Modulating expression of polypeptides via new gene switch expression systems

Abstract

To provide a composition for ligand-inducible control of heterologous gene expression.SOLUTION: A composition comprises a polynucleotide encoding gene switch polypeptides for ligand-inducible control of heterologous gene expression, wherein the gene switch polypeptides comprise: a) a first gene switch polypeptide comprising a DNA binding domain fused to a first nuclear receptor ligand binding domain, and b) a second gene switch polypeptide comprising a transactivation domain fused to a second nuclear receptor ligand binding domain, wherein the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is incorporated herein by reference in its entirety. U.S. Provisional Patent Application No. 62 / 444,775, filed on the 10th, and This application claims the benefit of U.S. Provisional Patent Application No. 62 / 464,958, filed February 28, 2019. do.

[0003] Reference to sequence listing This application has been filed electronically in ASCII format and is incorporated by reference in its entirety. Contains a sequence listing incorporated herein by reference in its entirety. The ASCII copy is named 50471_706_601_SL.txt and is located at 42 It is 1,163 bytes in size.

[0004] Incorporation by Reference All publications, patents, and patent applications mentioned herein are to be construed as separate entities each individually identified as such. Where any patent or patent application is specifically and individually indicated to be incorporated by reference, to the same extent as if each of the preceding claims were incorporated by reference herein. [Background technology]

[0005] Adoptive T cell immunization using chimeric antigen receptors (CARs) and T cell receptors (TCRs) The therapy has been shown to be successful in directly killing tumor cells. This innovative technology Although promising, administration of engineered T cells to tumor-bearing individuals raises safety concerns, e.g., tumor lysis. In addition, CAR or TCR monotherapy has not been performed without cytokine release syndrome. The expression of IL-2, IL-12, and IL-14 may not be sufficient for efficacy. Further expression of cytokines, such as IL-15 or IL-21, may also contribute to the efficacy of such treatment. However, this may pose additional safety issues. Therefore, after administration to patients, complete control of the expression of the therapeutic gene of interest is achieved. There is an interest in obtaining control. Summary of the Invention

[0006] In certain embodiments herein, systems and methods include gene switch polypeptides. or compositions, and polynucleotides encoding the same are disclosed.

[0007] Provided herein are gene switch polypeptides for ligand-induced control of heterologous gene expression. a composition comprising a polynucleotide encoding a gene switch polypeptide, (a) a DNA-binding peptide fused to a first nuclear receptor ligand-binding domain; (b) a first gene switch polypeptide comprising a second nuclear receptor linker domain; and A second gene switch containing a transactivation domain fused to a nucleic acid binding domain. a gene switch polypeptide, and a switch polypeptide linked by a linker.

[0008] Herein, we describe a gene switch system for ligand-induced control of heterologous gene expression. a composition comprising one or more polynucleotides that encode the gene switch system; (a) a first gene switch polypeptide comprising a transactivation domain; (b) A second gene switch containing a DNA-binding domain fused to a ligand-binding domain. and (c) at least one heterologous polypeptide, a first gene switch polypeptide, said second gene switch polypeptide, and One of the heterologous gene polypeptides is connected to the first gene by a polypeptide linker. a gene switch polypeptide, the second gene switch polypeptide, and the heterologous gene and a polypeptide linker linked to another one of the gene polypeptides, the polypeptide linker comprising: Compositions are presented that include cleavable linker or ribosomal skipping linker sequences. .

[0009] In some embodiments, the DNA-binding domain of any of the compositions presented herein The main components are GAL4 (GAL4 DBD), LexADBD, transcription factor DBD, and steroid Thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DB D, and at least one of the yeast DBDs. The DNA binding domain of any of the presented compositions may be selected from the group consisting of: In some embodiments, any of the compositions presented herein has a sequence The transactivation domain is a VP16 transactivation domain and a B42 acidic activator. In some embodiments, the protein comprises at least one of the transactivation domains described herein. The transactivation domain of any of the compositions presented herein may be selected from the group consisting of SEQ ID NO: 181. It has the sequence shown.

