CD19-targeted CAR T cell therapy

Abstract

Chimeric antigen receptors (CARs) capable of targeting antigens expressed on disease-associated cells such as tumor cells, e.g., CD19-specific CARs. Polynucleotides encoding CARs and, if necessary, i) miRNA silencer modules capable of inhibiting the expression of immune checkpoint proteins (e.g., PD-1); ii) cytokines (e.g., membrane-bound IL-15); and / or cell tags (e.g., HER-1 kill switch). Vectors comprising polynucleotides. Modified immunoeffector cells comprising CARs, polynucleotides, or vectors. Compositions and kits comprising CARs, polynucleotides, vectors, and / or modified immunoeffector cells. Use of CARs, polynucleotides, vectors, and / or modified immunoeffector cells in the manufacture of pharmaceuticals for the treatment of disease or disorder. Methods for treating a subject having a disease or disorder, comprising the step of administering CARs, polynucleotides, vectors, and / or modified immunoeffector cells to a subject in need thereof.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority rights to U.S. Provisional Patent Application No. 63 / 556,848, filed on 18 March 2024, and U.S. Provisional Patent Application No. 63 / 516,565, filed on 31 July 2023.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy created on July 25, 2024, is named 75594-410122_SL.xml and has a size of 784,341 bytes.

[0003] Field of Invention The present invention relates to chimeric receptor therapies, particularly chimeric antigen receptor (CAR) T-cell therapies, specifically those specific to CD19, for the treatment of diseases and disorders characterized by CD19 overexpression. [Background technology]

[0004] Chimeric receptor therapies, including chimeric antigen receptor T (CAR-T) cell 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, engineered cells bind to tumor cells and initiate an immune response that leads to the destruction of tumor cells. Similarly, in the treatment of autoimmune disorders, engineered cells can be made to direct towards specific immune cells that cause abnormal immune responses. By targeting and depleting these abnormal immune cells, chimeric receptor therapies have the potential to modulate the immune system and alleviate the symptoms of autoimmune diseases. This approach opens a promising avenue for treating both cancer and autoimmune conditions by leveraging the power of engineered immune cells to selectively eliminate harmful cells while preserving healthy tissue.

[0005] Antigens present on cancer cells, particularly those that may also be present on normal cells but are overexpressed on cancer cells, are promising targets for chimeric receptor therapy. Several targets for such therapies have been identified to date, and these targets include, but are not limited to, CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate-binding proteins, 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, MUC16, ROR1, and mesothelin are considered particularly promising targets for immunotherapy.

[0006] CD19 expression is used as a diagnostic marker for certain hematological malignancies. For example, CD19 expression is often used in combination with other markers to diagnose a variety of B-cell malignancies, including relapsed and refractory B-cell lymphoma, acute lymphoblastic leukemia, mantle cell lymphoma, chronic lymphocytic leukemia, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, and precursor B-cell acute lymphoblastic leukemia. Long-term follow-up data indicate that treatment with CD19-targeted CAR-T cells is effective in treating patients with these hematological malignancies as well as patients with other cancers associated with CD19 overexpression.

[0007] Ongoing research efforts aim to improve the persistence of responses after CAR-T cell therapy. One of the challenges in CAR-T cell design is maintaining their presence in the body for extended periods without exhaustion. This aspect plays a crucial role in their effectiveness and prevents relapse. Ensuring the sustained activity of CAR-T cells is extremely important for successful outcomes.

[0008] Immune checkpoint inhibitors, which prevent T cell switching and promote the activity of these cells, are considered promising. Immunotherapy utilizing blocking antibodies is widely evaluated in clinical practice and has been shown to improve tumor regression across multiple malignancies, particularly when administered in combination with CAR-T cells or cells expressing TCRs. Examples of checkpoint inhibitor targets, but not limited to, include 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 are considered the most promising targets. In particular, the PD-1 / programmed death ligand 1 (PD-L1) pathway plays a crucial role in how tumor cells evade the immune response, and therefore PD-1 and PD-L1 are especially promising targets.

[0009] However, adding systemic checkpoint inhibitors to conventional CAR-T therapy further complicates treatment and increases toxicity risks and costs. Furthermore, checkpoint inhibitor blocking antibodies do not function consistently across cancer types, may have limitations in entering the tumor microenvironment, require repeated doses, and may lose effectiveness over time. Genome editing is an alternative approach to checkpoint inhibition and has the advantage of limiting checkpoint inhibitors to only manipulated CAR-T cells. However, gene editing further complicates the manufacturing process, thereby increasing the time and cost of cell therapy.

[0010] Therefore, in this field, there continues to be a need for safer, more effective, and less expensive therapies for antigen-related diseases and conditions, including treatments that combine CAR-T and / or TCR therapies with systemic checkpoint inhibitors.

[0011] To address complex in vivo biological issues, such as loss of immune surveillance, genetic alteration of tumor antigen composition, and tumor heterogeneity (leading to differences in cancer cell phenotypes), it is also necessary to devise ways to diversify treatment regimens to target antigens from multiple angles.

[0012] In addition, the current state of traditional CAR-T cell production in centralized GMP facilities is burdened by high costs and labor-intensive processes. Dependence on viral vectors, as well as the need for ex vivo cell activation and large-scale expansion to obtain an appropriate number of cells for treatment, contribute to these inefficiencies. As a result, extended production times not only lead to treatment delays but also increase the risk of CAR-T cell exhaustion. Given these challenges, there is a need in this field for more efficient and rapid manufacturing processes to streamline CAR-T therapy production and improve patient outcomes.

[0013] This invention improves the efficacy and safety of CD19 CAR-T cell therapy and reduces the burdensome costs by eliminating the need for gene editing or combination with checkpoint inhibitors. [Overview of the project]

[0014] In part, this invention relates to a naturally occurring polynucleotide that codes for 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 a particular embodiment, 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 immune checkpoint proteins.

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

[0020] In a particular embodiment, the PD-1 silencing miRNA module is encoded by two individual miRNAs.

[0021] In a particular embodiment, 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 a nucleic acid sequence that can hybridize to a complementary sequence of SEQ ID NO: 348 under stringent hybridization conditions.

[0022] In a particular embodiment, 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 a nucleic acid sequence that can hybridize to a complementary sequence of SEQ ID NO: 349 under stringent hybridization conditions.

[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 a nucleic acid sequence that can hybridize to a complementary sequence of SEQ ID NO: 267 under stringent hybridization conditions. In other embodiments, the PD-1 silencing miRNA module is located within the 5'UTR. In certain embodiments, the 5'UTR is encoded by a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 944, or a nucleic acid sequence that can hybridize to a complementary sequence of SEQ ID NO: 944 under stringent hybridization conditions.

[0024] In certain embodiments, polynucleotides further encode cytokines.

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

[0026] In certain other 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, polynucleotides further encode cell tags.

[0029] In certain embodiments, cell tags act as safety or kill switches.

[0030] In certain embodiments, the cell tag includes 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 a particular embodiment, the cell tag is encoded by a polynucleotide having at least 90% identity with SEQ ID NO: 571.

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

[0033] In a particular embodiment, the cell tag is encoded by a polynucleotide having at least 90% 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 up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than SEQ ID NO: 836 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.

[0036] In a particular embodiment, the polynucleotide is CD19-specific V H Domain and CD19-specific V L Code the domain.

[0037] In a particular embodiment, the polynucleotide encodes a CD19-specific scFv.

[0038] In some embodiments, scFv comprises a polypeptide having at least 90% sequence identity with sequence number 959.

[0039] In certain embodiments, the polynucleotide encodes a CD8 hinge and a 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 the 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 a particular embodiment, the nucleic acid sequence encoding the chimeric receptor is flanked by transposon repeat sequence regions.

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

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

[0045] In a particular embodiment, the Sleeping Beauty transposon includes a nucleic acid sequence or a functional variant thereof having at least 90% sequence identity with sequence number 981 (for example, 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 sequence number 981, or a codon degenerate variant of sequence number 981).

[0046] In a particular embodiment, Sleeping Beauty includes a nucleic acid sequence or a functional variant thereof having at least 90% sequence identity with sequence number 982 (for example, 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 sequence number 982, or a codon degenerate variant of sequence number 982).

[0047] In part, this invention relates to modified immunoeffector cells containing the polynucleotides of the present invention.

[0048] The present invention relates in part to a composition comprising the polynucleotide of the present invention or modified immunoeffector cells of the present invention.

[0049] In part, the present invention relates to a kit comprising the polynucleotide or cells of the present invention.

[0050] In part, the present invention relates to a method for treating a disease or disorder, comprising the step of administering modified effector cells of the present invention to a subject in need of such treatment.

[0051] In a particular embodiment, the disease or disorder is cancer.

[0052] In certain embodiments, 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 an autoimmune disorder. In some embodiments, the autoimmune diseases or disorders are, among many others, rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), multiple sclerosis, myasthenia gravis (MG), type 1 diabetes, inflammatory bowel disease, psoriasis, and autoimmune thyroiditis. In certain embodiments, the autoimmune disease or disorder being treated is SLE.

[0054] In certain embodiments, the modified immunoeffector cells are neither proliferated nor activated before administration.

[0055] In part, the present invention relates to the use of modified immunoeffector cells in the manufacture of pharmaceuticals for the treatment of diseases or disorders.

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

[0057] In certain embodiments, additional therapeutic agents may be vaccines, interleukins (e.g., IL-12), immunotherapeutic agents, anticancer drugs, chemotherapeutic agents, or immune checkpoint inhibitors.

[0058] In certain embodiments, the chemotherapeutic agent is a histone deacetylase inhibitor. [Brief explanation of the drawing]

[0059] [Figure 1]Figure 1A is a schematic diagram of a single Sleeping Beauty transposon (or referred to herein as "Vector 1" or "CD19 UltraCAR-T") encoding a CD19-specific CAR, mbIL15, and HER1t. Figure 1B is a schematic diagram of a single Sleeping Beauty transposon (or referred to herein as "Vector 2" or "NextGen CD19 UltraCAR-T") encoding a CD19-specific CAR, mbIL15, HER1t, and two PD-1 silencing miRNAs. [Figure 2-1] Figures 2A-2C are sensorograms showing that CD19 binds to FMC63 scFv-Fc with a binding affinity of 4.5 nM (Figure 2A), CD19 binds to FMC63 IgG with a binding affinity of 7.9 nM (Figure 2B), and CD19 does not bind to the negative control scFv-Fc (Figure 2C). [Figure 2-2] Same as above. [Figure 3] Figures 3A and 3B show graphs illustrating the reduction in PD-1 expression in CD19 UltraCAR-T cells (produced using vector 2) with a PD-1 silencer compared to UltraCAR-T cells (produced using vector 1) without a PD-1 silencer, based on RT-qPCR (Figure 3A) and RNA-seq (Figure 3B) data. The values ​​represent the ratio of the change in expression in UltraCAR-T cells containing a PD-1 silencer compared to UltraCAR-T cells without a PD-1 silencer, based on samples generated from 10 healthy donors. [Figure 4]Figure 4A is a bar graph showing the multiplier of change in PD-1 targeting miRNA expression in CD19 UltraCAR-T (produced using vector 2) containing a PD-1 silencer compared to CD19 UltraCAR-T (produced using vector 1) without a PD-1 silencer. Figure 4B shows the alignment of small RNA reads to the PD-1 silencer module using the IGV browser. The mapped miRNAs match PD-1_1843 and PD-1_2061, which are intended PD-1s targeting the guide miRNAs, with little to no visible passenger miRNAs. The data are representative of one donor vector 2 sample. The X-axis shows nucleotide positions across the silencer module, and the gray vertical bars show read depth. [Figure 5] Figures 5A–5D show a series of bar graphs illustrating the specific cytotoxicity of CD19 UltraCAR-T cells containing the PD-1 silencer (produced using vector 2) against acute lymphoblastic leukemia cell lines (NALM-6 on day 3 (Figure 5A) and NLAM-6 / PD-L1 on day 3 (Figure 5B)) compared to CD19 UltraCAR-T cells without the PD-1 silencer (produced using vector 1). Various effector-to-target (E:T) ratios are shown. CD19 knockout NALM-6 (Figure 5C) and the CD19-nonexpressing cell line MOLM-13 (Figure 5D) are included as controls. [Figure 6] Figures 6A and 6B show bar graphs of pluripotency (Figure 6A) and pluripotency intensity index (PSI) (Figure 6B) in NALM-6 and NALM-6 / PDL1 target cell lines treated with Sleeping BeautyUltraCAR-T cells containing the PD-1 silencer (produced using vector 2) and those not containing the PD-1 silencer (produced using vector 1). [Figure 7]Figures 7A and 7B show bar graphs of pluripotency (Figure 7A) and pluripotency intensity index (PSI) (Figure 7B) in JEKO-1 / PDL1 target cell lines treated with Sleeping BeautyCAR-T cells containing the PD-1 silencer (produced using vector 2) and those not containing the PD-1 silencer (produced using vector 1). [Figure 8] This dot plot illustrates the cytotoxic activity of CAR-T cells after multiple rounds of stimulation with CD19+ tumor cells. Results are expressed as the ratio of target cell death percentage in each tumor cell addition round to the death capacity in the first round. Color represents the sustained cytotoxicity of CD19 CAR-T cells containing the PD-1 silencer (produced using vector 2; circles) under highly stimulated conditions compared to control CD19 UltraCAR-T cells without the PD-1 silencer (produced using vector 1; squares). [Figure 9-1] Figures 9A-9G are a series of Western blot images showing that CD19 UltraCAR-T cells containing a PD-1 silencer (produced using vector 2) and CD19 UltraCAR-T cells without a PD-1 silencer (produced using vector 1) express CD19-CAR (Figures 9A-9B), mbIL15 (Figures 9C-9D), and HER1t (Figure 9E), and that these UltraCAR-T cells containing a PD-1 silencer (vector 2) show reduced PD-1 expression upon stimulation with CD3 / CD28 beads (Figures 9F-9G). [Figure 9-2] Same as above. [Figure 10-1]Figure 10A is a bar graph showing the results from an antibody-dependent (anti-HER1 antibody-cetuximab) cytotoxicity assay involving CD19 UltraCAR-T cells containing a PD-1 silencer (produced using vector 2) and control UltraCAR-T cells without a PD-1 silencer (produced using vector 1), with the addition of either an anti-HER1 antibody (cetuximab) or a control anti-CD20 antibody (rituximab). Figure 10B is a graph of flow cytometry data from the antibody-dependent cytotoxicity assay. [Figure 10-2] Same as above. [Figure 11] This dot plot shows the results from cytokine depletion assays performed using CD19 UltraCAR-T cells (produced using vector 2) containing a PD-1 silencer and expressing or not expressing mbIL15 (cytokine), and CD19 UltraCAR-T cells (IGE-2503) that do not contain a PD-1 silencer and express or not expressing mbIL15 (cytokine). [Figure 12-1] Figure 12A is a dot plot of IVIS (tumor burden) in a NALM-6 tumor model using NSG mice treated with CD19 UltraCAR-T cells containing the PD-1 silencer (produced using vector 2) and control UltraCAR-T cells without the PD-1 silencer (produced using vector 1). Figure 12B is an image of in vivo bioluminescence imaging of treated CD19+ NALM-6 tumor-carrying NSG mice. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 12-5] Same as above. [Figure 12-6] Same as above. [Figure 13]Figure 13A is a dot plot showing relative body weight in NSG mice treated with CD19 UltraCAR-T cells containing the PD-1 silencer (produced using vector 2) and control CD19 UltraCAR-T cells without the PD-1 silencer module (produced using vector 1), using the NALM-6 tumor model. Figure 13B is a dot plot showing survival in these same mice. [Figure 14-1] Figures 14A–14E are dot plots resulting from flow cytometry characterization of peripheral blood from NSG mice (NALM-6 tumor model) treated with CD19 UltraCAR-T cells containing the PD-1 silencer module (produced using vector 2) and those not containing the PD-1 silencer module (produced using vector 1). Figures 14A–C are dot plots of HER1t+ cell concentration after treatment, Figure 14D is a dot plot of PD-1 expression after treatment, and Figure 14E is a dot plot of memory cell concentration after treatment. [Figure 14-2] Same as above. [Figure 15] Figures 15A and 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 (produced using vector 3) that do not express mbIL15 and do not have a PD-1 silencer; (ii) CD19 CAR-T cells (produced using vector 4) that contain a PD-1 silencer but do not express mbIL15; (iii) CD19 UltraCAR-T cells (produced using vector 1) that express mbIL-15 but do not have a PD-1 silencer; and (iv) CD19 UltraCAR-T cells (produced using vector 2) that express mbIL-15 and have a PD-1 silencer. Figures 15B and 16B are dot plots showing the relative body weight of NSG MHC class I / II KO mice treated (Tx) with (i), (ii), (iii), and (iv). [Figure 16] Same as above. [Figure 17] Figures 17A-17E are a series of dot plots showing the relative (%) change in body weight in NSG MHC class I / II KO mice (NALM-6 tumor model) treated with (i) CD19 CAR-T cells (produced using vector 3) that do not express mbIL-15 and do not have a PD-1 silencer (Figure 17B); (ii) CD19 CAR-T cells (produced using vector 4) that contain a PD-1 silencer but do not express mbIL-15 (Figure 17C); (iii) CD19 UltraCAR-T cells (produced using vector 1) that express mbIL-15 but do not have a PD-1 silencer (Figure 17D); (iv) CD19 UltraCAR-T cells (produced using vector 2) that express mbIL-15 and have a PD-1 silencer (Figure 17E); and (v) physiological saline (Figure 17A). [Figure 18-1] Figures 18A and 18B are bar graphs (day 21 of the study) showing the mbIL-15 expression level (Figure 18A) and PD-1 expression level (Figure 18B) in NSG MHC class I / II KO mice (NALM-6 tumor model) treated with (i) CD19 CAR-T cells (produced using vector 3) that do not express mbIL-15 and do not have a PD-1 silencer; (ii) CD19 CAR-T cells (produced using vector 4) that contain a PD-1 silencer but do not express mbIL-15; (iii) CD19 UltraCAR-T cells (produced using vector 1) that express mbIL-15 but do not have a PD-1 silencer; and (iv) CD19 UltraCAR-T cells (produced using vector 2) that express mbIL-15 and have a PD-1 silencer. Figures 18C-18F are dot plots showing the HER1t concentration in the blood after treatment in similarly treated mice. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 19]This is a dot plot of memory cell counts in Study 21 of NSG MHC class I / II KO mice (NALM-6 tumor model) treated with (i) CD19 CAR-T cells that do not express mbIL-15 and do not have a PD-1 silencer (produced using vector 3); (ii) CD19 CAR-T cells that have a PD-1 silencer but do not express mbIL-15 (produced using vector 4); (iii) CD19 UltraCAR-T cells that express mbIL-15 but do not have a PD-1 silencer (produced using vector 1); and (iv) CD19 UltraCAR-T cells that express mbIL-15 and have a PD-1 silencer (produced using vector 2). [Figure 20-1] Figures 20A-B and 21A-B are dot plots of tumor burden (dorsal and ventral IVIS) in NSG mice (Jeko-1 / PD-L1 tumor model) after treatment with 1 × 10⁶ (Figures 20A-B) and 5 × 10⁶ (Figures 21A-B) NextGen CD19 UltraCAR-T cells containing the PD-1 silencer (produced using vector 2) and control CD19 UltraCAR-T cells without the PD-1 silencer (produced using vector 1) cells. Figures 20C and 21C are in vivo bioluminescence imaging (tumor burden) images of treated CD19+Jeko-1 / PD-L1 tumor-bearing NSG mice. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 20-4] Same as above. [Figure 20-5] Same as above. [Figure 20-6] Same as above. [Figure 20-7] Same as above. [Figure 20-8] Same as above. [Figure 20-9] Same as above. [Figure 21-1] Same as above. [Figure 21-2] Same as above. [Figure 21-3]Same as above. [Figure 21-4] Same as above. [Figure 21-5] Same as above. [Figure 21-6] Same as above. [Figure 21-7] Same as above. [Figure 21-8] Same as above. [Figure 21-9] Same as above. [Figure 22] Figures 22A-22B are dot plots of relative body weight (%) in NSG mice (Jeko-1 / PD-L1 tumor model) after treatment with 1 × 10⁶ cells (Figure 22A) and 5 × 10⁶ cells (Figure 22B) containing NextGen CD19 UltraCAR-T cells (produced using vector 2) containing the PD-1 silencer and control CD19 UltraCAR-T cells (produced using vector 1) not containing the PD-1 silencer. [Figure 23-1]Figures 23A and 23B are dot plots showing HER1t concentrations in NSG mice (Jeko-1 / PD-L1 tumor model) after treatment with 1 × 10⁶ cells (Figure 23A) and 5 × 10⁶ cells (Figure 23B) of NextGen CD19 UltraCAR-T cells containing the PD-1 silencer (produced using vector 2) and control CD19 UltraCAR-T cells without the PD-1 silencer (produced using vector 1). Figures 23C and 23D are dot plots showing PD-1 levels in NSG mouse models (Jeko-1 / PD-L1 tumor model) after treatment with 1 × 10⁶ cells (Figure 23C) and 5 × 10⁶ cells (Figure 23D) of NextGen CD19 UltraCAR-T cells containing the PD-1 silencer (produced using vector 2) and control CD19 UltraCAR-T cells without the PD-1 silencer (produced using vector 1). Figures 23E and 23F are dot plots showing memory cell concentrations in NSG mice (Jeko-1 / PD-L1 tumor model) after treatment with 1 × 10⁶ cells (Figure 23E) and 5 × 10⁶ cells (Figure 23F) containing NextGen CD19 UltraCAR-T cells (produced using vector 2) and control CD19 UltraCAR-T cells (produced using vector 1) that do not contain the PD-1 silencer. [Figure 23-2] Same as above. [Figure 24] Figures 24A-24B are dot plots of tumor load (Figure 24A) and HER1t concentration (Figure 24B) in a Raji tumor model using NSG mice treated with saline (HBSS); CD19 CAR-T cells expressing HER1t but not expressing mbIL-15 and lacking a PD-1 silencer (produced using vector 5); control CD19 UltraCAR-T cells without a PD-1 silencer (produced using vector 1); or NextGen CD19 UltraCAR-T cells containing a PD-1 silencer (produced using vector 2). [Figure 25]Figures 25A–25C are dot plots of HER1t concentrations in the blood (Figure 25A), spleen (Figure 25B), and bone marrow (BM, Figure 25C) of humanized mouse models after treatment with physiological saline (HBSS) or NextGen CD19 UltraCAR-T cells (produced using vector 2) containing a PD-1 silencer. [Figure 26] Figures 26A–26C are dot plots of CD19+ B cell concentrations in the blood (Figure 26A), spleen (Figure 26B), and bone marrow (BM, Figure 26C) of a humanized mouse model after treatment with physiological saline (HBSS) or with NextGen CD19 UltraCAR-T cells (produced using vector 2) containing a PD-1 silencer. [Figure 27-1] Figures 27A–27H show the results of NSG MHC class I / II KO mice (NALM-6 tumor model) in initial challenge and rechallenge studies. Mice were treated with (i) CD19 CAR-T cells (produced using vector 3) that do not express mbIL-15 and do not have a PD-1 silencer (Figures 27A and 27C), or (ii) CD19 UltraCAR-T cells (produced using vector 2) that express mbIL-15 and have a PD-1 silencer (Figures 27B and 27D). Figures 27A–27D show dot plots of tumor burden as measured by total luminous flux. Figure 27E is a schematic diagram of an exemplary study protocol. Figures 27F–27G are dot plots of tumor burden after rechallenge with a NALM-6 tumor in naive mice (Figure 27F) or tumor-free mice previously treated with vector 2 (Figure 27G). Figure 27H shows the survival curves after tumor rechallenge for naive mice and tumor-free mice. [Figure 27-2] Same as above. [Figure 28-1]Figures 28A–28I show the production of NextGen CD19 UltraCAR-T cells (produced using vector 2) from SLE / LN donors and healthy donors. Figure 28A is a bar graph of T cell transfection efficiency. Figures 28B–28E are a series of graphs showing that NextGen CD19 CAR-T cells generated from SLE / LN donors and healthy donors exhibited similar T cell phenotypes when measured by (Figure 28B) CD4+ phenotype, (Figure 28C) CD8+ phenotype, (Figure 28D) CD4+ to CD8+ ratio, and (Figure 28E) memory cell phenotype (using CD45RA and CD62L markers). Figures 28F–28I are a series of bar graphs and dot plots showing that NextGen CD19 CAR-T cells (produced using vector 2) generated from SLE / LN donors and healthy donors induced complete autologous B cell depletion in vitro. Figure 28F illustrates the results of co-culture experiments of target cells (B cells) and effector cells (CAR-T cells) at various ratios, using bar graphs showing the percentage of CD19+ B cells killed by NextGen CD19 UltraCAR-T cells after electroporation and overnight incubation. Figure 28G is a dot plot showing the successful expansion of CAR-T cells by measuring the number of CAR-T cells over time after transfection. Figures 28H-28I are bar graphs showing the percentage of CD19+ B cells killed by NextGen CD19 UltraCAR-T cells after 3 weeks of B cell expansion and 24 hours (Figure 28H) or 48 hours (Figure 29I) of exposure to CAR-T cells. [Figure 28-2] Same as above. [Figure 29-1]Figures 29A–29M show that Nextgen CD19 CAR-T cells (Vector 2) generated from SLE / LN donors and healthy donors induced complete autologous B cell depletion in humanized MHC I / II double knockout (DKO) mice. Figure 29A is an exemplary schematic diagram of the study design. Figures 29B–29C are dot plots showing the number of B cells in blood at 7 and 14 days with and without autologous CAR-T treatment, prepared from either healthy donors (HD) (Figure 29B) or SLE / LN donors (Figure 29C). Figure 29D is a bar graph showing the frequency of CD19+ viable B cells in blood, used to calculate the in vivo death percentage. Figure 29E is a dot plot showing the expansion of HER1T+ CAR-T cells in blood at 7 days (Day 14 of the study) and 14 days (Day 21 of the study) after administration of NextGen CD19 UltraCAR-T cells. Figures 29F–29G are dot plots showing the number of B cells in the bone marrow at 7 and 14 days after administration of autologous CAR-T cells, with and without treatment, from either a healthy donor (HD) (Figure 29F) or an SLE / LN donor (Figure 29G). Figure 29H is a bar graph showing the frequency of CD19+ viable B cells in the bone marrow, used to calculate the in vivo death percentage. Figure 29I is a dot plot showing the expansion of HER1T+ CAR-T cells in the bone marrow (BM) at 7 days (day 14 of the study) and 14 days (day 21 of the study) after administration of NextGen CD19 UltraCAR-T cells. Figures 29J–29K are dot plots showing the number of B cells in spleen tissue at 7 and 14 days after administration of autologous CAR-T cells, with and without treatment, from either a healthy donor (HD) (Figure 29J) or an SLE / LN donor (Figure 29K). Figure 29L is a bar graph showing the frequency of CD19+ viable B cells in spleen tissue, which was used to calculate the in vivo death percentage. Figure 29M is a dot plot showing the expansion of HER1T+ CAR-T cells in spleen tissue 7 days (day 14 of the study) and 14 days (day 21 of the study) after administration of NextGen CD19 UltraCAR-T cells. [Figure 29-2]Same as above. [Figure 29-3] Same as above. [Figure 29-4] Same as above. [Modes for carrying out the invention]

