: (a) an antigen-binding domain comprising an amino acid sequence having at least 97% identity with SEQ ID NO: 959; (b) a 368  transmembrane domain comprising an amino acid sequence having at least 97% identity with SEQ ID NO: 812; (c) an intracellular signaling domain comprising an amino acid sequence having at least 97% identity with SEQ ID NO: 826 and an amino acid sequence having at least 97% identity with SEQ ID NO: 828; and (d) a stalk region comprising an amino acid sequence having at least 97% identity with SEQ ID NO:

816.

41. The polynucleotide of claim 1, wherein the CAR comprises: (a) an antigen-binding domain comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 959; (b) a transmembrane domain comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 812; (c) an intracellular signaling domain comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 826 and an amino acid sequence having at least 98% identity with SEQ ID NO: 828; and (d) a stalk region comprising an amino acid sequence having at least 98% identity with SEQ ID NO:

816.

42. The polynucleotide of claim 1, wherein the CAR comprises: (a) an antigen-binding domain comprising an amino acid sequence having at least 99% identity with SEQ ID NO: 959; (b) a transmembrane domain comprising an amino acid sequence having at least 99% identity with SEQ ID NO: 812; (c) an intracellular signaling domain comprising an amino acid sequence having at least 99% identity with SEQ ID NO: 826 and an amino acid sequence having at least 99% identity with SEQ ID NO: 828; and (d) a stalk region comprising an amino acid sequence having at least 99% identity with SEQ ID NO:

816.

43. The polynucleotide of claim 1, wherein the CAR comprises: (a) an antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 959 or one that differs therefrom by up to 5 conservative amino acid substitutions; (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 812 or one that differs therefrom by up to 5 conservative amino acid substitutions; (c) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 826, or one that differs therefrom by up to 5 conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 828, or one that differs therefrom by up to 5 conservative amino acid substitutions; and (d) a stalk region comprising the amino acid sequence of SEQ ID NO: 816 or one that differs therefrom by up to 5 conservative amino acid substitutions. 369  44. The polynucleotide of claim 1, wherein the CAR comprises: (a) an antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 959 or one that differs therefrom by up to 2 conservative amino acid substitutions; (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 812 or one that differs therefrom by up to 2 conservative amino acid substitutions; (c) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 826, or one that differs therefrom by up to 2 conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 828, or one that differs therefrom by up to 2 conservative amino acid substitutions; and (d) a stalk region comprising the amino acid sequence of SEQ ID NO: 816 or one that differs therefrom by up to 2 conservative amino acid substitutions.

45. The polynucleotide of claim 1, wherein the CAR comprises: (a) an antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 959 or one that differs therefrom by a single conservative amino acid substitution; (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 812 or one that differs therefrom by a single conservative amino acid substitution; (c) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 826, or one that differs therefrom by a single conservative amino acid substitution, and the amino acid sequence of SEQ ID NO: 828, or one that differs therefrom by a single conservative amino acid substitution; and (d) a stalk region comprising the amino acid sequence of SEQ ID NO: 816 or one that differs therefrom by a single conservative amino acid substitution.

46. The polynucleotide of claim 1, wherein the CAR comprises: (a) an antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 959; (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 812; (c) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 826 and the amino acid sequence of SEQ ID NO: 828; and (d) a stalk region comprising the amino acid sequence of SEQ ID NO:

816.

47. The polynucleotide of claim 46, wherein the first miRNA is encoded by a nucleic acid comprising SEQ ID NO: 72 and wherein the polynucleotide further encodes a second miRNA encoded by a nucleic acid comprising SEQ ID NO:

74.

48. The polynucleotide of claim 1, further encoding a cell tag. 370  49. The polynucleotide of claim 48, wherein the cell tag comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

571.