[0010] Optionally, the first nuclear receptor linker of any of the compositions presented herein. Ligand-binding domain, second nuclear receptor ligand-binding domain, and ligand binding At least one of the sex domains is involved in the ecdysone receptor (EcR), a ubiquitous receptor , orphan receptor 1, NER-1, steroid hormone nuclear receptor 1, retinoid X receptor receptor-interacting protein 15, liver X receptor β, steroid hormone receptor-like protein Protein, liver X receptor, liver X receptor α, farnesoid X receptor, receptor-interacting protein In other cases, the protein 14 and / or the farnesol receptor are included. The first nuclear receptor ligand binding domain of any of the compositions presented herein In, the second nuclear receptor ligand-binding domain, and the ligand-binding domain At least one of them has a sequence shown in any one of SEQ ID NOs: 185 to 186. In another embodiment, the first gene switch polypeptide is an EcR nuclear receptor ligand. the second gene switch polypeptide comprising a GAL4 DBD fused to a binding domain; The peptide is fused to the retinoid receptor X (RXR) nuclear receptor ligand-binding domain. In yet another case, the EcR nuclear receptor contains the VP16 transactivation domain. The Gal4 DBD fused to the ligand binding domain is shown in SEQ ID NOs: 185-186. or 187 to 188, and VP16 transactivation domain fused to the nuclear receptor (RXR) ligand-binding domain The gene has the sequence shown in SEQ ID NO:183.

[0011] In some cases, the linker of any of the compositions presented herein may be a cleavable linker. linker, ribosome skipping linker sequence, or IRES linker. Therefore, the linker is an IRES linker and is any one of SEQ ID NOs: 18 to 19. In other cases, the linker is a cleavable linker or a ribosomal linker. In some embodiments, the linker is a cleavable linker or ribosomal skipping linker sequence. The skipping linker sequences are 2A linker, p2A linker, T2A linker, F 2A linker, E2A linker, GSG-2A linker, GSG linker, SGSG linker ker, furinlink linker, and variants thereof and In other embodiments, the cleavable linker or the ribonucleotide may comprise one or more of the following derivatives: The enzyme skipping linker sequence is represented by any one of SEQ ID NOs: 146 to 162. The sequence is as follows:

[0012] In certain embodiments, the polynucleotide of any of the compositions presented herein The polynucleotide or polynucleotides further encode an antigen-binding polypeptide. In another embodiment, the antigen-binding polypeptide of any of the compositions presented herein The peptide binds to at least one of a chimeric antigen receptor (CAR) and a T cell receptor. In other embodiments, the antigen-binding polypeptide comprises a CAR, wherein the CAR comprises: CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, RO R1, CEA, EGP-2, EGP-40, HER2, HER3, folate-binding protein , GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-A1, h5T4, PSMA, TAG- 72, EGFRvIII, CD123, and VEGF-R2 In another embodiment, the antigen-binding polypeptide comprises a CAR. The CAR has a sequence shown in any one of SEQ ID NOs: 210 to 244.

[0013] In other embodiments, the polynucleotide of any of the compositions presented herein is The one or more polynucleotides further encode a cell tag. The cell tag is at least one of a truncated HER1 mutant and a truncated CD20 mutant. In other cases, the cell tag comprises any one of SEQ ID NOS: 189-202. It has the sequence shown in

[0014] In some embodiments, the first gene of any of the compositions presented herein a gene switch polypeptide, a second gene switch polypeptide, an antigen-binding polypeptide, and the expression of at least one of the cell tags is modulated by a promoter. The promoter may be a tissue-specific promoter or an EF1A promoter, or Optionally, the promoter is a functional variant of any of SEQ ID NOS: 58-60. EF1A promoter having a sequence shown in any one of In other cases, the promoter is a tissue-specific promoter containing a T cell-specific response element. In other cases, the tissue-specific promoter is a heterologous promoter. In yet another alternative, the NFAT response element comprises SEQ ID NO: It has a sequence shown in any one of Nos. 51 to 57.

[0015] In some examples, any of the compositions presented herein may contain a heterologous gene polypeptide. Optionally, the heterologous gene may further comprise a second polynucleotide encoding a heterologous gene. The peptides are a few of the following: cytokines, cell tags, and chimeric antigen receptors (CARs). Optionally, the cytokine comprises one cytokine, Cytokines are IL-1, IL-2, IL-15, IL-12, IL-21, IL-1 5, IL-15Rα, or a fusion of an IL-15 variant. In other cases, the cytokine is in a secreted form. In other cases, the cytokine is membrane-bound. In yet another embodiment, the cytokine is in a form selected from the group consisting of SEQ ID NOS: 203-209. It has the sequence shown in any one of the above.