[0060] Detailed description of the invention Embodiments of this disclosure will be described in detail by the following description and examples.

[0061] It should be understood that this disclosure is not limited to the specific embodiments described herein and is subject to modification. While various features of this disclosure may be described in the context of a single embodiment, features may also be provided separately or in any preferred combination. Those skilled in the art will recognize that variations and modifications of this disclosure exist that fall within their scope.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field relating to this disclosure.

[0063] Section headings used herein are for organizational purposes only and should not be construed as limitations on the subject matter described.

[0064] Various features of this disclosure may be described in the context of a single embodiment, but the features may also be provided separately or in any preferred combination. Conversely, this disclosure may be described herein in the context of separate embodiments for clarity, but this disclosure may also be implemented in a single embodiment.

[0065] definition The following definitions are supplementary to the definitions in the Art and apply to this application; they should not be attributed to any related or unrelated cases, such as any generally owned patent or application. Therefore, the technical terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting.

[0066] In this application, the use of the singular form includes the plural form unless specifically stated otherwise. It should be noted that when used in this specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise.

[0067] In this application, the use of "or" means "and / or" unless otherwise specified. The terms "and / or" and "any combination thereof", as well as their grammatical equivalents, can be used interchangeably when used in this specification. These terms can convey that any combination is specifically contemplated. Merely 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; as well as A, B, and C". The term "or" can be used conjunctively or disjunctively unless the context specifically indicates a disjunctive use.

[0068] The 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".

[0069] References to "some embodiments", "an embodiment", "one embodiment", or "other embodiments" in this specification mean that the particular features, structures, or characteristics described in connection with the embodiments are included in at least some embodiments of the present disclosure but not necessarily in all embodiments of the present disclosure.

[0070] As used herein and in the claims, the terms “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are inclusive or open-ended and do not exclude additional possible components, elements, or steps of the method. Any embodiment discussed herein is intended to be performed with respect to any method or composition of the Disclosure, and vice versa. Furthermore, compositions of the Disclosure may be used to achieve the methods of the Disclosure.

[0071] The term "approximately" refers to a range of values ​​that deviate slightly from a specific numerical value. This deviation generally encompasses the inherent experimental variability associated with biological assays and measurements commonly used in the field of biotechnology. The acceptable range for "approximately" can vary depending on the specific context and the parameter being measured. However, it should generally be understood as being within ±5 to 10% of the specified value. For example, the phrase "approximately 10 mg" could be interpreted as a range of 9.5 mg to 10.5 mg, acknowledging potential variability due to factors such as weighing error or instrument limitations. When the term "approximately" precedes a list of numbers or percentages, it indicates that the value can be an approximate value of any of the listed numbers or percentages. For example, the phrase "approximately 80%, 85%, 90%, 98%, or 99%" means "approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 98%, or approximately 99%."

[0072] The term "approximately" is similar to "about," but can imply a range of deviation slightly larger from a given value. It suggests a range within ±10-15% of a specified number. This term is often used when the exact value is not particularly important or when a wider tolerance range is expected due to biological variability. For example, "approximately 2 hours" for an incubation step might be acceptable even if the actual incubation time differs slightly between experiments due to factors such as temperature fluctuations. When the term "approximately" precedes a list of numbers or percentages, it indicates that the value can be an approximate value of any of the listed numbers or percentages. For example, the phrase "approximately 10, 15, 20, or 25" means "approximately 10, approximately 15, approximately 20, or approximately 25."

[0073] With respect to the numerical range limitations specified herein, each intervening number between them is explicitly intended to be of the same precision. For example, in the range of 6 to 9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range of 6.0 to 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 intended.

[0074] The term “isolated” and its grammatical equivalent, as used herein, refers to the removal of nucleic acids, proteins, polypeptides, cells, or other materials from their natural environment. The term “purified” and its grammatical equivalent, as used herein, refers to molecules or compositions whose purity has been increased, whether removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA), and / or amplified under laboratory conditions, where “purity” is a relative term, not “absolute purity.” However, it should be understood that nucleic acids and proteins may be formulated with diluents or adjuvants and still be isolated for practical purposes. For example, nucleic acids are typically mixed with an acceptable carrier or diluent when used for introduction into cells. The term “substantially purified” and its grammatical equivalent, as used herein, refers to nucleic acid sequences, polypeptides, proteins, or other compounds that essentially do not contain, i.e., no more than about 50%, no more than about 70%, or no more than about 90% of the polynucleotides, proteins, polypeptides, and other molecules to which the nucleic acid, polypeptide, protein, or other compound relates in nature.

[0075] "Nucleic acid," "nucleic acid molecule," "polynucleotide," "polynucleotide construct," "oligonucleotide," and their grammatical equivalents, as used herein, refer to polymeric forms of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of molecules. Therefore, this term includes double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA. This also includes polynucleotides modified, for example, by methylation and / or capping, as well as polynucleotides in their unmodified forms. This term also means that it includes molecules, including synthetic and semi-synthetic nucleotides and polynucleotides that do not exist in nature, as well as nucleotide analogs. When discussing the structure of a particular double-stranded DNA molecule, sequences may be described herein in accordance with the usual convention of giving the sequence only in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA). "Recombinant polynucleotide" is a polynucleotide that has undergone molecular biological manipulation. The polynucleotide sequences and vectors disclosed or intended herein can be introduced into cells, for example, by transfection, transformation, or transduction.

[0076] When applied to a polynucleotide or nucleic acid sequence, the term "fragment" refers to a nucleotide sequence that is shorter than a reference nucleic acid and, across its common portion, contains the same nucleotide sequence as the reference nucleic acid. Such a nucleic acid fragment according to the present invention may, where appropriate, be contained within a longer polynucleotide of which it is a component. Such fragments include, or alternatively consist of, oligonucleotides having a length range of 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 longer consecutive nucleotides of nucleic acid according to the present invention.

[0077] As used herein, “isolated polynucleotide” or “isolated nucleic acid fragment” refers to a polymer of RNA or DNA that is single-stranded or double-stranded, optionally containing synthetic, non-natural, or modified nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may constitute one or more segments of cDNA, genomic DNA, or synthetic DNA.

[0078] The term “gene” and its grammatical equivalent refer to polynucleotides containing nucleotides that code for functional molecules, including functional molecules produced solely by transcription (e.g., bioactive RNA species) or functional molecules produced by both transcription and translation (e.g., polypeptides). The term “gene” encompasses cDNA and genomic DNA nucleic acids. “Genes” also refer to nucleic acid fragments that express specific RNA, proteins, or polypeptides, including regulatory sequences preceding the coding sequence (5' non-coding sequence) and subsequent regulatory sequences (3' non-coding sequence). “Native gene” refers to a gene found naturally together with its own regulatory sequences. “Chimera gene” refers to any gene that is not a native gene and contains regulatory sequences and / or coding sequences that are not found together in nature. Thus, a chimeric gene may contain regulatory sequences and coding sequences from different origins, or regulatory sequences from the same origin but arranged in a different manner than those found in nature. A chimeric gene may contain coding sequences and / or regulatory sequences from different origins. “Endogenous gene” refers to a native gene in its natural location within the genome of an organism. An "external" gene or "other" gene refers to a gene that is not normally found in the host organism but has been introduced into the host organism through gene transfer. External genes can include native genes inserted into non-native organisms or chimeric genes. A "transgene" is a gene that has been introduced into the genome through a transformation procedure.

[0079] The term "genome" includes chromosomes, as well as mitochondria, chloroplasts, 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.

[0080] "Heterogeneous DNA" refers to DNA that is not naturally located in a cell or in a chromosomal region of a cell. Heterogeneous DNA may include exogenous genes. "Exogenous genes" means genes that are foreign to the subject, i.e., genes introduced into the subject by a transformation process, an unmutated version of an endogenous mutated gene, or a mutated version of an endogenous unmutated gene. Exogenous genes may be either native or synthetic genes introduced into the subject in the form of DNA or RNA that can function by a DNA intermediate, for example by reverse transcriptase. Such genes may be introduced into target cells, directly into the subject, or indirectly by the transfer of transformed cells into the subject.

[0081] A "primer" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double-stranded nucleic acid region that can function as an initiation site for DNA synthesis under favorable conditions. Such primers can be used in polymerase chain reactions or for DNA sequencing.

[0082] A DNA "coding sequence" or "coding region" refers to a double-stranded DNA sequence that codes for a polypeptide and, under the control of a suitable regulatory sequence, can be transcribed and translated into a polypeptide in cells, ex vivo, in vitro, or in vivo. A "suitable regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequence), internally, or downstream (3' non-coding sequence) of the coding sequence that influences transcription, RNA processing, stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. The boundaries of a coding sequence are determined by a 5' (amino) terminal start codon and a 3' (carboxy) terminal translation termination codon. Codextic sequences may include, but are not limited to, prokaryotic sequences, mRNA-derived cDNA, genomic DNA sequences, and synthetic DNA sequences. When a coding sequence is intended for expression in eukaryotic cells, the polyadenylation signal and transcription termination sequence are typically located at 3' of the coding sequence.

[0083] An "open reading frame," abbreviated as ORF, refers to a nucleic acid sequence of a certain length, whether DNA, cDNA, or RNA, that contains a translation start signal or start codon, e.g., ATG or AUG, and a stop codon, and is potentially translated into a polypeptide sequence.

[0084] The term "downstream" refers to a nucleotide sequence located 3' relative to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally refer to sequences that follow the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0085] The term "upstream" refers to a nucleotide sequence located 5' relative to a reference nucleotide sequence. In particular, upstream nucleotide sequences generally refer to sequences located 5' to the coding sequence or the transcription start site. For example, most promoters are located upstream of the transcription start site.

[0086] The term "response element" refers to one or more cis-acting DNA elements that confer responsiveness to a promoter mediated by interaction with the DNA-binding domain of a transcription factor. This DNA element may be palindromic (complete or incomplete) in its sequence, or it may consist of a sequence motif or half-regions separated by a variable number of nucleotides. The half-regions may be similar or identical and may be arranged as serial or reverse repeat sequences, as a single half-region, or as a serial multimer of adjacent half-regions. Depending on the nature of the cell or organism into which the response element is incorporated, the response element may include minimal promoters isolated from different organisms. The DNA-binding domain of the transcription factor binds to the DNA sequence of the response element, in the presence or absence of a ligand, to initiate or repress the transcription of downstream genes regulated by this response element.

[0087] The term “operably ligated,” as used herein, refers to the physical and / or functional ligation of a DNA segment to another DNA segment in such a manner that the segments function in their intended manner. A DNA sequence encoding a gene product is operably ligated to a regulatory sequence if it is ligated to a regulatory sequence, such as a promoter, enhancer, and / or silencer, in a manner that allows for the modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably ligated to a promoter if it is ligated to a promoter downstream of the transcription start site of the promoter in the correct reading frame relative to the transcription start site, allowing transcription elongation to proceed by the DNA sequence. An enhancer or silencer is operably ligated to a DNA sequence encoding a gene product if it is ligated to the DNA sequence in a manner that increases or decreases the transcription of the DNA sequence, respectively. Enhancers and silencers may be located upstream, downstream, or embedded within the coding region of the DNA sequence. The DNA for the signal sequence is operably ligated to the DNA encoding the polypeptide, if the signal sequence is expressed as a preprotein that participates in polypeptide secretion. Ligation of the DNA sequence to the regulatory sequence is typically achieved by ligation at a suitable restriction site or via an adapter or linker inserted into the sequence using a restriction endonuclease known to those skilled in the art.

[0088] As used herein, the term "codon degeneracy variant" refers to a modified nucleic acid sequence in which the specific nucleotides including the codon are different, but which encodes the same amino acid sequence as the original sequence. The genetic code is degenerate, which means that multiple codons can encode 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.

[0089]

Table 1

[0090] The following definitions supplement the definitions in the art and are intended for this application and should not be attributed to any related or unrelated case, for example, any commonly owned patent or application. Thus, the technical terms used herein are for the purpose of merely describing particular embodiments and are not intended to be limiting.

[0091] Furthermore, publicly available software resources are readily available for the “reverse translation” of polypeptide sequences, also known as computer-generated “reverse transcription,” which is the conversion of a polypeptide sequence into the nucleotide sequence that encodes it. See, for example, 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).

[0092] As used herein, codon degenerate variants may be utilized to optimize gene expression or enhance protein production. By modifying codons within a nucleic acid sequence, it is possible to utilize codons that are more frequently used or preferred by the translational mechanisms of the host organism. This may result in increased efficiency of protein expression or improved compatibility with specific host organisms.

[0093] As used herein, the term "expression" refers to the transcription and stable accumulation of sense RNA (mRNA) or antisense RNA derived from nucleic acids or polynucleotides. Expression may also refer to the translation of mRNA into proteins or polypeptides.