50. The polynucleotide of claim 1, further encoding a cytokine.

51. The polynucleotide of claim 50, wherein the cytokine is IL-15 or a functional variant thereof.

52. The polynucleotide of claim 1, further encoding a fusion protein comprising: IL-15, or a functional variant thereof, and IL-15Rα, or a functional variant thereof.

53. The polynucleotide of claim 52, wherein the fusion protein comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

523.

54. The polynucleotide of claim 1, comprising a promoter.

55. The polynucleotide of claim 54, wherein the promoter is an EF1a promoter.

56. A vector comprising the polynucleotide of any one of claims 1–55.

57. The vector of claim 56, wherein the vector is a plasmid, a viral vector, or a non-viral vector.

58. The vector of claim 56, wherein the vector is a non-viral vector.

59. The vector of claim 56, comprising a Sleeping Beauty transposon.

60. The vector of claim 56, wherein the polynucleotide is framed by a left transposon repeat region and a right transposon repeat region.

61. The vector of claim 60, wherein the left transposon repeat region comprises a nucleic acid of SEQ ID NO:

580.

62. The vector of claim 60, wherein the right transposon repeat region comprises a nucleic acid of SEQ ID NO:

581. 371  63. A system for use in expressing a CAR in a cell, the system comprising the vector of claim 59 and a transposase or a vector encoding the transposase.

64. The system of claim 63, wherein the transposase is salmonid-type Tc1-like transposase.

65. The system of claim 63, wherein the transposase is a SB11 or a SB100x transposase.

66. An engineered immune effector cell comprising the polynucleotide of any one of claims 1–55.

67. The engineered immune effector cell of claim 66, wherein the cell is a T cell or a NK cell.

68. An engineered immune effector cell comprising: (a) a first and a second miRNA that inhibit the expression of an immune checkpoint protein; and (b) a CD19-specific CAR.

69. A method for producing the engineered immune effector cell of claim 66, the method comprising introducing a vector comprising the polynucleotide into an immune effector cell.

70. A composition comprising the polynucleotide of any one of claims 1–55.

71. The composition of claim 70 for use in the manufacture of a medicament for the treatment of a disease or disorder.

72. A composition comprising the engineered immune effector cell of claim 66.

73. The composition of claim 72 for use in the manufacture of a medicament for the treatment of a disease or disorder.

74. A kit comprising the polynucleotide of any one of claims 1–55.

75. A kit comprising the engineered immune effector cell of claim 66.

76. A method of treating a disease or disorder, the method comprising administering the engineered immune effector cell of claim 66 to a subject in need thereof. 372  77. The method of claim 76, wherein the disease or disorder is associated with the overexpression of CD19.

78. The method of claim 76, wherein the disease or disorder is a cancer.

79. The method of claim 78, wherein the cancer is a hematological malignancy.

80. The method of claim 76, wherein the disease or disorder is relapsed and refractory B-cell lymphomas, acute lymphoblastic leukemia, mantle cell lymphoma, chronic lymphocytic leukemia, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, and B-cell precursor acute lymphoblastic leukemia.

81. The method of claim 76, wherein the disease or disorder is an autoimmune disorder.

82. The method of claim 76, wherein the disease or disorder is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), multiple sclerosis, myasthenia gravis (MG), type 1 diabetes, inflammatory bowel disease, psoriasis, and autoimmune thyroiditis.

83. The method of claim 76, further comprising the administration of an additional therapy.

84. The method of claim 76, wherein the engineered immune effector cell does not undergo propagation or activation before the administration to the subject.

85. The method of claim 76, further comprising a debulking procedure.

86. A use of the engineered immune effector cell of claim 66 in the manufacture of a medicament for the treatment of a disease or disorder.

87. The use of claim 86, wherein the disease or disorder is an autoimmune disorder.

88. The use of claim 86, wherein the disease or disorder is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), multiple sclerosis, myasthenia gravis (MG), type 1 diabetes, inflammatory bowel disease, psoriasis, and autoimmune thyroiditis. 373

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