[0016] In some embodiments, at least one of any of the compositions presented herein The expression of the heterologous gene polypeptide is modulated by an inducible promoter. In some embodiments, the inducible promoter is any of SEQ ID NOs: 40-64. In other embodiments, the inducible promoter has a sequence shown in a gene switch polypeptide, and at least one of a second gene switch polypeptide It's more modulated.

[0017] Also provided herein are polynucleotides of any of the compositions presented herein. Also provided is a vector comprising: In another embodiment, the vector is a non-viral vector. The virus vector is the Sleeping Beauty transposon.

[0018] Provided herein is a method for modulating expression of a heterologous gene in an effector cell, comprising: ) binding to said effector cells (i) DNA fused to a ligand-binding domain (ii) a first gene switch polypeptide comprising a transactivation domain; (iii) a second gene switch polypeptide encoded by the heterologous gene, and (iv) a cleavable linker sequence or ribosome. One or more polypeptides encoding a polypeptide linker containing a skipping linker sequence. introducing a polynucleotide, wherein the polypeptide linker is a gene switch polypeptide, said second gene switch polypeptide, and said heterologous one of the gene switch polypeptides, the first gene switch polypeptide, the second gene switch polypeptide, and another one of said heterologous gene polypeptides. (b) inducing expression of the heterologous gene in the effector cells. and contacting the antibody with a sufficient amount of ligand to

[0019] In some instances, the expression of heterologous genes in the effector cells provided herein may be regulated by wherein at least one of the one or more polynucleotides is an antigen-binding polynucleotide. Optionally, the antigen-binding polypeptide further encodes a target peptide. Optionally, the target cell is a mammalian cell. In other cases, the target cell is a tumor cell.

[0020] In some embodiments, the expression of heterologous genes in the effector cells provided herein In the method of modulating tumor growth, the predetermined cell surface protein is a tumor antigen. Prior to contacting the vector cells with the ligand, the antigen-binding polypeptide is selectively binds to a predetermined cell surface protein of a target cell. The cells may be incubated with the effector for at least 7 days prior to contacting the cells with the ligand. In other cases, the target cell is exposed to a specific cell surface protein of the target cell. Binding of the synthetic polypeptide to a selected cell surface protein activates effector cells. In some embodiments, the method further comprises activating the effector cells to the predetermined cell surface. The method further includes a step of co-culturing the cells with artificial antigen-presenting cells (aAPCs) that express the surface protein. and binding the antigen-binding polypeptide to the predetermined cell surface protein of the aAPC. Binding of the effector cells activates the effector cells. The trip may last for at least 7, 14, 21, or 28 days. aAPCs are transgenic K562 cells.

[0021] In other embodiments, expression of a heterologous gene in the effector cells provided herein is The antigen-binding polypeptides in the modulating method include chimeric antigen receptors (CARs) and T In another embodiment, the antigen-binding polypeptide comprises at least one of a cellular receptor. comprises a CAR, wherein the CAR is a fusion protein selected from the group consisting of CD19, CD33, BCMA, CD44, alpha-folate Receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2 , HER3, folate binding protein, GD2, GD3, IL-13R-α2, KDR, E DB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1, MUC-1 6, h5T4, PSMA, TAG-72, EGFRvIII, CD123, and VEG In yet another embodiment, the nucleotide sequence of ... The antigen-binding polypeptide is represented by any one of SEQ ID NOs: 210 to 244. In some embodiments, the heterologous polypeptide comprises a CAR having an antigen-binding sequence. It includes a cyclic polypeptide.

[0022] Optionally, modulate the expression of heterologous genes in the effector cells provided herein. In the method, at least one of the one or more polynucleotides is a cell type. Optionally, the cell tag further encodes a HER1 truncation mutant and a CD2 In other cases, the cell tag comprises at least one of SEQ ID NO: 1. 89 to 202. The polypeptide comprises a cell tag.

[0023] In other cases, the expression of heterologous genes in the effector cells provided herein is modulated. In the method, at least one of the one or more polynucleotides comprises a site Optionally, the cytokine further encodes 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 ... -15, IL-12, IL-21, IL-15, IL-15Rα, or IL-15 mutation In one embodiment, the cytokine comprises at least one of a secreted form and a fusion of a cytokine with a cytosine or a cytosine. In another embodiment, the cytokine is in a membrane-bound form. In the present invention, the cytokine has a sequence shown in any one of SEQ ID NOs: 203 to 209. In another embodiment, the heterologous polypeptide comprises a cytokine.