[0094] 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 by specific restriction sites or by homologous recombination. The DNA segment contains a polynucleotide encoding the polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in a proper reading frame for transcription and translation. A “transformation cassette” refers to a specific vector containing a polynucleotide encoding the polypeptide of interest, and having elements in addition to the polynucleotide that promotes transformation of a particular host cell. The cassettes, expression cassettes, gene expression cassettes, and transformation cassettes of the present invention may also include elements that enable enhanced expression of the polynucleotide encoding the polypeptide of interest in a host cell. These elements may include, but are not limited to, promoters, minimal promoters, enhancers, response elements, terminator sequences, and polyadenylation sequences. The expression cassettes described herein are approximately 500-10,000 bp, 1,000-5,000 bp, 1,500-4,500 bp, 1,800-4,400 bp, 2,000-4,500 bp, 2,100-4,400 bp, 2,200-4,300 bp, 2,300-4,200 bp, 2,400-4,100 bp, and 2,500-4,000 bp. The expression cassette may contain a total length of 0 bp, approximately 2,600 to 3,900 bp, approximately 2,700 to 3,800 bp, approximately 2,800 to 3,800 bp, approximately 2,900 to 3,700 bp, approximately 3,000 to 3,600 bp, approximately 3,100 to 3,500 bp, approximately 3,150 to 3,450 bp, approximately 3,200 to 3,400 bp, approximately 3,250 to 3,350 bp, or approximately 3,300 bp. Alternatively, the expression cassette may contain any number of base pairs within these ranges.For example, the expression cassettes are approximately 500 bp, 750 bp, 1,000 bp, 1,250 bp, 1,500 bp, 1,750 bp, 2,000 bp, 2,250 bp, 2,500 bp, 2,550 bp, 2,600 bp, 2,650 bp, 2,700 bp, 2,750 bp, 2,800 bp, 2,850 bp, 2,900 bp, 2,950 bp, 3,000 bp, 3,050 bp, 3,100 bp, 3,150 bp, 3,200 bp, 3,250 bp, and 3,300 bp. The expression may contain approximately 3,350 bp, approximately 3,400 bp, approximately 3,450 bp, approximately 3,500 bp, approximately 3,550 bp, approximately 3,600 bp, approximately 3,650 bp, approximately 3,700 bp, approximately 3,750 bp, approximately 3,800 bp, approximately 3,850 bp, approximately 3,900 bp, approximately 3,950 bp, approximately 4,000 bp, approximately 4,050 bp, approximately 4,100 bp, approximately 4,150 bp, approximately 4,200 bp, approximately 4,250 bp, approximately 4,300 bp, approximately 4,350 bp, approximately 4,400 bp, approximately 4,450 bp, or approximately 4,500 bp. In one embodiment, the expression cassette contains approximately 2,800 bp. In another embodiment, the expression contains 2,825 bp.

[0095] As used herein, the term “vector” refers to any vehicle for cloning and / or transferring nucleic acids into host cells. A vector may be a replicon to which another DNA segment can be attached, resulting in the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., is capable of replicating under its own control. The term “vector” includes both viral and nonviral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors known in the art can be used to manipulate nucleic acids, to incorporate response elements and promoters into genes, etc. Possible vectors include, for example, bacteriophages, e.g., lambda derivatives, or plasmids, e.g., pBR322 or pUC plasmid derivatives, or Bluescript vectors, e.g., plasmids or modified viruses. Another example of a vector useful in the present invention is the ULTRAVECTOR® Production System (Intrexon Corp., Blacksburg, VA), described in International Publication No. 2007 / 038276. For example, insertion of DN fragments corresponding to response elements and promoters into a suitable vector can be achieved by ligating a suitable DNA fragment into a selected vector having complementary adherent ends. Alternatively, the ends of the DNA molecule may be enzymatically modified, or any site may be generated by ligating a nucleotide sequence (linker) to the DNA ends. Such vectors may be engineered to contain a selection marker gene that provides selection of cells into which the marker has been incorporated into the cellular genome. Such markers enable the identification and / or selection of host cells that incorporate and express the protein encoded by the marker.

[0096] As used herein, the term “plasmid” refers to an extrachromosomal element, often containing genes, that is typically in the form of a circular double-stranded DNA molecule, rather than being part of the cell’s central metabolism. Such elements may be single-stranded or double-stranded DNA or RNA, of any origin, autologous replication sequences, genomic integration sequences, phages or nucleotide sequences, linear, circular, or higher-order coils, where some nucleotide sequences are conjugated to or recombined into a unique construct that can introduce into the cell a promoter fragment and DNA sequence for a selected gene product, along with a suitable 3’ untranslated sequence.

[0097] As used herein, the terms “cloning vector” and “replicon” refer to a unit-length nucleic acid, preferably DNA, such as a plasmid, phage, or cosmid, which replicates sequentially and contains an origin of replication, to which another nucleic acid segment may be attached in such a way that it results in replication of the attached segment. A cloning vector may be capable of replicating in one cell type and expressing in another cell type (a “shuttle vector”). A cloning vector may contain one or more sequences that can be used as a vector for insertion of a sequence of interest and / or for selection of cells containing one or more multiple cloning sites.

[0098] As used herein, the term “viral vector” refers to a virus, viral particle, or derivative thereof that can transfer nucleic acids into cells or into the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. Viral vectors, in particular retroviral vectors, are used in a wide variety of gene delivery applications in cells and living animals. Viral vectors that can be used include, but are not limited to, retroviruses, adeno-associated viruses, pox, baculoviruses, vaccinia, herpes simplex, Epstein-Barr, adenoviruses, geminiviruses, and karimovirus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cytofectins), DNA-protein complexes, and biopolymers. In addition to nucleic acids, vectors may also include one or more regulatory regions, and / or selection markers useful for selection, measurement, and monitoring of nucleic acid transfer outcomes (e.g., which tissues to transfer into, duration of expression).

[0099] As used herein, the terms “adenovirus” and “adenovirus vector” mean, as used herein, an adenovirus that retains the ability to participate in the adenovirus life cycle and / or has been physically inactivated by, for example, destruction (e.g., sonication), denaturation (e.g., using heat or a solvent), or crosslinking (e.g., via formalin crosslinking). The “adenovirus life cycle” includes (1) virus binding and entry into a cell, (2) transcription of the adenovirus genome and translation of adenovirus proteins, (3) replication of the adenovirus genome, and (4) construction of a viral particle (e.g., Fields Virology, 5 thSee, Knipe et al. (eds.), Lippincott Williams & Wilkins, Philadelphia, PA (2006). Adenoviruses may also be deficient in replication by the deletion of one or more portions of the naturally occurring viral genome (i.e., they do not retain the ability to participate in the adenovirus life cycle), as used and described herein. "Adenovirus" and "adenovirus vector" may include adenoviruses whose adenovirus genome has been engineered to accommodate nucleic acid sequences that are non-native with respect to the adenovirus genome. Typically, adenovirus vectors are constructed, for example, by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenovirus genome of an adenovirus to accommodate the insertion of a non-native nucleic acid sequence into the adenovirus for gene transfer.

[0100] As used herein, the terms “MOI” or “infection multiplicity” refer to the average number of viral particles (e.g., recombinant virus or control virus) that infect a single cell in a given experiment.

[0101] 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 is “transfected” with exogenous or heterologous RNA or DNA if such RNA or DNA is introduced into the cell. A cell is “transformed” with exogenous or heterologous RNA or DNA if the transfected RNA or DNA produces a phenotypic alteration. The RNA or DNA to be transformed may be incorporated (covalently linked) into the chromosomal DNA that makes up the cell’s genome.

[0102] As used herein, the term “transformation” refers to the introduction of a nucleic acid fragment into the genome of a host organism that results in genetically stable inheritance. A host organism containing a transformed nucleic acid fragment is referred to as a “transgenic,” “recombinant,” or “transformed” organism.

[0103] As used herein, the terms “inducing,” “inducing,” and their grammatical equivalents refer to an increase in nucleic acid sequence transcription, promoter activity, and / or expression, brought about by a transcription regulator, compared to some baseline level of transcription.

[0104] As used herein, the terms “promoter” and “promoter sequence” are interchangeable and refer to DNA sequences capable of controlling the expression of coding sequences or functional RNA. Generally, coding sequences are located 3' relative to promoter sequences. Promoters may be entirely derived from native genes, or they may consist of different elements derived from different promoters found in nature, or they may further include synthetic DNA segments. Those skilled in the art will understand that different promoters may direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that express genes in most cell types are generally referred to as “constitutive promoters.” Promoters that express genes in specific cell types are generally referred to as “cell-specific promoters” or “tissue-specific promoters.” Promoters that express genes at specific stages of development or cell differentiation are generally referred to as “development-specific promoters” or “cell differentiation-specific promoters.” Promoter induces and triggers gene expression after exposure or treatment of cells with virulence factors, biomolecules, chemicals, ligands, light, etc., which induce the promoter; these are generally referred to as "inducible promoters" or "regulatory promoters." It is further recognized that, in most cases, the precise boundaries of regulatory sequences are not fully defined, and DNA fragments of different lengths may have the same promoter activity.

[0105] The promoter sequence typically extends upstream (5' direction) at its 3' end, bordered by the transcription start site, to contain the minimum number of bases or elements necessary to initiate transcription at a level detectable above the background. Within the promoter sequence, the transcription start site (conveniently defined, e.g., by mapping to a nuclease SI) and the protein-binding domain (consensus sequence) that leads to RNA polymerase binding are found.

[0106] The origin of the promoter inserted into the gene switch may be natural or synthetic, and the origin of the promoter should not limit the scope of the invention as described herein. In other words, the promoter may be cloned directly from a cell, or it may have been previously cloned from a different origin, or it may be synthetic.

[0107] As used herein, the term “transcriptional regulator” refers to a biological element that acts to prevent or inhibit the transcription of promoter-driven DNA sequences under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein), or to allow or stimulate the transcription of promoter-driven DNA sequences under certain environmental conditions (e.g., an inducer or enhancer).

[0108] As used herein, the term “enhancer” refers to a DNA sequence that increases transcription, for example, of a operably linked nucleic acid sequence. Enhancers can be located several kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, the pattern of DNA methylation, or changes in DNA structure. Numerous enhancers from various different origins are well known in the art and are available as cloned polynucleotides or within them (e.g., from contract laboratories, e.g., ATCC, and other commercial or personal origins). Some polynucleotides containing promoters (e.g., the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, within, or downstream of the coding sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region mapped within an immunoglobulin (Ig) gene locus (such enhancers include, for example, heavy chain (mu) 5' enhancers, light chain (kappa) 5' enhancers, kappa and mu intron enhancers, and 3' enhancers (see, in general, Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353–363; and U.S. Patent No. 5,885,827)).

[0109] When RNA polymerase transcribes the coding sequence into mRNA, it is "under the control" of transcriptional and translational regulatory sequences in the cell, which are then trans-spliced ​​(if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.

[0110] "Transcriptional and translational regulatory sequences" refer to DNA regulatory sequences, such as promoters, enhancers, and terminators, that provide expression of coding sequences in host cells. In eukaryotic cells, polyadenylation signals are regulatory sequences. Enhancers that may be used in embodiments of the present invention include, but are not limited to, SV40 enhancers, cytomegalovirus (CMV) enhancers, elongation factor 1 (EF 1) enhancers, yeast enhancers, and viral gene enhancers.

[0111] The terms “3' non-coding sequence” and “3' untranslated region (UTR)” refer to DNA sequences located downstream (3') of the coding sequence and may include polyadenylation [poly(A)] recognition sequences and other sequences that encode regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are typically characterized by influencing the addition of polyadenylate tracts to the 3' end of mRNA precursors.

[0112] 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 a sequence that is naturally responsible for the expression of a particular nucleic acid (homologous region), or it may include a sequence of a different origin that is responsible for the expression of a different protein or even a synthetic protein (heterologous region). In particular, the sequence may be a sequence of a prokaryotic, eukaryotic, or viral gene, or an induced sequence, that stimulates or represses the transcription of a gene in a specific or nonspecific manner, and in an inducible or non-inducible manner. A regulatory region may include an origin of replication, an RNA splice site, a promoter, an enhancer, a transcription termination sequence, and a signal sequence that directs a polypeptide to the secretory pathway of a target cell.

[0113] As used herein, the term “modulate” means to induce, reduce, or inhibit nucleic acid or gene expression, resulting in the induction, reduction, or inhibition of protein or polypeptide production, respectively.

[0114] As used herein, the term "CAP" or "cap" refers to a modified nucleotide, generally 7-methylguanosine (7meG-ppp-G) ligated at the 5' end of eukaryotic mRNA, typically from 3' to 5', which plays a necessary element in the normal translation initiation pathway during protein expression from that mRNA.

[0115] As used herein, the term “Sleeping Beauty (SB) transposon system” refers to a synthetic Tc1 / Mariner transposon system for introducing DNA sequences into cells or vertebrate chromosomes. Some exemplary embodiments of the system are described, for example, in U.S. Patent Nos. 6,489,458, 8,227,432, 9,228,180 and International Publication No. 2016 / 145146. The Sleeping Beauty transposon system comprises a Tc1 / Mariner transposase called Sleeping Beauty (SB) transposase and an SB transposon. In embodiments, the Sleeping Beauty transposon system may include an SB11 transposon system, an SB100X transposon system, or an SB110 transposon system.

[0116] As used herein, the terms “transposon” or “transposition element” (TE) refer to vector DNA sequences that can alter their position within the genome, sometimes by producing or reversing mutations, thereby changing the size of the cell’s genome. Transposition often results in duplication of TEs. Class I TEs are copied in two steps: first, they are transcribed from DNA to RNA, and the resulting RNA is then reverse-transcribed back into DNA. This copied DNA is then inserted into the genome at the new location. 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 those of retroviruses, e.g., HIV. The cut-and-paste transposition mechanism of Class II TEs does not involve an RNA intermediate. Transposition is catalyzed by several transposase enzymes. Some transposases bind nonspecifically to any target site in DNA, while others bind to specific DNA sequence targets. Transposases perform alternating cuts at the target site, resulting in a single-stranded 5' or 3' DNA overhang (sticky end). This step excises the DNA transposon, which is then ligated to a new target site. This process involves the activity of DNA polymerase to fill the gap and DNA ligase to close the sugar-phosphate backbone. This results in duplication of the target site. The insertion site of a DNA transposon can be identified by a short series of repeat sequences that can be created by alternating cuts and filling by DNA polymerase in the target DNA, followed by a series of reverse repeat sequences that are crucial for subsequent transposase-mediated TE excision. Cut-and-paste TEs can overlap if their transposition occurs during the S phase of the cell cycle, when the donor site has already replicated but the target site has not yet. Transpositions can be classified as either autonomous or non-autonomous in both class I and class II TEs. Autonomous TEs can move on their own, while non-autonomous TEs require the presence of another TE to move. This is often because non-autonomous TEs lack transposases (for class II) or reverse transcriptases (for class I).

[0117] As used herein, the term “transposase” refers to an enzyme that binds to the end of a transposon and catalyzes the transposon’s movement to another part of the genome via a cut-and-paste mechanism or a replication-transposition mechanism.

[0118] As used herein, the terms “polypeptide,” “peptide,” “polypeptide construct,” and “peptide construct,” as well as their grammatical equivalents, refer to polymer compounds composed of covalently linked amino acid residues. “Mature protein” is a full-length protein, optionally including glycosylation or other modifications typical of a protein in a given cellular environment. Embodiments of the present invention, as disclosed herein, include the HPV antigen / antigenic polypeptides, peptides, and mature proteins described herein, and also include polynucleotides (DNA or RNA) encoding them. Polypeptides and proteins disclosed herein (including their functional fragments and functional variants) may contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indoline. Examples include -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, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0119] As used herein, the term “polypeptide fragment” means a polypeptide whose amino acid sequence is shorter than that of a reference polypeptide and which contains the same amino acid sequence throughout the entire portion having these reference polypeptides. Such fragments may, where appropriate, be contained within a larger polypeptide of which they are part. Such fragments of polypeptides according to the present invention may have amino acid lengths 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 longer.

[0120] As used herein, the terms “isolated polypeptide,” “isolated peptide,” or “isolated protein” refer to polypeptides or proteins that are substantially free from compounds normally associated with them in their natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). “Isolated” does not mean the exclusion of artificial or synthetic mixtures with other compounds, or the presence of impurities that do not impede biological activity, for example, resulting from incomplete purification, the addition of stabilizers, or formulation into pharmaceutically acceptable preparations.

[0121] As used herein, the terms “identical” or “sequence identity” in the context of two nucleic acid or amino acid sequences of polypeptides refer to residues in two sequences that are identical when aligned for maximum match across a defined comparison window. “Comparison window,” as used herein, refers to a segment of at least about 20, typically about 50 to about 200, and more commonly about 100 to about 150 consecutive positions over which the sequences can be compared to the same number of consecutive positions of a reference sequence after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art.The optimal sequence alignment for comparison can be performed 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 similarity search method of Pearson and Lipman, Proc. Nat. Acad Sci USA., 85:2444 (1988); or by computer execution of these algorithms (including, but not limited to, CLUSTAL in the PC / Gene program from Intelligentics, Mountain View Calif, Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA, GAP, BESTFIT, BLAST, FASTA, and TFASTA); the CLUSTAL program is described in Higgins and Sharp, Gene, 73:237-244. Alignment is also frequently performed by laboratory and manual alignment. (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).

[0122] 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 fragment thereof, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters, for example. Similarly, nucleic acids can also be described by reference to an initiating nucleic acid, for example, a nucleic acid may 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 fragment thereof, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters, for example. When a molecule is said to have a certain percentage of sequence identity with a larger molecule, this means that, when the two molecules are optimally aligned, the percentage of residues in the smaller molecule will find matching residues in the larger molecule in the order in which the two molecules are optimally aligned.

[0123] As used herein, the term “identity percentage” is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing their sequences, as is known in the art. In the art, “identity” also means the degree of sequence relevance between polypeptide or polynucleotide sequences, determined by matching strings of such sequences. "Identity" and "similarity" are not limited to those described above, or can be readily calculated by known methods, including, for example, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, 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 for determining identity and similarity are systematized in publicly available computer programs. Sequence alignment and identity percentage calculations may be performed using sequence analysis software, such as the MegAlign (or more recently, MegAlign Pro) program from the LASERGENE Bioinformatics Computer Suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of sequences may be performed using Clustal's alignment method (Higgins et al., CABIOS. 5:151 1989) with default parameters (gap penalty=10, gap length penalty=10). Default parameters for pairwise alignment using Clustal's method may be selected: KTUPLE 1, gap penalty=3, window=5, and DIAGONALS SAVED=5.

[0124] As used herein, the term “substantially similar” and its grammatical equivalents, applied to nucleic acids or amino acid sequences, mean that the nucleic acid or amino acid sequence has sequence identity of at least 90% or higher relative to a reference sequence, e.g., at least 95%, at least 98%, at least 99%, 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%, relative to a reference sequence, using standard parameters and comparison programs described above, e.g., BLAST. The term “substantially identical” and its grammatical equivalent, applied to nucleic acids or amino acid sequences, means that a nucleic acid or amino acid sequence contains a sequence that, using standard parameters and a comparison program described above, e.g., BLAST, has at least 99% sequence identity with respect to a reference sequence, e.g., 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%. For example, the BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and comparisons of both strands. For amino acid sequences, the BLASTP program uses, by default, a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). The percentage of sequence identity is determined by comparing two optimally aligned sequences across a comparison window, where portions of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences.The percentage is calculated by determining the number of positions that give the number of matched positions, where the same nucleic acid base or amino acid residue is present in both sequences, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. In embodiments, substantial identity exists over a region of sequence that is at least about 50 residues long, 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 region.

[0125] Nucleic acid molecules that hybridize to the disclosed sequences are also intended and included herein. Hybridization conditions may be mild, moderate, or stringent, depending on the circumstances. Suitable stringency conditions that promote DNA hybridization, e.g., washing with 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing with 2× SSC at 50°C, are known and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6. As used herein, the term “stringent hybridization conditions” refers to conditions including washing with a salt concentration of about 1.0 M NaCl in 50% formamide at a temperature of about 37°C for about 4–12 hours, followed by washing with 0.1× SSC at about 60–65°C.

[0126] As used herein, the term “functional fragment” or its grammatical equivalent is used herein to mean a portion, fragment, or segment of a biomolecule that retains essential functional characteristics or activity of the original biomolecule.

[0127] The term “functional variant” or its grammatical equivalent is used herein to mean a modified form of a biomolecule that retains essential functional characteristics or activity of the original molecule, while exhibiting some degree of variation. This includes biomolecules that have been modified, for example, by genetic engineering or mutagenesis techniques, to introduce specific changes while preserving the overall functionality of the biomolecule. Functional variants 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. Techniques for obtaining these variants, including genetic (repression, deletion, mutation, etc.), chemical, and enzymatic techniques, are known to those skilled in the art. In one embodiment, a biological variant comprises at least about 14 monomers (e.g., nucleotides or amino acids). In certain embodiments, a functional variant of a reference amino acid sequence is a variant of the reference amino acid sequence that 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 reference amino acid sequence and / or is a conservatively substituted variant of the reference amino acid sequence. When used in reference to nucleic acids, the term “functional variant” refers to a nucleic acid that is different from the reference nucleic acid but encodes a polypeptide that has the same primary function as the polypeptide encoded by the reference nucleic acid. In certain embodiments, a 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 a reference nucleic acid sequence, or hybridizes with a complementary sequence of the reference nucleic acid sequence under stringent hybridization conditions, or is a codon degenerate variant of the nucleic acid.

[0128] As used herein, the term “homology” in all its grammatical and spelling variations refers to the percentage of identity between two polynucleotides or two polypeptide segments. The degree of sequence agreement between one segment and another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of sequence information between two polypeptide molecules by aligning the sequence information using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form a stable double helix between homologous regions, followed by digestion by a single-strand specific nuclease and sizing of the digested fragments.

[0129] When used in the context of amino acid sequences, the term "substitution" refers to a variation in an amino acid sequence in which one amino acid is replaced by another. The nomenclature used to represent amino acid substitutions follows a standard format. For example, in "L50G," "L" represents the original amino acid leucine (abbreviated as "L"), "50" indicates the position of the amino acid in the amino acid sequence relative to its N-terminus (in this case, the amino acid is the 50th amino acid from the N-terminus of the sequence), and "G" indicates the substituted amino acid, in this example, glycine (abbreviated as "G"). Therefore, "L50G" represents a substitution in which leucine at position 50 (relative to its N-terminus) of the amino acid sequence is replaced by glycine.

[0130] As used herein, the terms “conservative amino acid substitution” or “conservative mutation” refer to the substitution of one amino acid with another amino acid that shares common properties. A functional method for defining the common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (see Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, a group of amino acids can be defined as those within a group in which the amino acids preferentially exchange with each other, and therefore their effects on the overall protein structure are most similar to each other (Schulz, GE and Schirmer, RH, above). Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, e.g., lysine and arginine, and vice versa, in which a positive charge can be maintained; glutamic acid and aspartic acid, and vice versa, in which a negative charge can be maintained; serine and threonine, in which free -OH can be maintained; and glutamine and asparagine, in which free -NH2 can be maintained. Examples of conservative amino acid substitutions are shown in the chart below.

[0131] [Table 2]

[0132] An amino acid sequence that differs from a reference amino acid sequence solely through conservative amino acid substitutions is referred to herein as a “conservatively substituted variant” of the reference sequence. Given the established knowledge and well-known techniques in protein science, determining the functional impact of a “conservatively substituted variant” compared to a reference amino acid sequence is well within the skill of those skilled in the art.

[0133] 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 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 substituted 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 substitution.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.

[0134] An amino acid sequence that differs from a reference amino acid sequence by at least one non-conservative amino acid substitution is 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 amino acid substitutions between different groups, such as the substitution of tryptophan by lysine or serine by phenylalanine. In this case, it is preferable that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, resulting in an increased biological activity of the functional variant compared to the homologous parent protein. The substitutability of amino acids is discussed in more detail, for example, LY 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, determining the functional effect of non-conserved amino acid substitutions in a functional variant compared to a reference amino acid sequence is well within the skill of those skilled in the art.

[0135] In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by at least one non-conserved 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-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 10 to 20 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 21 to 30 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 31 to 40 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 41 to 50 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 51 to 60 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 61 to 70 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 71 to 80 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 81 to 90 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 91 to 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.

[0136] As used herein, the term "antibody" refers to monoclonal or polyclonal antibodies. The term "monoclonal antibody" as used herein refers to an antibody produced by a single clone of B cells that binds to the same epitope. In contrast, a "polyclonal antibody" refers to a population of antibodies produced by different B cells that bind to different epitopes of the same antigen. An intact antibody typically consists of four polypeptides: two identical copies of the heavy (H) chain polypeptide and two identical copies of the light (L) chain polypeptide. Each heavy chain contains one N-terminal variable domain (V H region, also called the V H domain) and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable domain (V L region, also called the V L domain) and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The V H domain and the V L domain have a similar general structure, and each region contains four framework regions, the sequences of which are relatively conserved. The framework regions are connected by three complementarity-determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody, which is responsible for antigen binding.

[0137] As used herein, the terms “functional antibody fragment” and “functional fragment of an antibody” or their grammatical equivalents are used interchangeably to mean a portion, fragment, or segment of an antibody that retains essential functional characteristics or activity of the original antibody. In one embodiment, the activity is the ability to specifically bind to an antigen (see Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005) in general). A functional antibody fragment may include, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or a combination thereof. A non-limiting example of a functional antibody fragment is (i)V L Domain, V H (ii) an antigen-binding fragment (Fab) consisting of a monovalent fragment comprising a domain, a CL domain, and a CH1 domain; (ii) a bivalent fragment F(ab')2 fragment containing two Fab fragments linked by disulfide crosslinking in the stalk region; (iii) a single arm of the antibody V L and V H A variable fragment ("Fv") consisting of domains; (iv) Two domains of the Fv fragment joined by a synthetic linker that enables the synthesis of a single polypeptide chain (i.e., V L and V H A monovalent molecule consisting of a domain, single-chain Fv(scFv) (see, for example, 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) each polypeptide chain having V on the same polypeptide chain H Domain and V L The peptide linker is too short to allow pairing between domains. L V connected to the domain H Including the domain, thereby different V H -V LExamples include dimers of polypeptide chains, called dibodies, in which pairing between complementary domains on a domain polypeptide chain is driven to produce a dimer molecule having two functional antigen-binding sites. Functional antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent No. 8,603,950.

[0138] As used herein, the term “antibody-like molecule” can be, for example, a protein that is a member of the Ig superfamily capable of selectively binding to 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 is modified to increase its MHC binding affinity.

[0139] As used herein, the terms “antigen-recognizing moiety” or “antigen-recognizing domain” refer to a molecule or part of a molecule that specifically binds to an antigen. In one embodiment, the antigen-recognizing moiety is an antibody, an antibody-like molecule, or a fragment thereof, and the antigen is a tumor antigen.

[0140] 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).

[0141] 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 their cell surface. The “TCR” is a protein molecule found on the surface of T cells, and it is a type of leukocyte involved in adaptive immune responses. The variable domain of the TCR contains a highly pleomorphic loop called the complementarity-determining region (CDR), which is responsible for binding to peptide-presenting MHC. There are two main forms of TCR: αβ TCR and γδ TCR. Both forms consist of two protein chains, known as alpha (α) and beta (β) chains for αβ TCR and gamma (γ) and delta (δ) chains for γδ TCR. These chains together form a heterodimer structure. The majority of T cells in the human immune system express αβ TCR. The α and β chains of αβ TCRs are encoded by separate gene segments, which undergo recombination during T cell development to produce diverse TCR specificities. The α and β chains contain variable (V), diverse (D), and conjugating (J) gene segments, respectively, similar to the antibody gene rearrangement process. The combination of the V, D, and J gene segments contributes to the unique antigen-binding specificity of αβ TCRs. αβ TCRs recognize antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. In contrast to αβ TCRs, γδ TCRs are less dominant in the immune system but still play an important role. 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 differs from that of αβ TCRs. γδ T cells often exhibit tissue-specific distributions and are found in epithelial tissues, such as the skin and intestines. γδ TCRs can recognize various antigens, including certain peptides and non-peptide molecules, independently of MHC presentation. Both αβ TCRs and γδ TCRs participate in and respond to immune surveillance, but they have different functions and specificities.While αβ TCRs are primarily involved in the recognition of peptides presented by major histocompatibility complex (MHC) molecules, γδ TCRs may possess a wider range of antigen recognition capabilities.

[0142] TCRs and constructs encoding TCRs that recognize MHC-antigen complexes can be created and introduced into T cells (known as TCR T cells), and the following TCR-peptide-MHC interactions can be utilized to induce an immune response. Greenbaum et al., Cancer Immunol Res 1 November 2021; 9 (11): 1252-1261. There is interest in the use of TCRs with higher affinity than the normal range for peptide-MHC antigens (type I), referred to as high-affinity TCRs, to generate soluble TCRs that can be used directly against target cells, either 1) to drive the activity of CD4 helper T cells (which lack the CD8 coreceptor), or 2) by attaching to "effector" molecules (e.g., antibody Fc region, toxic drug, or antibody scFv for forming bispecific proteins, e.g., anti-CD3 antibody) (Ashfield and Jakobsen, IDrugs, 9, 554-9 (2006); Foote and Eisen Proc Natl Acad Sci USA, 97:10679-81 (2000); Holler et al., Proc Natl Acad Sci USA, 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 due to their T cells not expressing TCRs with sufficient specificity and binding affinity to basic tumor antigens. For example, more than 300 MHC-restricted T cells that define tumor antigens have been identified (Cheever et al., Clin Cancer Res. 2009;15(17):5323-5337). These tumor antigens include mutated peptides, differentiation antigens, and overexpressed antigens, all of which function as targets for therapy.Since most cancer antigens described to date are derived from intracellular proteins that can only be targeted on the cell surface in the context of MHC molecules, TCRs are ideal candidates for therapy because they have evolved to recognize this class of antigens. Similarly, TCRs can detect peptides derived from viral proteins that are processed naturally in infected cells and displayed on the cell surface by MHC molecules. However, patients with these diseases may not have optimized TCRs that bind to and destroy infected cells. Finally, in a highly specific manner, TCRs can be used as receptor antagonists for autoimmune targets or as delivery agents to immunosuppress local immune cell responses, thereby avoiding general immunosuppression.

[0143] As used herein, the term “helper T cells” (TH or Th cells) refers to cells that assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide 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, TH17, TH22, or TFH (follicular helper T cells), which secrete different cytokines that promote different types of immune responses. Signaling from APCs directs T cells to specific subtypes.

[0144] As used herein, the terms “cytotoxic T cells” (TC cells, or CTLs) or “cytotoxic T lymphocytes” refer to cells that destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. CD8+ cells can be inactivated into an anerogenic state that prevents autoimmune diseases by IL-10, adenosine, and other molecules secreted by regulatory T cells.

[0145] As used herein, the term “memory T cells” refers to a subset of antigen-specific T cells that persist for a long period after an infection has resolved. They rapidly expand into a large number of effector T cells upon re-exposure to their congener antigens, thus providing an immune system with memory of past infections. Memory T cells include 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.

[0146] As used herein, the term “regulatory T cells” (Treg cells), previously known as suppressor T cells, refers to T cells that play a role in maintaining immune tolerance. Their primary roles are to shut down T cell-mediated immunity toward termination of the immune response and to suppress autoreactive T cells that have evaded the process of negative selection in the thymus.

[0147] As used herein, the term “natural killer T cell” (NKT cell – not to be confused with natural killer cells of the innate immune system) refers to cells that bridge the adaptive immune system to the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by molecules called CDs. When activated, these cells can perform functions attributed to both helper T cells (TH) and cytotoxic T cells (TC) (i.e., cytokine production and release of cytotoxic / death-causing molecules). They can also recognize and eliminate certain tumor cells and cells infected with herpesviruses.

[0148] As used herein, the term “proliferative disorders” refers to a unified concept in which excessive cell proliferation and / or turnover of the intracellular matrix significantly contribute to the pathogenesis of the disease, including cancer.

[0149] Where used herein, “patient” or “subject” refers to a mammalian subject that has been diagnosed with, has, or is suspected of having, a disease or disorder, such as cancer. In some embodiments, the term “patient” refers to a mammalian subject that has a higher-than-average likelihood of developing a proliferative disorder, such as cancer. Exemplary patients may be humans, apes, dogs, pigs, cattle, cattle, horses, goats, sheep, rodents, and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients may be male and / or female. “Patient in need of it” or “subject in need of it” is used herein to refer to a patient who has been diagnosed with, or is suspected of having, a disease or disorder, but is not limited to, for example, human papillomavirus (HPV) infection.

[0150] "Administering" is used herein to refer to providing one or more compositions described herein to a patient or subject. For example, but not limited to, administration of a composition, e.g., by injection, may be carried out by intravenous, subcutaneous, intradermal, intraperitoneal, or intramuscular injection. One or more such routes may be used. Parenteral administration may be, for example, by bolus injection or by progressive perfusion over time. Alternatively, or in combination, administration may be by an oral route. In addition, administration may also be by surgical deposition or placement of a medical device. Pharmaceutical compositions may include the compositions of the present invention described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline or phosphate-buffered saline; carbohydrates, such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0151] As used herein, the term “therapeutic product” refers to a therapeutic polypeptide or therapeutic polynucleotide that confers a beneficial function to a host cell on which such product is expressed. Therapeutic polypeptides may include, but are not limited to, small peptides of approximately three amino acids in length, single-stranded or multi-stranded proteins, and fusion proteins. Therapeutic polynucleotides may include, but are not limited to, antisense oligonucleotides, small interfering RNAs, ribozymes, and RNA external guide sequences. Therapeutic products may include naturally occurring sequences, synthetic sequences, or combinations of natural and synthetic sequences.

[0152] As used herein, the terms “treatment,” “to treat,” or their grammatical equivalents refer to obtaining a desired pharmacological and / or physiological effect. In embodiments, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptoms or pathological signs that may contribute to the disease. For this purpose, the method of the present invention comprises the step of administering a therapeutically effective amount of a composition of the present invention expressing the nucleic acid sequence of the present invention, or a vector comprising the nucleic acid sequence of the present invention.

[0153] As used herein, “treatment interval” refers to a treatment cycle, for example, a course of administration of a therapeutic agent that may be repeated on a regular schedule. In some embodiments, the dosage regimen may have one or more periods during the treatment interval in which no therapeutic agent is administered.

[0154] As used herein, “dosage regimen” or “medication regimen” includes a treatment regimen based on a determined set of doses. The terms “dose” and “medicate” as used herein refer to the administration of a substance to achieve a therapeutic objective (e.g., treatment of a tumor).

[0155] The terms “administered in combination,” “simultaneously administered,” “to administer simultaneously,” or “to provide simultaneously,” as used herein, mean that two (or more) different treatments are delivered to a subject during the course of the subject’s suffering due to a disease or disorder, for example, that two or more treatments are delivered after the subject has been diagnosed with a disease or disorder and before the disease or disorder is cured or eliminated, or before the treatments are discontinued for any other reason. In some embodiments, the delivery of one treatment is still taking place when the delivery of a second treatment is initiated, resulting in an overlap in the duration of administration. This is sometimes referred to herein as “simultaneous” or “simultaneous delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment is initiated. In some embodiments of either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is observed with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be observed if the second treatment were administered in the absence of the first treatment, or a similar situation is observed with the first treatment. In some embodiments, the delivery is such that the reduction of symptoms or other parameters relating to the impairment is greater than that observed with a treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, entirely additive, or more than additive. The delivery may be such that the effect of the delivered first treatment is still detectable when the second treatment is delivered.

[0156] In some embodiments of the present invention, the first and second treatments may be administered simultaneously (e.g., at the same time) or sequentially, using the same or separate compositions. Sequential administration refers to the administration of one treatment prior to the administration of an additional (e.g., secondary) treatment (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 hours or longer before; 4, 5, 6, 7, 8, 9 days or longer before; or 1, 2, 3, 4, 5, 6, 7, 8 weeks or longer before). The order of administration of the first and secondary treatments may also be reversed.

[0157] The terms “therapeutic effective dose,” “therapeutic dose,” “immunological effective dose,” “antiotumor effective dose,” and “tumor inhibitory effective dose,” or their grammatical equivalents, refer to an effective dose, which is the amount and duration of medication required to achieve the desired therapeutic outcome. The therapeutic effective dose may vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the compositions described herein to induce a desired response in one or more subjects.

[0158] Alternatively, the pharmacological and / or physiological effects of administering one or more compositions described herein to a patient or subject may be “preventive,” meaning the effect completely or partially prevents the disease or its symptoms. “Preventive effective dose” refers to the effective dose and duration required to achieve the desired preventive outcome (e.g., prevention of disease or prevention of signs of a target condition).

[0159] miRNA As used herein, “miR”, “mir”, and “miRNA” refer to microRNAs, a class of small non-coding RNA molecules that can influence the expression of a gene (“target gene”) by modulating the translation of messenger RNA transcribed from it (increasing or decreasing gene expression) and / or destabilizing such messenger RNA.

[0160] Primary miRNA, abbreviated as "pri-miRNA," refers to miRNA containing at least one RNA hairpin. The RNA hairpin is cleaved from the pri-miRNA in the cell nucleus to form one or more miRNA precursors ("pre-miRNAs"). These pre-miRNAs are transported to the cytoplasm, where their stem-loop structure is cleaved, generating a double-stranded miRNA containing 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 Argonaut protein then binds to the double-stranded miRNA, releasing one of the strands (either the miRNA-5p or miRNA-3p sequence). The remaining bound strand becomes the "guide strand," while the released strand is known as the "passenger strand" and is preferably degraded. The guide strand then interacts with messenger RNA derived from the target gene, thereby influencing its translation.

[0161] Both the miRNA-5p and miRNA-3p sequences will be referred to as “mature miRNA” sequences in this specification. The remaining parts of the pri-miRNA or pre-miRNA (the 5' portion of the miRNA-5p sequence, the 3' portion of the miRNA-3p sequence, and the stem-loop sequence between the miRNA-5p and miRNA-3p sequences) will be collectively referred to as the miRNA backbone sequence. The term “5' backbone sequence” will be used here to refer to the backbone sequence at the 5' end of the miRNA-5p sequence in pri- or pre-miRNA. The term “3' backbone sequence” will be used here to refer to the backbone sequence at the 3' end of the miRNA-3p sequence in pri- or pre-miRNA. The term “loop sequence” will refer to the backbone sequence between the miRNA-5p and miRNA-3p sequences in pri- or pre-miRNA.

[0162] The term "miRNA," unless otherwise specified, generally refers to the mature, primary, and precursor forms of specific microRNAs and their functional fragments and variants.

[0163] miRNAs may not exist in nature. The terms “not found in nature,” “non-natural,” “synthetic,” and “artificial” are used interchangeably herein and refer to miRNAs that have sequences not found in nature.

[0164] The present invention relates in part to a ribonucleic acid comprising two non-natural pre-miRNA sequences, each pre-miRNA sequence comprising 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, e.g., three, four, five, six, seven, eight, nine, ten, or more such sequences. It is understood that each guide miRNA may target the same gene or different genes. In embodiments in which two or more guide miRNAs target the same gene, such guide miRNAs may target the same region of such gene or different regions.

[0165] In certain embodiments, each non-natural pre-miRNA sequence in ribonucleic acid forms a stem-loop secondary structure which is distinctly different from and non-complementary to those formed by different non-natural pre-miRNAs in ribonucleic acid. In certain embodiments, the non-natural pre-miRNA sequences have sequence identity of less than approximately 95%, less than approximately 90%, less than approximately 85%, less than approximately 80%, less than approximately 75%, less than approximately 70%, less than approximately 65%, less than approximately 60%, less than approximately 55%, less than approximately 55%, or less than 50%.