[0024] In some embodiments, expression of a heterologous gene in the effector cells provided herein can be achieved by In a regulated manner, expression of the heterologous gene polypeptide is controlled by an inducible promoter. In certain embodiments, the inducible promoter is selected from the group consisting of SEQ ID NOs: 40-6. 4.

[0025] In another embodiment, the expression of a heterologous gene in the effector cells provided herein is The DNA binding domain in the regulating method is GAL4 (GAL4 DBD), Lex ADBD, transcription factor DBD, steroid / thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DBD, and yeast DBD. In yet another embodiment, the DNA binding domain has the sequence set forth in SEQ ID NO: 184. It has.

[0026] In yet another embodiment, the expression of a heterologous gene in an effector cell as provided herein is The transactivation domain in the method for regulating expression is the VP16 transactivation domain. and at least one of the B42 acidic activator transactivation domains. Optionally, the transactivation domain has the sequence set forth in SEQ ID NO:181. In another embodiment, the first gene switch polypeptide and the second gene switch At least one of the polypeptides is capable of binding to the DNA binding domain. The nucleic acid further comprises a responsive element capable of binding to a target gene.

[0027] In some embodiments, the expression of heterologous genes in the effector cells provided herein The ligand binding domain in the method for modulating the ecdysone receptor (EcR), ubiquitin cytoplasmic receptor, orphan receptor 1, NER-1, steroid hormone nuclear receptor 1, Liver X receptor-interacting protein 15, liver X receptor β, steroid hormone receptor liver X receptor-like protein, liver X receptor α, farnesoid X receptor, receptor interaction The receptors include at least one of the following: agonist protein 14, farnesol receptor, and farnesol receptor. In certain embodiments, the ligand binding domain is selected from the group consisting of SEQ ID NOs: 185-186. In certain embodiments, the first gene switch promoter has a sequence as set forth in any one of The peptide was a GAL4 DB fused to the EcR nuclear receptor ligand binding domain. D, and the second gene switch polypeptide is a retinoid receptor X (RXR) nuclear receptor. It contains the VP16 transactivation domain fused to a receptor ligand binding domain. In some embodiments, the Gal4 DBD fused to EcR is SEQ ID NOs: 185-186 and a retinoid receptor X (RXR) nuclear receptor having a sequence shown in any one of The VP16 transactivation domain fused to a ligand binding domain is shown in SEQ ID NO: It has the sequence shown in No. 183.

[0028] In certain embodiments, the expression of a heterologous gene in an effector cell as provided herein The ligand in the method for modulating expression is (2S, 3R, 5R, 9R, 10R, 13R, 14S,17R)-17-[(2S,3R)-3,6-dihydroxy-6-methylhepta 2,3,14-trihydroxy-10,13-dimethyl-2,3,4, 5,9,11,12,15,16,17-Decahydro-1H-cyclopenta[a]phena N'-(3,5-dimethylbenzoyl)-N'-[(3R)-2, 2-Dimethyl-3-hexanyl]-2-ethyl-3-methoxybenzohydrazide;5-Me 2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5 -dimethyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydroxide 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2 -dimethyl-propyl)-hydrazide;5-methyl-2,3-dihydro-benzo[1,4 ]dioxine-6-carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3, 5-Dimethyl-benzoyl)-hydrazide; 5-Methyl-2,3-dihydro-benzo[1 ,4]dioxine-6-carboxylic acid N'-(1-tert-butyl-butyl)-N'-( 3,5-Dimethoxy-4-methyl-benzoyl)-hydrazide; 5-Ethyl-2,3-di Hydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethyl-benzoyl) 5-Ethyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide 2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5- Dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propanol Pyr)-hydrazide; 5-ethyl-2,3-dihydro-benzo[1,4]dioxin-6 -carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethyl- 5-Ethyl-2,3-dihydro-benzo[1,4]dioxin N'-(1-tert-butyl-butyl)-N'-(3,5-dimethoxy-6-carboxylic acid) 3,5-Dimethylbenzoic acid N-(1-ethoxybenzoyl)hydrazide ethyl-2,2-dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl )-hydrazide;3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2 -dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazine 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3- Methoxy-2-methyl-benzoyl)-hydrazide;3,5-Dimethoxy-4-methyl- Benzoic acid N-(1-tert-butyl-butyl)-N'-(3-methoxy-2-methyl- benzoyl)-hydrazide;3,5-dimethyl-benzoic acid N-(1-ethyl-2,2-di Methyl-propyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propionyl) (2-ethyl-3-methoxybenzoyl)hydrazide;3,5-dimethyl N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methylbenzoate 3,5-Dimethoxy-4-methyl-benzoic acid N-(1 -tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)- 2-Methoxy-nicotinic acid N-(1-tert-butyl-pentyl)-N'- (4-Ethyl-benzoyl)-hydrazide; 3,5-dimethyl-benzoic acid N-(2,2- Dimethyl-1-phenyl-propyl)-N'-(4-ethyl-benzoyl)-hydrazide ;3,5-Dimethyl-benzoic acid N-(1-tert-butyl-pentyl)-N'-(3- Methoxy-2-methyl-benzoyl)-hydrazide; and 3,5-dimethoxy-4-methyl- N-(1-tert-butyl-pentyl)-N'-(3-methoxy-2-methylbenzoate methyl-benzoyl)-hydrazide.