[0166] In certain embodiments, the secondary structure of each non-natural pre-miRNA is sufficiently similar to that of a naturally occurring pre-miRNA sequence to reduce or prevent cellular RNAi-based anti-pathogen toxicity. In certain such embodiments, the nucleic acid sequence of the non-natural pre-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%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.1%, at least about 99.5%, at least about 99.9%, or at least about 99.99% sequence identity with that of a naturally occurring pre-miRNA, and / or can hybridize with a naturally occurring pre-miRNA under stringent hybridization conditions.

[0167] In certain embodiments, the secondary structure of each pri-miRNA containing a non-natural pre-miRNA (hereinafter referred to as "non-natural pri-miRNA") is sufficiently similar to that of a naturally occurring pri-miRNA sequence to reduce or prevent cellular RNAi-based antipathogen toxicity. In certain such embodiments, the nucleic acid sequence of the 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 99.1%, at least about 99.5%, at least about 99.9%, or at least about 99.99% of sequence identity with that of a naturally occurring pri-miRNA, and / or can hybridize with a naturally occurring pri-miRNA under stringent hybridization conditions.

[0168] The non-natural pre-miRNAs of the present invention can be produced by removing native mature miRNA sequences from naturally occurring pre-miRNAs and replacing them with non-natural mature miRNA sequences, one of which can serve as a guide miRNA targeting a target gene.

[0169] In certain embodiments, each non-natural pre-miRNA includes a skeletal sequence derived from a naturally occurring pre-miRNA, for example, one present in mouse, rat, or human. In certain embodiments, the skeletal sequences of the non-natural pre-miRNA (3' skeletal sequence, 5' skeletal sequence, and loop sequence) 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%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.1%, at least about 99.5%, at least about 99.9%, or at least about 99.99% sequence identity with the corresponding skeletal sequence of the naturally occurring pre-miRNA, and / or can hybridize with such corresponding skeletal segments under stringent hybridization conditions. In certain embodiments, the skeletal segment of a non-natural pre-miRNA sequence is identical to the corresponding skeletal segment of a naturally occurring pre-miRNA. In certain embodiments, the naturally occurring pre-miRNAs are 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-miRNAs are miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In certain embodiments, the naturally occurring pre-miRNAs are miR16, miR21, miR22, miR204, or miR206.

[0170] In certain embodiments, each non-natural pri-miRNA includes a skeletal sequence derived from a naturally occurring pri-miRNA, for example, one present in mouse, rat, or human. In certain embodiments, the skeletal sequences of the non-natural pri-miRNA (3' skeletal sequence, 5' skeletal sequence, and loop sequence) 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%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.1%, at least about 99.5%, at least about 99.9%, or at least about 99.99% sequence identity with the corresponding skeletal sequence of the naturally occurring pri-miRNA, and / or can hybridize with such corresponding skeletal segments under stringent hybridization conditions. In certain embodiments, the skeletal segment of a non-natural pri-miRNA sequence is identical to the corresponding skeletal segment of a naturally occurring pri-miRNA. In certain embodiments, the naturally occurring pri-miRNAs are 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-miRNAs are miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In certain embodiments, the naturally occurring pre-miRNAs are miR16, miR21, miR22, miR204, or miR206.

[0171] While miRNA-5p and miRNA-3p sequences hybridize with each other, they are not necessarily strictly complementary. In the design of miRNAs that do not exist in nature, compensatory mutations may be introduced into the miRNA-5p and / or miRNA-3p sequences 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 complementary sequence encoding the miRNA-5p sequence, or can hybridize with the sequence encoding the miRNA-5p sequence under stringent hybridization conditions.

[0172] To a certain particular extent, two non-natural pre-miRNA sequences are separated from each other by at least approximately 1, at least approximately 2, at least approximately 3, at least approximately 4, at least approximately 5, at least approximately 6, at least approximately 7, at least approximately 8, at least approximately 9, at least approximately 10, at least approximately 15, at least approximately 20, at least approximately 25, at least approximately 30, at least approximately 35, at least approximately 40, at least approximately 45, at least approximately 50, at least approximately 60, at least approximately 70, at least approximately 80, at least approximately 90, at least approximately 100, at least approximately 110, at least approximately 120, at least approximately 130, at least approximately 140, at least approximately 150, at least approximately 160, at least approximately 170, at least approximately 180, at least approximately 190, at least approximately 200, at least approximately 210, at least approximately 220, at least approximately 230, at least approximately 240, or at least approximately 250 nucleotides. In a particular embodiment, the two non-natural pre-miRNA sequences are approximately 5-250 nucleotides, approximately 10-250 nucleotides, approximately 10-200 nucleotides, approximately 10-150 nucleotides, approximately 10-100 nucleotides, approximately 10-50 nucleotides, approximately 10-40 nucleotides, approximately 10-30 nucleotides, approximately 10-20 nucleotides, approximately 16-250 nucleotides, approximately 16-200 nucleotides, approximately 16-150 nucleotides, approximately 16-100 nucleotides, approximately 16-50 nucleotides, and approximately 16-4 They are separated by 0 nucleotides, approximately 16-30 nucleotides, approximately 16-20 nucleotides, approximately 20-200 nucleotides, approximately 20-150 nucleotides, approximately 20-100 nucleotides, approximately 20-50 nucleotides, approximately 20-45 nucleotides, approximately 20-40 nucleotides, approximately 20-35 nucleotides, approximately 20-30 nucleotides, approximately 20-25 nucleotides, approximately 30-200 nucleotides, approximately 30-150 nucleotides, approximately 30-100 nucleotides, approximately 30-50 nucleotides, or approximately 30-40 nucleotides.In a particular embodiment, two non-natural pre-miRNA sequences are at least approximately 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, 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, 13 8, 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, 19 They are separated by 6, 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.

[0173] In a particular embodiment, two non-natural pri-miRNA sequences are adjacent to each other, with the 3' nucleotide of one pri-miRNA directly linked to the 5' nucleotide of the other pri-miRNA. In such an embodiment, the nucleotides separating each non-natural pre-miRNA contained within each pri-miRNA form a part of the pri-miRNA.

[0174] In certain embodiments, the non-natural pre-miRNA comprises a mature miRNA sequence that can bind to mRNA, thereby interfering with its translation and / or promoting its degradation. mRNA may be produced from the expression of a target gene.

[0175] In certain embodiments, the target gene encodes an immune checkpoint protein. Thus, the pre-miRNA sequence inhibits the expression of the immune checkpoint protein by targeting the gene that expresses it. 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 embodiments, the immune checkpoint protein is CTLA4, CD70, PD-1, TIGIT, TIM3, LAG3, GITR, or PIK3IP1. In certain embodiments, the immune checkpoint protein is CD70, PD-1, or TIGIT. In certain embodiments, the immune checkpoint protein is PD-1.

[0176] In a particular embodiment, each non-natural pre-miRNA targets a different gene, or a specific different region of the same gene.

[0177] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets PD-1; and (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets PD-1.

[0178] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets PD-1; and (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets PD-1.

[0179] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets PD-1; and (b) a pre-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets PD-1.

[0180] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets PD-1; and (b) a pri-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets PD-1.

[0181] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets TIGIT.

[0182] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets TIGIT.

[0183] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR21 and a guide miRNA that targets TIGIT.

[0184] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR21 and a guide miRNA that targets TIGIT.

[0185] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT.

[0186] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT.

[0187] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT.

[0188] In certain embodiments, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT.

[0189] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR142 and a guide miRNA that targets TIGIT.

[0190] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR142 and a guide miRNA that targets TIGIT.

[0191] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets TIGIT.

[0192] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets TIGIT.

[0193] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR21 and a guide miRNA that targets TIGIT.

[0194] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR22 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR21 and a guide miRNA that targets TIGIT.

[0195] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR21 and a guide miRNA that targets TIGIT.

[0196] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR21 and a guide miRNA that targets TIGIT.

[0197] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR142 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets TIGIT.

[0198] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR142 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets TIGIT.

[0199] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR142 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT.

[0200] In certain embodiments, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR142 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT.

[0201] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR142 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR21 and a guide miRNA that targets TIGIT.

[0202] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR142 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR21 and a guide miRNA that targets TIGIT.

[0203] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets TIGIT.

[0204] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets TIGIT.

[0205] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT.

[0206] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR16 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA that targets TIGIT.

[0207] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting CD70.

[0208] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting CD70.

[0209] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR16 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a skeletal sequence from miR22 and a guide miRNA targeting CD70.

[0210] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR16 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a skeletal sequence from miR22 and a guide miRNA targeting CD70.

[0211] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR26a1 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting CD70.

[0212] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR26a1 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting CD70.

[0213] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR26a1 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a skeletal sequence from miR16 and a guide miRNA targeting CD70.

[0214] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR26a1 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a skeletal sequence from miR16 and a guide miRNA targeting CD70.

[0215] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a skeletal sequence from miR26a1 and a guide miRNA targeting CD70.

[0216] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a skeletal sequence from miR26a1 and a guide miRNA targeting CD70.

[0217] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting CD70; and (b) a pre-miRNA comprising a skeletal sequence from miR16 and a guide miRNA targeting CD70.

[0218] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting CD70; and (b) a pri-miRNA comprising a skeletal sequence from miR16 and a guide miRNA targeting CD70.

[0219] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR150 and a guide miRNA targeting TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1.

[0220] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR150 and a guide miRNA that targets TIGIT; and (b) a pie-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets PD-1.

[0221] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pre-miRNA comprising a skeletal sequence from miR17 and a guide miRNA targeting TIGIT.

[0222] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pri-miRNA comprising a skeletal sequence from miR17 and a guide miRNA targeting TIGIT.

[0223] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR17 and a guide miRNA that targets TIGIT; and (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets PD-1.

[0224] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR17 and a guide miRNA that targets TIGIT; and (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA that targets PD-1.

[0225] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pre-miRNA comprising a skeletal sequence from miR150 and a guide miRNA targeting TIGIT.

[0226] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pri-miRNA comprising a skeletal sequence from miR150 and a guide miRNA targeting TIGIT.

[0227] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pre-miRNA comprising a skeletal sequence from miR16 and a guide miRNA targeting CD70.

[0228] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pri-miRNA comprising a skeletal sequence from miR16 and a guide miRNA targeting CD70.

[0229] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pre-miRNA comprising a skeletal sequence from miR22 and a guide miRNA targeting CD70.

[0230] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pri-miRNA comprising a skeletal sequence from miR22 and a guide miRNA targeting CD70.

[0231] In a particular embodiment, the ribonucleic acid comprises (a) a pre-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pre-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pre-miRNA comprising a skeletal sequence from miR22 and a guide miRNA targeting CD70.

[0232] In a particular embodiment, the ribonucleic acid comprises (a) a pri-miRNA comprising a skeletal sequence from miR204 and a guide miRNA targeting PD-1; (b) a pri-miRNA comprising a skeletal sequence from miR206 and a guide miRNA targeting PD-1; and (c) a pri-miRNA comprising a skeletal sequence from miR22 and a guide miRNA targeting CD70.

[0233] The present invention also relates in part to deoxyribonucleic acid encoding any of the ribonucleic acids described above.

[0234] Examples of deoxyribonucleic acid sequences encoding skeletal sequences that may be used in carrying out the present invention include, but are not limited to, those listed in Table 3 below. In Table 3, the symbols "X" and "Y" represent nucleic acid sequences encoding guide miRNA (which may be either miRNA-5p or miRNA-3p) and passenger miRNA (which may be either miRNA-5p or miRNA-3p), respectively, while the symbol "n" represents the number of nucleotides in such a sequence, for example, 16 to 30, preferably 18 to 25. In some embodiments, n may 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 a particular embodiment, the deoxyribonucleic acid encoding the skeletal sequence hybridizes with one of the complementary sequences listed in Table 3 under stringent hybridization conditions.

[0235] [Table 3]

[0236] In any of the embodiments described above, the sequence encoding pre-miRNA is: Sequence ID 1, Sequence ID 2, and Sequence ID 3, respectively; Sequence ID 4, Sequence ID 5, and Sequence ID 6, respectively; Sequence ID 7, Sequence ID 8, and Sequence ID 9, respectively; Sequence ID 10, Sequence ID 11, and Sequence ID 12, respectively; Sequence ID 13, Sequence ID 14, and Sequence ID 15, respectively; Sequence ID 16, Sequence ID 17, and Sequence ID 18, respectively; Sequence ID 19, Sequence ID 20, and Sequence ID 21, respectively; Sequence IDs 22, 23, and 24, respectively; Sequence IDs 25, 26, and 27, respectively; Sequence IDs 28, 29, and 30, respectively; Sequence ID 31, Sequence ID 32, and Sequence ID 33, respectively; Sequence ID 34, Sequence ID 35, and Sequence ID 36, respectively; Sequence ID 37, Sequence ID 38, and Sequence ID 39, respectively; Sequence ID 40, Sequence ID 41, and Sequence ID 42, respectively; Sequence ID 43, Sequence ID 44, and Sequence ID 45, respectively; Sequence ID 46, Sequence ID 47, and Sequence ID 48, respectively; Sequence ID 49, Sequence ID 50, and Sequence ID 51, respectively; Sequence ID 52, Sequence ID 53, and Sequence ID 54, respectively; Sequence IDs 55, 56, and 57, this time; Sequence ID 58, Sequence ID 59, and Sequence ID 60, respectively; Sequence ID 61, Sequence ID 62, and Sequence ID 63, respectively; Sequence IDs 338, 339, and 340, respectively; Sequence ID 341, Sequence ID 342, and Sequence ID 343, respectively; or Sequence IDs 344, 345, and 346, respectively; Alternatively, a sequence having at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any of the sequences mentioned above, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of such a sequence. Includes.

[0237] Table 4 lists non-exclusive examples of nucleic acid sequences encoding guide miRNAs that target genes encoding such checkpoint inhibitors. Table 4 also lists sequences encoding passenger strands. As previously discussed, the guide and passenger strands are not necessarily complementary. The passenger strand is intended to also be useful for targeting messenger RNA associated with the target gene. Sequences that hybridize with complementary sequences of the sequences listed in Table 4 under stringent hybridization conditions are also intended to be used. The mature miRNAs used may be combined with specific pri-miRNA backbones. Table 4 also lists backbones that may be combined with the mature guide and passenger miRNAs listed therein.

[0238] [Table 4-1]

[0239] [Table 4-2]

[0240] [Table 4-3]

[0241] In a particular embodiment, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80% sequence identity with any one of sequence numbers 64-83, 85, 87-171, 293-322, and 704-713, or a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 64-83, 85, 87-171, 293-322, and 704-713. In a particular embodiment, the present invention relates to sequence numbers 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, 15 6, 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, with at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% Nucleic acid sequences having sequence identity, or sequence numbers 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, 1 This relates to deoxyribonucleic acid, comprising a nucleic acid sequence that can hybridize under stringent hybridization conditions to any one of the complementary sequences among 56, 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.

[0242] In a particular embodiment, the sequences encoding the guide miRNA sequence are sequence numbers 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, 29 It has at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of 3, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 704, 705, 709, and 710, or can hybridize to a complementary sequence of any one of such sequences under stringent hybridization conditions.

[0243] In a particular embodiment, the sequences encoding the passenger miRNA sequence are sequence numbers 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 have at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity to any one of such sequences, or can hybridize to a complementary sequence of any one of such sequences under stringent hybridization conditions.

[0244] 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 a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence 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 sequence numbers 64, 66, 68, and 70, or a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 64, 66, 68, and 70.

[0245] 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 a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence 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% sequence identity with any one of sequence numbers 72, 74, 76, 78, 80, 82, 704, 705, 709, and 710, or a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 72, 74, 76, 78, 80, 82, 704, 705, 709, and 710.

[0246] 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 sequence numbers 88-145, or a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 88-145. In certain embodiments, the present invention has sequence identity of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% with any one of sequence numbers 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. The present invention relates to a nucleic acid sequence, or a nucleic acid sequence that can be hybridized under stringent hybridization conditions to any one complementary sequence among sequence numbers 64, 66, 68 and 70, 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.

[0247] 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 a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence 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 sequence numbers 146, 148, 150, 152, 154, and 156, or a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 146, 148, 150, 152, 154, and 156.

[0248] 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 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 a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence 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 either SEQ ID NO: 158 or 160, or a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of either SEQ ID NO: 158 or 160.

[0249] 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 a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence 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 either SEQ ID NO: 162 or 164, or a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of either SEQ ID NO: 162 or 164.

[0250] 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 a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence 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 sequence numbers 166, 168, and 170, or a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 166, 168, and 170.

[0251] In certain embodiments, the deoxyribonucleic acid encodes a pre-miRNA that targets 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 a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of SEQ ID NOs. 293-322. In a particular embodiment, 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 sequence numbers 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, and 321, or a nucleic acid sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, and 321.

[0252] In a particular embodiment, the present invention provides that each sequence encoding a pre-miRNA is, a) Sequence encoding the 5' miRNA skeletal sequence; b) Sequences encoding guide miRNA sequences; c) An array that codes for a stem-loop array; d) A sequence that codes for a passenger miRNA sequence; and e) Sequence encoding the 3' skeletal structure This concerns deoxyribonucleic acid, including deoxyribonucleic acid.

[0253] In a particular embodiment, the sequence encoding pre-miRNA is: a) Sequence IDs 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, 17 A guide miRNA sequence having at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with any one of 0, 704, 705, 709, and 710, or a guide miRNA sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of such sequences; and b) Sequence IDs 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, 30 Passenger sequences having at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity to any one of 6, 308, 310, 312, 314, 316, 318, 320, 322, 706-708, and 711-713, respectively, or passenger sequences that can hybridize under stringent hybridization conditions to a complementary sequence of any one of such sequences. Includes.

[0254] Deoxyribonucleic acids encoding exemplary non-natural pre-miRNA sequences that target specific checkpoint inhibitors are listed in Table 5. In certain embodiments, the deoxyribonucleic acids may include a sequence that can hybridize with any one complementary sequence from those listed in Table 5 under stringent hybridization conditions.

[0255] [Table 5-1]

[0256] [Table 5-2]

[0257] [Table 5-3]

[0258] 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 sequence numbers 347 to 447, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 347 to 447.

[0259] 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 sequence numbers 178-263 and 323-337, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 178-263 and 323-337.

[0260] In certain embodiments, the miRNA targets CTLA4. In certain such embodiments, the present invention relates to deoxyribonucleic acids 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 a sequence that can hybridize under stringent hybridization conditions to a complementary sequence 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 sequence numbers 178 and 250-252, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 178 and 250-252.

[0261] In certain embodiments, the pre-miRNA targets PD-1. In certain such embodiments, the present invention relates to deoxyribonucleic acids 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 sequence numbers 348, 349, and 410-418, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 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 sequence numbers 179, 180, and 241-249, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 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 either SEQ ID NO: 348 or 349, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of either SEQ ID NO: 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 either SEQ ID NO: 179 or 180, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of either SEQ ID NO: 179 or 180.

[0262] 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 a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of nucleic acid comprising SEQ ID NO: 179 and SEQ ID NO: 180.

[0263] In certain embodiments, the pre-miRNA targets TIGIT. In certain such embodiments, the present invention relates to deoxyribonucleic acids 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 sequence numbers 350-377 and 404-409, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 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 sequence numbers 181-208 and 235-240, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 181-208 and 235-240.

[0264] In certain embodiments, the pre-miRNA targets TIM3. In certain such embodiments, the present invention relates to deoxyribonucleic acids 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 sequence numbers 378-389, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 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 sequence numbers 209-220, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 209-220.

[0265] In certain embodiments, the pre-miRNA targets LAG3. In certain such embodiments, the present invention relates to deoxyribonucleic acids 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 sequence numbers 390-396, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 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 sequence numbers 221-227, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 221-227.

[0266] In certain embodiments, the pre-miRNA targets GITR. In certain such embodiments, the present invention relates to deoxyribonucleic acids 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 sequence numbers 397-403, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 397-403. 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 sequence numbers 228-234, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 228-234.

[0267] In certain embodiments, the pre-miRNA targets PIK3IP1. In certain such embodiments, the present invention relates to deoxyribonucleic acids 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 sequence numbers 422-424, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 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 sequence numbers 253-255, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 253-255.

[0268] In certain embodiments, the pre-miRNA targets CD70. In certain such embodiments, the present invention relates to deoxyribonucleic acids 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 sequence numbers 433-447, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 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 sequence numbers 323-337, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 323-337.

[0269] In embodiments of the present invention, two or more pre-miRNAs encoded by deoxyribonucleic acid may each contain a guide miRNA sequence that targets the same target gene, or various guide miRNAs that may target different genes. In addition, each pre-miRNA design of the pri-miRNAs containing them may be based on different native miRNA backbones to reduce the likelihood of misfolding between one miRNA and another. Table 6 provides examples of deoxyribonucleic acid encoding two or more pri-miRNAs.

[0270] [Table 6-1]

[0271] [Table 6-2]

[0272] 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. 267-290, 448-460, and SEQ ID NO. 944, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of SEQ ID NOs. 267-290, 448-460, and SEQ ID NO. 944.

[0273] 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 sequence numbers 267, 282, and 944, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 267, 282, and 944. In certain embodiments, the present invention relates to 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 a sequence that can hybridize to a complementary sequence of SEQ ID NO: 267 under stringent hybridization conditions.

[0274] 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 sequence numbers 269-274, 287, 288, and 290, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 269-274, 287, 288, and 290.

[0275] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs targeting PD-1 and a pre-miRNA targeting 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 sequence numbers 275-280, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 275-280.

[0276] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting PD-1 and a pre-miRNA targeting 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 sequence numbers 281, 283, and 284, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 281, 283, and 284.

[0277] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting TIGIT and a pre-miRNA targeting 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 sequence numbers 285, 286, and 289, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 285, 286, and 289.

[0278] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs targeting PD-1 and a pre-miRNA targeting 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 a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of SEQ ID NO: 448 or 451.

[0279] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting PD-1 and two pre-miRNAs targeting 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, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of SEQ ID NO: 449.

[0280] In certain such embodiments, the deoxyribonucleic acid encodes a pre-miRNA targeting PD-1 and a pre-miRNA targeting 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 a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of SEQ ID NO: 450.

[0281] In certain such embodiments, the deoxyribonucleic acid encodes two pre-miRNAs, 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 sequence numbers 452-460, or a sequence that can hybridize under stringent hybridization conditions to a complementary sequence of any one of sequence numbers 452-460.

[0282] Chimeric antigen receptor (CAR) The polynucleotides of this disclosure may further encode chimeric receptors, such as chimeric antigen receptors (CARs). Therefore, in some embodiments, the polynucleotides of this disclosure may encode the miRNAs and CARs described herein. In any embodiment of this disclosure, modified immunoeffector cells may include the chimeric antigen receptors described herein.

[0283] CARs are engineered receptors that impart exogenous specificity to immune effector cells. In some examples, a CAR comprises an extracellular domain (ectodomain) containing an antigen-binding domain, a transmembrane domain, and an intracellular domain (endodomain). The intracellular domain contains an intracellular signaling domain. In certain embodiments, the extracellular domain further includes a spacer between the antigen-binding domain and the transmembrane domain.

[0284] A. Antigen-binding domain The extracellular domain of a CAR includes an antigen-binding domain that can recognize and bind to an epitope on a target antigen.

[0285] The antigen-binding domain may include a complementation-determining region (CDR) that binds to an epitope on the target antigen, for example, a monoclonal CDR that binds to the antigen. The complementation-determining region (CDR) is a short amino acid sequence found in various domains of antigen-binding antigen receptor (e.g., immunoglobulin and T cell receptor) proteins, and therefore gives the receptor its specificity for a particular antigen. Each polypeptide chain of an antigen receptor may contain three CDRs (CDR1, CDR2, and CDR3).

[0286] In certain embodiments, the antigen-binding domain includes an Fv, Fab, Fab2, Fab', F(ab')2, or F(ab')3 fragment of an antibody that binds to the antigen.

[0287] In a particular embodiment, the antigen-binding domain is a variable domain (V) of the antibody's heavy chain that binds to the antigen. H (domain) and / or variable domain of the antibody light chain (V L The domain, or a functional fragment or variant thereof. In certain embodiments, the functional fragment or variant includes a CDR that binds to an antigen. For example, V H A functional fragment or variant of a domain is V H The domain may include CDR1, CDR2 and CDR3; and / or V LA functional fragment or variant of a domain is V L This may include CDR1, CDR2, and CDR3 for the domain.

[0288] In certain embodiments, the antigen-binding domain includes scFv, sc(Fv)2, dsFv, diabody, minibody, nanobody, or a binding fragment thereof. In certain embodiments, the antigen-binding domain further includes an Fc fragment of an antibody, which may include, for example, an scFv linked to the Fc fragment.

[0289] In some embodiments, CARs target antigens that are elevated in cancer cells or autoimmune cells. Autoimmune diseases may include graft-versus-host disease, rheumatoid arthritis, lupus, lupus nephritis (LN), myasthenia gravis (MG), celiac disease, Crohn's disease, Sjögren's syndrome, polymyalgia rheumatica, multiple sclerosis, neuromyelitis optica, ankylosing myelitis, type 1 diabetes mellitus, alopecia areata, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris, and autoimmune uveitis.

[0290] In some embodiments, the CARs described herein are 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, C EACAM6, 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 antigen, HER1, HER2, HER3, HER4, HER1 / HER3, h5T4, HPV antigen, HVEM, IGF1R, Ig kappa 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, Mucin, e.g., MUC1, MUC-4 or MUC16, NGFR, NKG2D, Notch-1-4, NY-ESO-1, O-acetyl GD2, O-acetyl It contains an antigen-binding domain that binds to an epitope on GD3, 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, TGF beta, TGF beta-II, TGFBR1, TGFBR2, titin, TLR7, TLR9, TNFR1, TNFR2, TNFRSF4, TRBC1, TWEAK-R, VEGF, VEGF-R2, or WT-1.

[0291] In some embodiments, the CAR described herein includes an antigen-binding domain that binds to an epitope on CD19.

[0292] Antigen binding can be evaluated by flow cytometry, cell-based assays, or any other equivalent assay. Cell-based assays can evaluate antigen binding using cell types that express the antigen of interest on their surface. Antigens or fragments of antigens expressed as soluble proteins can be used to evaluate antigens using flow cytometry or similar assays. Enhanced antigen binding can be directly evaluated by functional measurements of the antigen-binding domain or chimeric receptor. For example, enhanced antigen binding of the chimeric receptor or CAR described herein can be measured by increased specific cytotoxicity to target cells expressing the antigen.

[0293] The cell surface expression levels of the polypeptides of this disclosure can be evaluated, for example, using a flow cytometry-based assay. The increase in antigen-binding polypeptide expression can be measured as the percentage of analytical cells expressing the antigen-binding polypeptide, or alternatively, as the average density of the antigen-binding polypeptide on the cell surface. Additional preferred methods that may be used to evaluate the cell surface expression of the antigen-binding polypeptides described herein include Western blotting or any other equivalent assay.

[0294] CD19 is a cell surface glycoprotein of the immunoglobulin superfamily. In some cases, 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 differentiation antigen group 19 protein, an antigenic determinant commonly expressed on B cells and during various B-cell malignancies. CD19 is a transmembrane protein that plays a crucial role in B-cell development, activation, and differentiation. 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 number P15391) and its corresponding nucleotide sequence (e.g., GenBank accession number NM_001178098), as well as their counterparts in other species. The term "CD19" also includes any of the following: (a) naturally occurring allele 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) variants or derivatives of CD19, whether naturally occurring or artificially produced, that retain characteristic CD19 function or antigenic properties; (e) fusion proteins containing CD19 or a portion thereof; and (f) any CD19 sequence having conserved or non-conserved amino acid substitutions, deletions, or insertions.

[0295] In some embodiments, the antigen-binding domain of the CARs described herein is specific to CD19. When expressed on the cell surface, CD19-specific CARs can redirect the specificity of T cells to human CD19.

[0296] In some embodiments, the antigen-binding domain includes a CDR that binds to CD19. In a particular embodiment, the antigen-binding domain is V L The domains include CDR1, CDR2, and CDR3. In certain embodiments, the antigen domain is V HThe domains include CDR1, CDR2, and CDR3. In certain embodiments, the antigen-binding domain is V L Domain CDR1, CDR2 and CDR3, and V H Includes CDR1, CDR2, and CDR3 for the domain.

[0297] In some embodiments, V coupled to CD19 L Domains, for example, barrier domain light chains from CD19-specific monoclonal anti-CD19 antibodies, or V L An antigen that binds to a functional fragment or variant of a domain. In certain embodiments, V L A functional fragment or variant of a domain is V L Includes CDR1, CDR2, and CDR3 for the domain.

[0298] In some embodiments, the antigen-binding domain binds to CD19. H Domains, for example, barrier domain heavy chains from CD19-specific monoclonal anti-CD19 antibodies, or V H Includes a functional fragment or variant of a domain. In a particular embodiment, V H Functional fragments or variants of the domain include CDR1, CDR2, and CDR3 of the VH domain.

[0299] In some embodiments, the antigen-binding domain is a variable domain light chain (V) that binds to CD19. L Domain) and variable domain heavy chain (V H Domain), for example, V from a CD19-specific antibody L and V H Domain, or such V L and V H Contains a single-chain antibody fragment (scFv) containing a functional fragment or variant of the domain. H and V LThe domains may be joined by linkers, such as flexible linkers, such as glycine-serine linkers or Whitlow linkers. In embodiments, scFv is SJ25Cl and / or FMC63. In embodiments, scFv is humanized. In some embodiments, the antigen-binding moieties are linked in a directional manner. H and V L Domains, for example, from the N-terminus to the C-terminus, V H Domain-Linker-V L Domain or V L Domain-Linker-V H It may include a domain name.

[0300] 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 comprises either the sequence encoded by SEQ ID NO: 536, a stringent hybridization of the complementary sequence of SEQ ID NO: 536, or a codon-degenerate version of SEQ ID NO: 536.

[0301] In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 527, or an amino acid sequence comprising one, two, three, four, or five amino acid modifications to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 1023, 1024, 1025, or 1026. In some embodiments, the linker is encoded by a polynucleotide sequence that is 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 that is 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 sequence number 536.

[0302] 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).

[0303] In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD19 antibody described in U.S. Patent Application Publication No. 2016 / 0152723.

[0304] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19 (Kite Pharma, Inc.). Some embodiments described herein include CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by KTE-C19.

[0305] In some embodiments, the CD19-specific CAR-T cells described herein include an anti-CD antibody, or a fragment or derivative thereof, as described in International Publication No. 2015 / 187528.

[0306] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019 (Novartis). Some embodiments described herein include CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by CTL019.

[0307] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19(Cellectis). Some embodiments described herein include CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by UCART19.

[0308] In some embodiments, the antigen-binding domain recognizes an epitope on CD19 that is also recognized by BPX-401 (Bellicum). Some embodiments described herein include CD19-specific CAR-T cells in which the antigen-binding domain recognizes an epitope on CD19 that is also recognized by BPX-401.

[0309] In some cases, the antigen-binding domain recognizes an epitope on CD19, which is also recognized by blinatumomab (Amgen), coltuximablavtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-551 (Medimmune), denintuzumab mahodotin (Seattle Genetics), B4 (or DI-B4) (Merck Serono), taplitumomabpaptox (National Cancer Institute), XmAb 5871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex), or AFM11 (Affimed).

[0310] In addition to the above, V is described as being able to target CD19 in the art. H and V L Exemplary anti-CD19 specific CARs, including an antigen-binding domain containing a domain, or a functional fragment or variant thereof, can be used. Such CD19 specific CARs, antigen-binding domains, and V H and V LExamples of domains include U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 20170368098 specification, US Patent Application Publication No. 20160145337 specification, US Patent No. 9701758 specification, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848 specification, US Patent Application Publication No. 20190135894 specification, US Patent No. 10774388 specification, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 10864 This is described in U.S. Patent No. 2, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985, the entire contents of each of these are incorporated herein by reference.

[0311] In certain embodiments, the antigen-binding domain is specified in U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Publication No. 20170368098, U.S. Patent Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent V LIncludes a domain. In certain embodiments, the functional fragment is up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than any one of the sequences described above.In certain embodiments, the functional variant is as described in U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 2017036809 Specifications No. 8, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International A 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 sequences disclosed in Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985, and / or a conservatively substituted variant thereof.

[0312] In certain embodiments, the antigen-binding domain is specified in U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Patent Publication No. 20170368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2 U.S. Patent No. 020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669 V is encoded by a polynucleotide comprising any one of the nucleic acid sequences disclosed in Patent No. 549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985 or a functional fragment or variant thereof. LIncludes domains. In certain embodiments, functional variants include U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 2017 Specifications 0368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. 1 Specification No. 0765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with one of the sequences disclosed in National Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985;U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 20170368098 Specification, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549 , hybridizes under stringent hybridization conditions with any one complementary sequence from among the sequences disclosed in International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985;Alternatively, U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 20 Specifications No. 170368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 1 It is one codon degenerate variant of the sequences disclosed in U.S. Patent No. 08642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985.

[0313] In certain embodiments, the antigen-binding domain is specified in U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. National Patent Application Publication No. 20170368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, National International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent V includes any one of the amino acid sequences disclosed in Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985, or a functional fragment or variant thereof. HIncludes a domain. In certain embodiments, the functional fragment is up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than any one of the sequences described above.In certain embodiments, the functional variant is as described in U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 2017036809 Specifications No. 8, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International A 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 sequences disclosed in Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985, and / or a conservatively substituted variant thereof.

[0314] In certain embodiments, the antigen-binding domain is specified in U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Patent Publication No. 20170368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2 U.S. Patent No. 020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669 V is encoded by a polynucleotide comprising any one of the nucleic acid sequences disclosed in Patent No. 549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985 or a functional fragment or variant thereof. HIncludes domains. In certain embodiments, functional variants include U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 2017 Specifications 0368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. 1 Specification No. 0765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with one of the sequences disclosed in National Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985;U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 20170368098 Specification, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549 , hybridizes under stringent hybridization conditions with any one complementary sequence from among the sequences disclosed in International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985;Alternatively, U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 201 70368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 1086 It is a codon degenerate variant of any one of the sequences disclosed in U.S. Patent No. 42, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985.

[0315] In certain embodiments, the antibody-binding domain is (i) U.S. Patent Publication No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent Publication No. 10457730, International Publication No. 2019 / 159193, U.S. Patent Publication No. 10287350, U.S. Patent Publication No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 20170368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 2019 U.S. Patent No. 0135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 1 U.S. Patent Application No. 08644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,V, includes any one of the amino acid sequences disclosed in Specification No. 985, or a functional fragment or variant thereof, for example, a functional fragment that is up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than any of the aforementioned sequences, or 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, and / or a conservatively substituted variant thereof. LDomain; and (ii) U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 2 Patent No. 01794, U.S. Patent Application Publication No. 20170368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 2019013589 Specification No. 4, U.S. Publication No. 10774388, International Publication No. 2020 / 180882, U.S. Publication No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 1086 44, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,V, includes any one amino acid sequence, or a functional fragment or variant thereof, of any one of the amino acid sequences disclosed in Specification No. 985, for example, a functional fragment that is up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than any one of the aforementioned sequences, or 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 one of the aforementioned sequences, and / or a conservatively substituted variant thereof. H Includes the domain.

[0316] In certain embodiments, the antibody-binding domain is (i) U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 20170368 Specifications No. 098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701 International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, Country One of the nucleic acid sequences, or a functional fragment or variant thereof, disclosed in International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985, for example, one of the aforementioned sequences in combination with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99%.V is encoded by a polynucleotide containing a functional variant that has 99% sequence identity, hybridizes with a complementary sequence of any of the aforementioned sequences under stringent hybridization conditions, or is a codon degenerate variant of any of the aforementioned sequences. LDomain; and (ii) U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 20170368098, U.S. National Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, U.S. Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication One of the nucleic acid sequences, or a functional fragment or variant thereof, disclosed in U.S. Patent No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, International Publication No. 2019 / 246546, and U.S. Patent Application No. 18 / 259,985, for example, one of the aforementioned sequences in combination with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99%.V is encoded by polynucleotides containing functional variants that have 99% sequence identity, hybridize under stringent hybridization conditions with a complementary sequence of any of the aforementioned sequences, or are codon degenerate variants of any of the aforementioned sequences. H Includes the domain.

[0317] In some embodiments, V H The domain consists of three complementary determination regions (CDRs): V H CDR1, V H CDR2, and V H Includes CDR3. In some embodiments, V H The domain is V, indicated by sequence number 947. H CDR1, V H CDR2, and V H Includes CDR3, or a functional fragment or variant thereof. In some embodiments, V H The amino acid sequence of CDR1 includes the amino acid sequence of SEQ ID NO: 954, or a functional fragment or variant thereof (for example, an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 954); V H The amino acid sequence of CDR2 includes the amino acid sequence of SEQ ID NO: 955, or a functional fragment or variant thereof (for example, an amino acid sequence having up to 5, 4, 3, 2, or 1 conservative amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 955); V H The amino acid sequence of CDR3 includes the amino acid sequence of SEQ ID NO: 956, or a functional fragment or variant thereof (for example, an amino acid sequence having up to 5, 4, 3, 2, or 1 conserved amino acid substitutions to the amino acid sequence of SEQ ID NO: 956). In some embodiments, V H The amino acid sequence of CDR1 includes the amino acid sequence of SEQ ID NO: 954; V H The amino acid sequence of CDR2 includes the amino acid sequence of SEQ ID NO: 955; V H The amino acid sequence of CDR3 includes the amino acid sequence of SEQ ID NO: 956.

[0318] In some embodiments, V L domain includes three complementarity-determining regions (CDRs): V L CDR1, V L CDR2, and V L CDR3. In some embodiments, V L includes the V of SEQ ID NO: 946 L CDR1, V L CDR2, and V L CDR3, or a functional fragment or variant thereof. In some embodiments, the amino acid sequence of V L CDR1 includes 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 relative to the amino acid sequence of SEQ ID NO: 948); the amino acid sequence of V L CDR2 includes 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 relative to the amino acid sequence of SEQ ID NO: 949); the amino acid sequence of V L CDR3 includes 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 relative to the amino acid sequence of SEQ ID NO: 950). In some embodiments, the amino acid sequence of V L CDR1 includes the amino acid sequence of SEQ ID NO: 948; the amino acid sequence of V L CDR2 includes the amino acid sequence of SEQ ID NO: 949; the amino acid sequence of V L CDR3 includes the amino acid sequence of SEQ ID NO: 950.

[0319] In some embodiments, the V H domain includes an amino acid sequence that is a functional fragment or variant of SEQ ID NO: 947. In certain such embodiments, V HThe domain is a functional variant of SEQ ID NO: 947, comprising a CDR having an amino acid sequence 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 substitution relative 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 substitution relative 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 substitution relative to the amino acid sequence of SEQ ID NO: 956). In some embodiments, V H The domain has an amino acid sequence of any one of SEQ ID NOs: 947, 1031, 1032, 1033, or 1034.

[0320] In some embodiments, V L The domain comprises an amino acid sequence that is a functional fragment or variant of SEQ ID NO: 946. In certain such embodiments, V L The domain is a functional variant of SEQ ID NO: 946, comprising a CDR having an amino acid sequence 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 substitution relative 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 substitution relative 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 substitution relative to the amino acid sequence of SEQ ID NO: 950). In some embodiments, V[[ID=,11]] L The domain has an amino acid sequence of SEQ ID NOs: 946, 1027, 1028, 1029, or 1030.

[0321] In some embodiments, VH The amino acid sequence of CDR1 includes the amino acid sequence of SEQ ID NO: 954; V H The amino acid sequence of CDR2 includes the amino acid sequence of SEQ ID NO: 955; V H The amino acid sequence of CDR3 includes the amino acid sequence of SEQ ID NO: 956; V L The amino acid sequence of CDR1 includes the amino acid sequence of SEQ ID NO: 948; V L The amino acid sequence of CDR2 includes the amino acid sequence of SEQ ID NO: 949; V L The amino acid sequence of CDR3 includes the amino acid sequence of SEQ ID NO: 950.