[0029] Optionally, modulate the expression of heterologous genes in the effector cells provided herein. In the method, expression of the heterologous gene is induced in the absence of a ligand and in the presence of a ligand. In certain cases, the expression of the variant is reduced or eliminated compared to the expression in the variant. Expression of the seed gene is restored by administering additional amounts of the ligand.

[0030] In other cases, the first gene switch polypeptide and the second gene switch polypeptide Expression of at least one of the peptides in the effector cells provided herein A method for regulating the expression of a heterologous gene, comprising: The promoter may be a tissue-specific promoter or the EF1A promoter, or In certain cases, the promoter is a functional variant of any of SEQ ID NOS: 58-60. EF1A promoter having a sequence shown in any one of the above, or a functional variant thereof In other cases, the promoter is a tissue-specific promoter. In other cases, tissue-specific promoters contain T cell-specific response elements. The motor comprises one or more NFAT response elements. The AT response element has a sequence shown in any one of SEQ ID NOs: 50 to 57. do.

[0031] In certain cases, expression of heterologous genes in the effector cells provided herein In the method for regulating the expression of a gene, one or more polynucleotides are contained within a vector. Thus, the vector may be a lentiviral vector, a retroviral vector, or a non-viral vector. In certain cases, non-viral vectors are Sleeping Beauty transposon.

[0032] As used herein, ligand-inducible expression of heterologous genes comprising one or more expression cassettes is a gene switch system for regulating the expression of a gene of interest, the one or more expression cassettes comprising: (a) a a nuclear receptor ligand-binding domain of a first fusion protein comprising a DNA-binding domain fused to the nuclear receptor ligand-binding domain of a first fusion protein; (b) a sequence encoding a second nuclear receptor ligase; A second gene switch containing a transactivation domain fused to a nucleotide-binding domain. a sequence encoding a first gene switch polypeptide; and the second gene switch polypeptide are linked by a polypeptide linker. , a gene switch system is presented.

[0033] As used herein, ligand-inducible expression of heterologous genes comprising one or more expression cassettes is a gene switch system for regulating the expression of a gene, the one or more expression cassettes comprising: (a) a a sequence encoding a first gene switch polypeptide comprising a transactivation domain; b) a second gene comprising a DNA-binding domain fused to a ligand-binding domain (c) a sequence encoding a heterologous gene polypeptide; the transactivation domain fused to the ligand binding domain, comprising a sequence wherein one of the polypeptide, the DNA binding domain, and the heterologous gene polypeptide is a polypeptide the transactivator fused to the ligand-binding domain by a peptide linker. a DNA-binding domain, and another one of said heterologous gene polypeptides. and wherein the polypeptide linker is linked to a cleavable linker sequence or a ribosomal linker sequence. A gene switch system is presented that includes a musk-skipping linker sequence.

[0034] In some embodiments, expression of one or more of the gene switch systems presented herein The cassette further comprises a sequence encoding a heterologous gene polypeptide. The one or more expression cassettes may comprise: (a) one or more recombinase junctions; and (b) a sequence encoding a serine recombinase. In other embodiments, the one or more expression cassettes comprise: (a) a non-inducible promoter; and (b) an inducible promoter.