[0322] In some embodiments, V H The amino acid sequence of CDR1 is derived from the amino acid sequence of SEQ ID NO: 954; V H The amino acid sequence of CDR2 is derived from the amino acid sequence of SEQ ID NO: 955; V H The amino acid sequence of CDR3 is derived from the amino acid sequence of SEQ ID NO: 956; V L The amino acid sequence of CDR1 is derived from the amino acid sequence of SEQ ID NO: 948; V L The amino acid sequence of CDR2 is derived from the amino acid sequence of SEQ ID NO: 949; V L The amino acid sequence of CDR3 is the same as the amino acid sequence of SEQ ID NO: 950.

[0323] In some embodiments, V H The domain contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 947. In some embodiments, V H The domain contains the amino acid sequence of sequence number 947. In some embodiments, V H The domain consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 947. In some embodiments, V H The domain consists of the amino acid sequence of sequence number 947.

[0324] In some embodiments, V L The domain contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 946. In some embodiments, V L The domain contains the amino acid sequence of sequence number 946. In some embodiments, V L The domain consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 946. In some embodiments, V L The domain consists of the amino acid sequence of sequence number 946.

[0325] In some embodiments, V H The domain contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 947; V L The domain contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 946. In some embodiments, V H The domain contains the amino acid sequence of sequence number 947; V L The domain contains the amino acid sequence of sequence number 946. In some embodiments, V H The amino acid sequence of the domain is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 947; V L The domain consists of a sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 946. In some embodiments, V H The amino acid sequence of the domain consists of the amino acid sequence of sequence number 947; V L The amino acid sequence of the domain consists of the amino acid sequence of sequence number 946.

[0326] In some embodiments, V L and V H The domains are linked by a linker, such as a Whitlow linker, or a functional fragment or variant thereof. In some embodiments, the linker contains an amino acid sequence that is 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 contains the amino acid sequence of SEQ ID NO: 527. In some embodiments, the amino acid sequence of the linker consists of a sequence that is 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 a particular embodiment, V L The domain is located on the N-terminal side of the linker, and V is located opposite it. H The domain is located on the C-terminal side of the linker.

[0327] In some embodiments, the CD19-binding domain includes an amino acid sequence that is 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 includes 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 that is 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 of the amino acid sequence of SEQ ID NO: 958. In some embodiments, the amino acid sequence of the CD19-binding domain includes one, two, three, four, or five 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 is 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.

[0328] In some embodiments, the CD19-binding domain includes an amino acid sequence that is 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 CD19-binding domain includes 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 that is 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 of the amino acid sequence of SEQ ID NO: 959. In some embodiments, the amino acid sequence of the CD19-binding domain includes one, two, three, four, or five 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 is 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.

[0329] In some embodiments, the CD19-binding domain includes an amino acid sequence that is 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 includes 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 that is 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 of the amino acid sequence of SEQ ID NO: 960. In some embodiments, the amino acid sequence of the CD19-binding domain includes one, two, three, four, or five 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 is 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.

[0330] In some embodiments, the CD19-binding domain includes an amino acid sequence that is 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 includes 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 that is 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 sequence of the CD19-binding domain consists of the amino acid sequence of SEQ ID NO: 961. In some embodiments, the amino acid sequence of the CD19-binding domain includes one, two, three, four, or five 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 is 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.

[0331] In some embodiments, the CD19-binding domain includes an amino acid sequence that is 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 includes 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 that is 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 of the amino acid sequence of SEQ ID NO: 962. In some embodiments, the amino acid sequence of the CD19-binding domain includes one, two, three, four, or five 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 is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 962.

[0332] In some embodiments, the CD19-binding domain includes an amino acid sequence that is 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 includes 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 that is 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 of the amino acid sequence of SEQ ID NO: 963. In some embodiments, the amino acid sequence of the CD19-binding domain includes one, two, three, four, or five 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 is 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.

[0333] In some embodiments, V H The domain is encoded by the polynucleotide sequence of SEQ ID NO: 970, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 970, V H CDR1; Encoded by the polynucleotide sequence of SEQ ID NO: 971, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 971, V H CDR2; Encoded by the polynucleotide sequence of SEQ ID NO: 972, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 972, V H Includes CDR3. In some embodiments, V HThe domain is V, which is encoded by the polynucleotide sequence of sequence number 970. H CDR1; V encoded by the polynucleotide sequence of sequence number 971 H V encoded by the polynucleotide sequence of CDR2; and sequence number 972 H Includes CDR3.

[0334] In some embodiments, V L The domain is encoded by the polynucleotide sequence of SEQ ID NO: 967, or by a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 967, V L CDR1; Encoded by the polynucleotide sequence of SEQ ID NO: 968, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 968, V L CDR2; Encoded by the polynucleotide sequence of SEQ ID NO: 969, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 969, V L Includes CDR3. In some embodiments, V L The domain is V, which is encoded by the polynucleotide sequence of sequence number 967. L CDR1; V encoded by the polynucleotide sequence of sequence number 968 L V encoded by the polynucleotide sequence of CDR2; and SEQ ID NO: 969 L Includes CDR3.

[0335] In some embodiments, V H The domain is encoded by the polynucleotide sequence of SEQ ID NO: 970, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 970, V HCDR1; Encoded by the polynucleotide sequence of SEQ ID NO: 971, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 971, V H CDR2; Encoded by the polynucleotide sequence of SEQ ID NO: 972, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 972, V H CDR3 Includes;V L The domain is encoded by the polynucleotide sequence of SEQ ID NO: 967, or by a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 967, V L CDR1; Encoded by the polynucleotide sequence of SEQ ID NO: 968, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 968, V L CDR2; Encoded by the polynucleotide sequence of SEQ ID NO: 969, or a polynucleotide sequence containing up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide modification of the polynucleotide sequence of SEQ ID NO: 969, V L Includes CDR3.

[0336] In some embodiments, V H The domain is V, which is encoded by the polynucleotide sequence of sequence number 970. H CDR1; V encoded by the polynucleotide sequence of sequence number 971 H V encoded by the polynucleotide sequence of CDR2; and sequence number 972 H CDR3 Includes;V L The domain is V, which is encoded by the polynucleotide sequence of sequence number 967. LCDR1; V encoded by the polynucleotide sequence of sequence number 968 L V encoded by the polynucleotide sequence of CDR2; and SEQ ID NO: 969 L Includes CDR3.

[0337] In some embodiments, V H The domain is encoded by a polynucleotide sequence that is 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 sequence number 966. In some embodiments, V H The domain is encoded by the polynucleotide sequence of sequence number 966.

[0338] In some embodiments, V L The domain is encoded by a polynucleotide sequence that is 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 sequence number 965. In some embodiments, V L The domain is encoded by the polynucleotide sequence of sequence number 965.

[0339] In some embodiments, V H The domain is encoded by a polynucleotide sequence that is 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 sequence number 966; V L The domain is encoded by a polynucleotide sequence that is 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 sequence number 965. In some embodiments, V H The domain is encoded by the polynucleotide sequence of sequence number 966; V LThe domain is encoded by the polynucleotide sequence of sequence number 965.

[0340] In some embodiments, the CD19-binding domain is encoded by a polynucleotide sequence that is 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 sequence number 974. In some embodiments, the CD19-binding domain is encoded by a polynucleotide sequence that is 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 sequence number 975. In some embodiments, the CD19-binding domain is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of sequence number 976. In some embodiments, the CD19-binding domain is encoded by a polynucleotide sequence that is 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 sequence number 977. In some embodiments, the CD19-binding domain is encoded by a polynucleotide sequence that is 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 sequence number 978. In some embodiments, the CD19-binding domain is encoded by a polynucleotide sequence that is 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 sequence number 979.

[0341] B. Transmembrane domain The transmembrane domain of CARs is involved in the placement of CARs on the cell surface of manipulated T cells.

[0342] The transmembrane domain may originate from either a natural or synthetic source. If the origin is natural, the domain may originate from, for example, any membrane-bound or transmembrane protein. Preferred transmembrane domains include those from TCR-alpha, TCR-beta, TCR-γ1, TCR-δ, TCR-zeta, 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 functional fragments or variants thereof. Alternatively, the transmembrane domain may be synthetic and may contain hydrophobic residues, such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at one or both ends of the synthetic transmembrane domain. In some embodiments, the transmembrane domain includes a CD8α transmembrane domain, a CD152 (CTLA-4) transmembrane domain, a TCRγ1, a TCRδ or a CD3ζ transmembrane domain.

[0343] Optionally, in some embodiments, the transmembrane domain and intracellular signaling domain of the CAR may be linked by a short oligopeptide or polypeptide linker between 2 and 10 amino acids in length. In some embodiments, the linker is a glycine-serine linker.

[0344] In some embodiments, the transmembrane domain includes a CD8α transmembrane domain, a CD3ζ transmembrane domain, or a functional fragment or variant thereof.

[0345] In certain embodiments, the transmembrane domain comprises the 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 up to about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than the amino acid sequence of SEQ ID NO: 812 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.

[0346] In certain embodiments, the CD8α transmembrane domain, or a 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, or hybridizes with a complementary sequence of SEQ ID NO: 813 under stringent hybridization conditions, or is a codon degenerate variant of SEQ ID NO: 813.

[0347] In certain embodiments, the transmembrane domain comprises the 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 up to about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than the amino acid sequence of SEQ ID NO: 814. 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.

[0348] In certain embodiments, the CD28 transmembrane domain, or a functional fragment or variant thereof, is encoded by SEQ ID NO: 815 or its functional fragment or variant. 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, or hybridizes with a complementary sequence of SEQ ID NO: 815 under stringent hybridization conditions, or is a codon degenerate variant of SEQ ID NO: 815.

[0349] C. Spacer In some embodiments, the CAR of the Disclosure includes 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 be oriented in different directions to facilitate antigen recognition.

[0350] In certain embodiments, CARs containing a spacer exhibit enhanced functional activity compared to otherwise identical CARs lacking the spacer. In certain embodiments, CARs containing a spacer exhibit increased expression on the cell surface compared to otherwise identical CARs lacking the spacer. In certain embodiments, CARs containing a spacer are polypeptides that, without the spacer, would not be expressed on the cell membrane surface and / or would be unable to bind to their targets due to insufficient proximity or steric hindrance.

[0351] In certain embodiments, the spacer includes a stalk region, for example, a hinge region from an antibody. In some embodiments, the stalk region may be about 20 to about 300 amino acids in length. In some cases, the stalk region may 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, or 60 amino acids or more in length. In other cases, the stalk region may be about 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids in length. In some cases, the stalk region may be less than 20 amino acids in length.

[0352] In some examples, the stalk region includes the hinge region from IgG, e.g., IgG1. In alternative examples, the stalk region includes the CH2CH3 region of the immunoglobulin and, optionally, a portion of CD3.

[0353] In some embodiments, the stalk region includes a CD8α hinge domain, e.g., the sequence of SEQ ID NO: 816; an IgG4-Fc 12-amino acid hinge region, e.g., the sequence of SEQ ID NO: 818; a CD28 hinge domain; a CTLA-4 hinge domain; or a functional or variant thereof.

[0354] In certain embodiments, the stalk region comprises the 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 up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than the amino acid sequence of SEQ ID NO: 816. 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 is a variant of the amino acid sequence of SEQ ID NO: 816 that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity and / or a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 816. In some embodiments, the amino acid sequence of the spacer includes one, two, or three conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 816. In some embodiments, the spacer may include an amino acid sequence that has 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.

[0355] In certain embodiments, the CD8α hinge region, or a functional fragment or variant thereof, is coded by sequence number 817 or its functional fragment or variant. 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 sequence number 817, or hybridizes with a complementary sequence of sequence number 817 under stringent hybridization conditions, or is a codon degenerate variant of sequence number 817.

[0356] In certain embodiments, the stalk region comprises the 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 up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than the amino acid sequence of SEQ ID NO: 818. 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 is a variant of the amino acid sequence of SEQ ID NO: 818 that has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity and / or a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 818. In some embodiments, the amino acid sequence of the spacer includes one, two, or three conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 818. In some such embodiments, the spacer includes an amino acid sequence that has 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.

[0357] In some embodiments, the stalk region may be able to dimerize with the homologous stalk region of the second CAR.

[0358] In certain embodiments, in addition to the stalk region, the spacer may include one or more stalk extension regions. In certain embodiments, the stalk extension region is a polypeptide homologous to the stalk region. For example, it may include at least one amino acid residue substitution compared to the stalk region. In some embodiments, the stalk extension region includes a sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% identity with respect to the stalk region to which it is attached, e.g., the CD8α hinge domain, the CD28 hinge domain, or the CTLA-4 hinge domain.

[0359] In some embodiments, the spacer includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 stalk extension regions.

[0360] In certain embodiments, the stalk region may be connected to the stalk extension region by a linker.

[0361] In certain embodiments, the stalk extension region may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the length of the stalk region when measured by the number of amino acids.

[0362] In some embodiments, the stalk region includes at least one dimerization site. In certain embodiments, the stalk region may include one or more dimerization sites to form a homodimerized or heterodimerized chimeric polypeptide. In other embodiments, the stalk region or one or more stalk extension regions may contain mutations that completely eliminate the dimerization site.

[0363] In certain embodiments, the stalk extension region has at least one fewer dimerization site compared to the stalk region. For example, if the stalk region contains two dimerization sites, the stalk extension region may contain one or zero dimerization sites. As another example, if the stalk region contains one dimerization site, the stalk extension region may contain zero dimerization sites. In some examples, the stalk extension region lacks dimerization sites. In some cases, one or more dimerization sites in the spacer may be located proximal to the membrane (for example, the spacer includes a stalk region containing dimerization sites that is proximal to the membrane, and a stalk extension region that does not contain dimerization sites that is distal to the membrane). In other cases, one or more dimerization sites may be located distal to the membrane (for example, a spacer includes a stalk region containing dimerization sites that is distal to the membrane, and a stalk extension region that does not contain dimerization sites that is proximal to the membrane).

[0364] In certain embodiments, the dimerization site is a cysteine ​​residue capable of forming a disulfide bond. In certain embodiments, the stalk extension region can form fewer disulfide bonds compared to the stalk region. For example, if the stalk region can form two disulfide bonds, the stalk extension region may be capable of forming one disulfide bond or not forming any disulfide bonds at all. As another example, if the stalk region can form one disulfide bond, the stalk extension region may not be capable of forming such a bond.

[0365] Each of the stalk extension regions is approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 amino acids in length, or may be longer.

[0366] 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 up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than the amino acid sequence of SEQ ID NO: 820. 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 includes one, two, or three conservative amino acid modifications to the amino acid sequence of SEQ ID NO: 820. In some such embodiments, the spacer includes an amino acid sequence that has 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.

[0367] In certain embodiments, the stalk extension region is coded by any one of sequence numbers 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 sequence numbers 821-823, or hybridizes with a complementary sequence of any one of sequence numbers 821-823 under stringent hybridization conditions, or is a codon degenerate variant of any one of sequence numbers 821-823.

[0368] In a particular embodiment, the spacer includes a stalk region and one to three stalk extension regions. In a particular such embodiment, the spacer includes a stalk region and two stalk extension regions, for example, a CD8α hinge region and two stalk extension regions, each of which is homologous to the CD8α hinge region.

[0369] In some embodiments, each of the stalk region and stalk extension regions may originate from at least one of the following: the CD8α hinge domain, the CD28 hinge domain, the CTLA-4 hinge domain, the LNGFR extracellular domain, the IgG1 hinge, the IgG4 hinge, and the CH2-CH3 domain. The stalk region and stalk extension regions may individually originate from any combination of the CD8α hinge domain, the CD28 hinge domain, the CTLA-4 hinge domain, the LNGFR extracellular domain, the IgG1 hinge, the IgG4 hinge, or the CH2-CH3 domain. For example, the stalk region may originate from the CD8α hinge domain, and at least one stalk extension region may originate from the CD28 hinge domain, thereby creating a hybrid spacer. As another example, the stalk region may originate from the IgG1 hinge or the IgG4 hinge, and at least one stalk extension region may originate from the CH2-CH3 domain of IgG.

[0370] 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.

[0371] In certain embodiments, the spacer is coded by sequence number 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 sequence number 825, or hybridizes with the complementary sequence of sequence number 825 under stringent hybridization conditions, or is a codon degenerate variant of sequence number 825.

[0372] D. Intracellular signaling domain The intracellular signaling domain of a CAR may be involved in the activation of at least one of the normal effector functions of the immune cell in which the CAR is located. The term “effector function” refers to a specialized function of the cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including cytokine secretion. Usually, the entire intracellular signaling domain can be utilized, but in many cases it is not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain if it transmits an effector function signal. In some embodiments, the intracellular domain further includes a signaling domain for T cell activation.

[0373] In some embodiments, the intracellular signaling domain interacts with T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), or regulatory T cells.

[0374] The intracellular domains are 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, This may include amino acid sequences derived from 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 functional fragments or variants thereof. In some cases, the signaling domain for T cell activation includes a domain derived from CD3ζ, or a functional fragment or variant thereof.

[0375] In certain embodiments, the intracellular signaling domain comprises the 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 up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than SEQ ID NO: 826 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.

[0376] In certain embodiments, the CD3ζ intracellular signaling domain, or a functional fragment or variant thereof, is encoded by a nucleic acid containing 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, or hybridizes with a complementary sequence of SEQ ID NO: 827 under stringent hybridization conditions, or is a codon degenerate variant of SEQ ID NO: 827.

[0377] The intracellular signaling domain may further include one or more co-stimulatory domains. Exemplary co-stimulatory domains include, but are not limited to, the CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), and CD3-zeta co-stimulatory domains, as well as their functional fragments or variants. In some examples, the CARs described herein include one, more, or two or more co-stimulatory domains selected from the CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, and OX40 (CD134) co-stimulatory domains, as well as their functional fragments or variants. In some examples, the CARs described herein include one or more, or more, of the co-stimulatory domains selected from the CD27, CD28, 4-1BB (CD137), ICOS, and OX40 (CD134) co-stimulatory domains, as well as their functional fragments or variants. In some examples, the CARs described herein include one or more, or more, of the co-stimulatory domains selected from the CD8, CD28, 4-1BB (CD137), DAP10, and DAP12 co-stimulatory domains, as well as their functional fragments or variants. In some examples, the CARs described herein include one or more, or more, of the co-stimulatory domains selected from the CD28 and 4-1BB (CD137) co-stimulatory domains, as well as their functional fragments or variants. In some examples, the CARs described herein include the CD28 and 4-1BB (CD137) co-stimulatory domains, or their respective functional fragments or variants. In some examples, the CARs described herein include the CD28 and OX40 (CD134) costimulatory domains, or functional fragments or variants thereof. In some examples, the CARs described herein include the CD8 and CD28 costimulatory domains, or functional fragments or variants thereof. In some examples, the CARs described herein include the CD28 costimulatory domain, or a functional fragment or variant thereof.In some cases, the CARs described herein include the 4-1BB(CD137) costimulatory domain, or a functional fragment or variant thereof. In some cases, the CARs described herein include the OX40(CD134) costimulatory domain, or a functional fragment or variant thereof. In some cases, the CARs described herein include the CD8 costimulatory domain, or a functional fragment or variant thereof. In some cases, the CARs described herein include the DAP10 costimulatory domain, or a functional fragment or variant thereof. In some cases, the CARs described herein include the DAP12 costimulatory domain, or a functional fragment or variant thereof.

[0378] In certain embodiments, the intracellular signaling domain includes a CD28 costimulatory domain, or a functional fragment or variant thereof. In certain such embodiments, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 828, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than the amino acid sequence of SEQ ID NO: 828. 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.

[0379] In certain embodiments, the CD28 costimulatory domain, or a functional fragment or variant thereof, is encoded by a nucleic acid containing sequence number 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 sequence number 829, or hybridizes with a complementary sequence of sequence number 829 under stringent hybridization conditions, or is a codon degenerate variant of sequence number 829.

[0380] In certain embodiments, the intracellular signaling domain comprises a 4-1BB costimulatory 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 up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than the amino acid sequence of SEQ ID NO: 830. 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.

[0381] In certain embodiments, the 4-1BB costimulatory domain, or a 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, or hybridizes with a complementary sequence of SEQ ID NO: 831 under stringent hybridization conditions, or is a codon degenerate variant of SEQ ID NO: 831.

[0382] In certain embodiments, the intracellular signaling domain includes the DAP10 costimulatory domain, or a functional fragment or variant thereof. In certain embodiments, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 832, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than the amino acid sequence of SEQ ID NO: 832. 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.

[0383] In certain embodiments, the DAP10 costimulatory domain, or a 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, or hybridizes with a complementary sequence of SEQ ID NO: 833 under stringent hybridization conditions, or is a codon degenerate variant of SEQ ID NO: 833.

[0384] In certain embodiments, the intracellular signaling domain includes the DAP12 costimulatory domain, or a functional fragment or variant thereof. In certain embodiments, the intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 834, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than the amino acid sequence of SEQ ID NO: 834. 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.

[0385] In certain embodiments, the DAP12 costimulatory domain, or a functional fragment or variant thereof, is encoded by the sequence of SEQ ID NO: 835 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: 835, or hybridizes with a complementary sequence of SEQ ID NO: 835 under stringent hybridization conditions, or is a codon degenerate variant of SEQ ID NO: 835.

[0386] In certain embodiments, the intracellular signaling domain includes both the CD28 costimulatory domain and the 4-1BB costimulatory domain, or functional fragments or variants of each.

[0387] In a particular embodiment, the intracellular signaling domain includes a CD3ζ intracellular signaling domain, for example, the sequence of SEQ ID NO: 826 or a functional fragment or variant thereof, and a CD28 co-stimulatory domain, for example, the sequence of SEQ ID NO: 828 or a functional fragment or variant thereof.

[0388] E. signal peptide In one embodiment, the signal peptide directs the newly synthesized CAR protein to the endoplasmic reticulum. This is, for example, when the receptor is glycosylated and tethered within the cell membrane. Any eukaryotic signal peptide sequence is expected to function. Generally, a signal peptide that naturally attaches to the protein, or to the component closest to the N-terminus in the case of a fusion protein, is used (e.g., V is located closest to the N-terminus). L In the case of scFv having a domain, the native signal of the light chain is used. In some embodiments, the signal peptide is native to GM-CSFRa (SEQ ID NO: 836) or Ig kappa (IgK) (SEQ ID NO: 838), immunoglobulin E (IgE) (SEQ ID NO: 834), or a functional fragment or variant thereof. Other signal peptides that may be used are native to CD8α (SEQ ID NO: 842) and CD28. In some embodiments, the signal peptide is native to mouse IgV H Native to region 3 (SEQ ID NO: 844), β2M signal peptide (SEQ ID NO: 846), azulocidine (SEQ ID NO: 848), human serum albumin signal peptide (SEQ ID NO: 850), A2M receptor-related 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), TVB2 (T21A) signal peptide (SEQ ID NO: 860), CD52 signal peptide (SEQ ID NO: 862), low affinity nerve growth factor receptor (LNGFR, TNFRSF16) signal peptide (SEQ ID NO: 864), or their functional fragments or variants.

[0389] In certain embodiments, the CAR is ligated 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 embodiments, the functional fragment is up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than SEQ ID NO: 836 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.

[0390] In certain embodiments, the GM-CSFRa signal peptide, or a functional fragment or variant thereof, is encoded by a nucleic acid containing 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, or hybridizes with a complementary sequence of SEQ ID NO: 837 under stringent hybridization conditions, or is a codon degenerate variant of SEQ ID NO: 837.

[0391] F. Exemplary CD19 CAR structure Simply as an example, though not limited to, CD19 CAR is (a) signal peptide; (b) V H Domain and / or V L The domain may include (b) a transmembrane domain and (c) an intracellular signaling domain. In certain embodiments, the CD19 CAR further includes a spacer domain.

[0392] 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 up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than SEQ ID NO: 836 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.

[0393] In a particular embodiment, the CD19-specific CAR construct is V H Domain and / or V L Includes domains. In some embodiments, the CAR construct is V H Domain and V L Includes both domains. In a further embodiment, the CAR construct is V H Domain, linker, and V L The domain scFv structure is included. In some embodiments, the scFv includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 959, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than SEQ ID NO: 959 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.

[0394] In certain embodiments, the CD19-specific CAR includes a CD8α transmembrane domain, or a functional fragment or variant thereof. In some embodiments, the CD8α transmembrane domain, or a functional fragment or variant thereof, includes an amino acid sequence, or a functional fragment or variant thereof, having at least 90% sequence identity with SEQ ID NO: 812. In certain embodiments, the functional fragment is up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than SEQ ID NO: 812 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.

[0395] In certain embodiments, the spacer comprises a CD8α hinge domain, or a functional fragment or variant thereof. In some embodiments, the CD8α hinge domain, or a functional fragment or variant thereof, comprises an amino acid sequence, or a functional fragment or variant thereof, having at least 90% sequence identity with SEQ ID NO: 816. In certain embodiments, the functional fragment is up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than SEQ ID NO: 816 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 includes a CD3ζ signaling domain, or a functional fragment or variant thereof. In some embodiments, the CD3ζ signaling domain, or a functional fragment or variant thereof, includes an amino acid sequence, or a functional fragment or variant thereof, having at least 90% sequence identity with SEQ ID NO: 826. In certain embodiments, the functional fragment is up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than SEQ ID NO: 826 at the N and / or C-terminus. In certain embodiments, the functional variant is a conservatively substituted variant of SEQ ID NO: 826, 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: 826.

[0396] In certain embodiments, the intracellular signaling domain further comprises a CD28 co-stimulatory signaling domain, or a functional fragment or variant thereof. In some embodiments, the CD28 co-stimulatory signaling domain, or a functional fragment or variant thereof, comprises an amino acid sequence, or a functional fragment or variant thereof, having at least 90% sequence identity with SEQ ID NO: 828. In certain embodiments, the functional fragment is up to 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than SEQ ID NO: 828 at the N and / or C-terminus. In certain embodiments, the functional variant is a conservatively substituted variant of SEQ ID NO: 828, 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: 828.

[0397] In a particular embodiment, the CD19-specific CAR construct is encoded by a nucleic acid or a functional variant of sequence number 939 having at least 90% sequence identity (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 sequence number 939, or a codon degenerate variant of sequence number 939).

[0398] In some embodiments, the CD19-specific CAR contains an amino acid sequence that is 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 contains an amino acid sequence that is 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 contains an amino acid sequence that is 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 contains an amino acid sequence that is 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 contains an amino acid sequence that is 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, CAR includes an amino acid sequence that is 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, CAR includes an amino acid sequence that is 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, CAR includes an amino acid sequence that is 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 contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 990.In some embodiments, the CAR contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 991. In some embodiments, the CAR contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to the amino acid sequence of sequence number 992.

[0399] In some embodiments, the CAR is encoded by a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 99.99%, or 100% identical to the polynucleotide sequence of sequence number 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, or 1004. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 993. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 994. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 995. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 996. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 997.In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 998. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 999. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 1000. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 1001. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 1002. In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 1003.In some embodiments, the CAR is encoded by a polynucleotide sequence that is 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 sequence number 1004.

[0400] In some embodiments, CAR includes the amino acid sequence of CAR CTL019. In some embodiments, CAR is CAR CTL019. In some embodiments, CAR includes the amino acid sequence of CAR expressed by CAR T cell tisagenecleucel. In some embodiments, CAR is CAR expressed by CAR T cell tisagenecleucel. In some embodiments, CAR includes the amino acid sequence of CAR expressed by CAR T cell KYMRIAH®. In some embodiments, CAR is CAR expressed by CAR T cell KYMRIAH®. In some embodiments, CAR includes the amino acid sequence of CAR KTE-C19. In some embodiments, CAR is CAR KTE-C19. In some embodiments, CAR includes the amino acid sequence of CAR expressed by CAR T cell axicapbutagen silolucel. In some embodiments, CAR is CAR expressed by CAR T cell axicapbutagen silolucel. In some embodiments, CAR includes the amino acid sequence of CAR expressed by CAR T cell YESCARTA®. In some embodiments, the CAR is a CAR expressed by CAR T cells called YESCARTA®.

[0401] CAR and CAR construction and composition are also described in the following literature, for example: ● 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. Vertes, 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); ● International Publication No. 2020 / 209934 (PCT / US2020 / 017794) - Novel chimeric antigen receptors and libraries (MIT); ● International Publication No. 2020 / 037142 (PCT / US2019 / 046691) - Compositions and methods for high-throughput activation screening to boost t-cell effector function (Yale); ● International Publication No. 2015 / 123642 (PCT / US2015 / 016057) - Chimeric antigen receptors and methods of making (Univ.TX); ● International Publication No. 2019 / 079486 (PCT / US2018 / 056334) - Polypeptide compositions comprising spacers (Precigen) ● International Publication No. 2017 / 214333 (PCT / US2017 / 036440) - Cd33 specific chimeric antigen receptors (Precigen) ● International Publication No. 2016 / 126608 (PCT / US2016 / 015978) - Car-expressing cells against multiple tumor antigens and uses thereof ● U.S. Patent Application Publication No. 2020 / 0377589 - Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy ● U.S. Patent No. 10,800,840 - Compositions and methods for generating a persistent population of T cells useful for the treatment of cancer ● Chinese Patent No. 109400713 - Use of novel chimeric antigen receptor modified T cells for the treatment of cancer ● U.S. Patent Application No. 18 / 259,985 - Recombinant Vectors Comprising Polycistronic Expression Cassettes and Methods of Use Thereof

[0402] Each of the above documents is a U.S. Patent No. 89006682, International Publication No. 2019 / 213282, U.S. Patent Application Publication No. 20200268860, International Publication No. 2020 / 227177, U.S. Patent No. 10457730, International Publication No. 2019 / 159193, U.S. Patent No. 10287350, U.S. Patent No. 10221245, U.S. Patent Application Publication No. 20190125799, International Publication No. 2018 / 201794, U.S. Patent Application Publication No. 20170368098, U.S. Patent Application Publication No. 20160145337, U.S. Patent No. 9701758, International Publication No. 2014 / 153270, International Publication No. 2012 / 079000, International Publication No. 2019 / 160956, International Publication No. 2019 / 161796, International Publication No. 2020 / 222176, International Publication No. 2020 / 219848, U.S. Patent Application Publication No. 20190135894, United States Japanese Patent No. 10774388, International Publication No. 2020 / 180882, U.S. Patent No. 10765701, International Publication No. 2020 / 172641, International Publication No. 2020 / 172440, International Publication No. 2016 / 149578, International Publication No. 2020 / 124021, International Publication No. 2020 / 108646, International Publication No. 2020 / 108643, International Publication No. 2020 / 113188, International Publication No. 2020 / 108644, International Publication No. 2 Together with 020 / 108645, International Publication No. 2020 / 108642, U.S. Patent No. 10669549, International Publication No. 2020 / 102770, U.S. Patent No. 10501539, International Publication No. 2020 / 069409, U.S. Patent No. 10603380, U.S. Patent No. 10533055, International Publication No. 2020 / 010235, and International Publication No. 2019 / 246546, these are incorporated herein by reference. Accordingly, any one of the CARs described in each of the above documents, in particular any one of the CD19-specific CARs disclosed therein, may include the expression cassettes described herein.

[0403] Cytokine In some embodiments, the modified immune effector cells of the present invention may contain cytokines. Cytokines are encoded, for example, by polynucleotides of the present disclosure. For example, polynucleotides may encode miRNA, CAR and cytokine; miRNA and cytokine; or CAR and cytokine.

[0404] In some cases, the cytokines include at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or a variant or combination thereof. In certain embodiments, the cytokines are interferon, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factor, hGH, and / or ligands for human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, IFN-alpha, IFN-beta, or IFN-gamma.

[0405] For certain uses, cytokines are interleukins. In some cases, interleukins are 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 functional variants or fragments thereof.

[0406] In certain embodiments, the cytokine may be IL-12, or a functional fragment or variant thereof. In some embodiments, IL-12 may be single-stranded IL-12 (scIL-12), protease-sensitive IL-12, destabilized IL-12, membrane-bound IL-12, or intercalated IL-12. In some examples, IL-12 variants are as described in International Publications 2015 / 095249, 2016 / 048903, and 2017 / 062953.

[0407] In certain embodiments, the cytokine may be IL-15, or a functional fragment or variant thereof. In certain embodiments, IL-15, or a functional fragment or variant thereof, may be membrane-bound. Such a form may exist 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 include 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").

[0408] In certain embodiments, IL-15, or a functional fragment or variant thereof, comprises the amino acid sequence of SEQ ID NO: 519, or a functional fragment or variant thereof. In certain embodiments, the functional fragment is up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter at the N and / or C-terminus than the amino acid sequence of SEQ ID NO: 519. 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.

[0409] In certain embodiments, IL-15, or a functional fragment or variant thereof, is encoded by a nucleic acid or a functional fragment or v...

Claims

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

2. The polynucleotide according to 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 according to claim 1, wherein the immune checkpoint protein is PD-1.

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

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

6. The polynucleotide according to claim 4, wherein the first miRNA is encoded by a nucleic acid containing sequence number 72.

7. The polynucleotide according to claim 4, wherein the first miRNA is encoded by a nucleic acid having at least 90% identity with sequence number 348.

8. The polynucleotide according to claim 4, wherein the first miRNA is encoded by a nucleic acid having at least 90% identity with sequence number 179.

9. The polynucleotide according to claim 4, wherein the first miRNA is encoded by a nucleic acid containing sequence number 74.

10. The polynucleotide according to claim 4, wherein the first miRNA is encoded by a nucleic acid having at least 90% identity with sequence number 349.

11. The polynucleotide according to claim 4, wherein the first miRNA is encoded by a nucleic acid having at least 90% identity with sequence number 180.

12. The polynucleotide according to claim 4, wherein the first and second miRNAs are encoded by nucleic acids having at least 90% identity with SEQ ID NO:

267.

13. The CD19-specific CAR is (a) (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) an antigen-binding domain comprising 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) Transmembrane domains; and (c) Intracellular signal transduction domain The polynucleotide according to claim 1, comprising:

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

15. (i) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 948 or a sequence that differs therefrom by up to two conservative amino acid substitutions, the amino acid sequence of SEQ ID NO: 949 or a sequence that differs therefrom by up to two conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 950 or a sequence that differs therefrom by up to two 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 two conservative amino acid substitutions, the amino acid sequence of SEQ ID NO: 955 or a sequence that differs therefrom by up to two conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 956 or a sequence that differs therefrom by up to two conservative amino acid substitutions, according to claim 1.

16. (i) The first polypeptide comprises the amino acid sequence of SEQ ID NO: 948 or a sequence different therefrom by a single amino acid substitution, the amino acid sequence of SEQ ID NO: 949 or a sequence different therefrom by a single amino acid substitution, and the amino acid sequence of SEQ ID NO: 950 or a sequence different 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 different therefrom by a single amino acid substitution, the amino acid sequence of SEQ ID NO: 955 or a sequence different therefrom by a single amino acid substitution, and the amino acid sequence of SEQ ID NO: 956 or a sequence different therefrom by a single amino acid substitution, according to claim 1.

17. (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, according to claim 1.

18. (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, according to claim 1.

19. (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, according to claim 1.

20. (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, according to claim 1.

21. (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, according to claim 1.

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

24. (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, according to claim 1.

25. The polynucleotide according to 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 according to 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 according to 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 according to 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 according to claim 1, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 959 or differs from it by up to five conservative amino acid substitutions.

30. The polynucleotide according to claim 1, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 959 or differs from it by up to two conservative amino acid substitutions.

31. The polynucleotide according to claim 1, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 959 or differs from it by a single amino acid substitution.

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

959.

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

812.

35. The polynucleotide according to 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 according to claim 1, wherein the intracellular signaling domain includes a co-stimulatory domain.

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

816.

39. The polynucleotide according to 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 according to 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 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 according to 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 according to 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 according to claim 1, wherein the CAR comprises (a) an antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 959 or a sequence thereof differing from it by up to five conservative amino acid substitutions; (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 812 or a sequence thereof differing from it by up to five conservative amino acid substitutions; (c) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 826 or a sequence thereof differing from it by up to five conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 828 or a sequence thereof differing from it by up to five conservative amino acid substitutions; and (d) a stalk region comprising the amino acid sequence of SEQ ID NO: 816 or a sequence thereof differing from it by up to five conservative amino acid substitutions.

44. The polynucleotide according to claim 1, wherein the CAR comprises (a) an antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 959 or a sequence thereof differing from it by up to two conservative amino acid substitutions; (b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 812 or a sequence thereof differing from it by up to two conservative amino acid substitutions; (c) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 826 or a sequence thereof differing from it by up to two conservative amino acid substitutions, and the amino acid sequence of SEQ ID NO: 828 or a sequence thereof differing from it by up to two conservative amino acid substitutions; and (d) a stalk region comprising the amino acid sequence of SEQ ID NO: 816 or a sequence thereof differing from it by up to two conservative amino acid substitutions.

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

46. The polynucleotide according to 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 according to claim 46, wherein the first miRNA is encoded by a nucleic acid including sequence number 72, and the polynucleotide further encodes a second miRNA encoded by a nucleic acid including sequence number 74.

48. The polynucleotide according to claim 1, further encoding a cell tag.

49. The polynucleotide according to claim 48, wherein the cell tag comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

571.

50. The polynucleotide according to claim 1, further encoding a cytokine.

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

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

523.

54. The polynucleotide according to claim 1, comprising a promoter.

55. The polynucleotide according to claim 54, wherein the promoter is the EF1a promoter.

56. A vector comprising a polynucleotide according to any one of claims 1 to 55.

57. The vector according to claim 56, which is a plasmid, a viral vector, or a nonviral vector.

58. The vector according to claim 56, which is a non-viral vector.

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

60. The vector according to claim 56, wherein the polynucleotide is sandwiched between a left transposon repeat sequence region and a right transposon repeat sequence region.

61. The vector according to claim 60, wherein the left transposon repeat sequence region contains the nucleic acid of sequence number 580.

62. The vector according to claim 60, wherein the right-side transposon repeat sequence region contains the nucleic acid of sequence number 581.

63. A system for use in expressing CAR in cells, comprising the vector according to claim 59 and a transposase, or a vector encoding the transposase.

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

65. The system according to claim 63, wherein the transposase is SB11 or SB100x transposase.

66. Engineered immunoeffector cells comprising the polynucleotide described in any one of claims 1 to 55.

67. The manipulated immune effector cell according to claim 66, which is a T cell or an NK cell.

68. Engineered immune effector cells comprising (a) first and second miRNAs that inhibit the expression of immune checkpoint proteins, and (b) a CD19-specific CAR.

69. A method for producing manipulated immune effector cells according to claim 66, comprising the step of introducing a vector containing the polynucleotide into immune effector cells.

70. A composition comprising the polynucleotide described in any one of claims 1 to 55.

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

72. A composition comprising the manipulated immunoeffector cells described in claim 66.

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

74. A kit comprising the polynucleotide described in any one of claims 1 to 55.

75. A kit comprising the manipulated immunoeffector cells described in claim 66.

76. A method for treating a disease or disorder, comprising the step of administering the manipulated immunoeffector cells described in claim 66 to a subject in need thereof.

77. The method according to claim 76, wherein the disease or disorder is related to the overexpression of CD19.

78. The method according to claim 76, wherein the disease or disorder is cancer.

79. The method according to claim 78, wherein the cancer is a hematological tumor.

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

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

82. The method according to 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 mellitus, inflammatory bowel disease, psoriasis, and autoimmune thyroiditis.

83. The method according to claim 76, further comprising administering an additional therapy.

84. The method according to claim 76, wherein the manipulated immune effector cells are neither proliferated nor activated before administration to the subject.

85. The method according to claim 76, further comprising a weight loss procedure.

86. Use of the manipulated immunoeffector cells according to claim 66 in the manufacture of a pharmaceutical product for the treatment of a disease or disorder.

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

88. The use according to 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 mellitus, inflammatory bowel disease, psoriasis, and autoimmune thyroiditis.

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