[0035] In certain embodiments, the DNA-binding domain of the gene switch system provided herein The amino acid sequence of the present invention is as set forth in SEQ ID NO: 184. The transactivation domain of the gene switch system has the sequence shown in SEQ ID NO: 181. In another embodiment, a first nuclear receptor ligand binding domain and a second nuclear receptor ligand binding domain are At least one of the receptor ligand-binding domains, and the ligand-binding domain The specific embodiment has a sequence shown in any one of SEQ ID NOs: 185 to 186. In embodiments, the non-inducible promoter is any one of SEQ ID NOs: 40-64. In certain embodiments, the inducible promoter has a sequence selected from the group consisting of SEQ ID NOs: 40-6. In certain embodiments, the polypeptide has a sequence as set forth in any one of SEQ ID NO:4. The drinker has a sequence shown in any one of SEQ ID NOs: 146 to 162. In some embodiments, the polypeptide linker is selected from the group consisting of SEQ ID NOs: 18-19 and 146-16. Cleavable linker with a sequence shown in any one of 2, ribosome skipping linker, or IRES linker.

[0036] In some examples, one or more expression cassettes of the gene switch systems presented herein The kit comprises an expression cassette containing a sequence encoding a chimeric antigen receptor (CAR), The expression of the antigen receptor is modulated by a non-inducible promoter. CARs bind to CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate Synthetic protein, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelial , CD22, EGFR, MUC-1, MAGE-A1, MUC-16, h5T4, PS MA, TAG-72, EGFRvIII, CD123, and VEGF-R2 It is possible to combine at least one

[0037] In other examples, the non-inducible promoter of the gene switch system provided herein is EF 1A or a variant thereof. In certain cases, the expression cassette comprises SEQ ID NO: 13 1. In other cases, the expression cassette has a sequence encoding a cell tag. wherein the cell tag is linked to the CAR by a linker. The cell tag is at least one of a truncated HER1 mutant and a truncated CD20 mutant. Includes one.

[0038] In another example, the expression cassette for the gene switch system provided herein is SEQ ID NO: 13 2. In some embodiments, the expression cassette has the sequence shown in a sequence encoding a gene switch polypeptide and a sequence encoding a second gene switch polypeptide The first gene switch polypeptide and the second gene switch polypeptide further comprise a sequence One of the peptides is linked to the CAR by a linker.

[0039] In some embodiments, the first gene switch of the gene switch system presented herein The polypeptide and the second gene switch polypeptide are connected to the polypeptide linker. and the polypeptide linker is a cleavable linker. In some embodiments, the expression cassette has the sequence set forth in SEQ ID NO: 133. The first gene switch polypeptide and the second gene switch polypeptide are polypeptides. The polypeptide linker is an IRES linker. do.

[0040] In certain embodiments, the expression cassette of the gene switch system presented herein comprises: In another embodiment, one or more expression cassettes The vector comprises an expression cassette containing a sequence encoding a heterologous gene polypeptide, Expression of the gene polypeptide is modulated by an inducible promoter. In some embodiments, the heterologous polypeptide comprises a cytokine. are IL-1, IL-2, IL-15, IL-12, IL-21, IL-15, IL-1 IL-15 fusions, including at least one of IL-15 fusions with IL-15, IL-5Rα, or IL-15 variants.

[0041] In another embodiment, the expression cassette of the gene switch system provided herein is In some embodiments, the expression cassette has the sequence shown in SEQ ID NO:135. a second heterologous gene polypeptide comprising a sequence encoding a heterologous gene polypeptide, and a second heterologous gene polypeptide comprising a cell tag In certain cases, the cell tag includes a truncated HER1 mutant and a truncated CD20 mutant. In other cases, the cell tag comprises at least one of the variants. Alternatively, the expression cassette may be linked to a cytokine signaling vector as set forth in SEQ ID NO: 136. It has a sequence that can be

[0042] In some cases, the gene switch systems provided herein can be used to transfer heterologous genes into host cells. a gene switch system for integrating a gene into a host cell, the system comprising: a serine recombinase; contacting one or more expression cassettes in the presence of one or more recombinase junction sites; When the heterologous gene is introduced into the host cell, it is integrated into the host cell. The catch system further comprises a ligand, and when the host cell is contacted with the ligand, the heterologous gene is expressed. In certain cases, the host cell is a T cell or an NK cell. In certain cases, one or more recombinase junction sites are located within the phage genome assembly. The recombinant junction site (attP) or the bacterial genome recombinant junction site (attB) may be included. Alternatively, the serine recombinase may be SF370.

[0043] In certain instances, the inducible promoter of the gene switch system presented herein is , activated by a transactivation domain. In certain cases, the system may comprise one or more In certain cases, the system is contained within one vector. .

[0044] Encoding one or more of the components of the gene switch system presented herein Also disclosed herein are polynucleotides that can be used to identify the gene switches presented herein. Also disclosed are vectors containing polynucleotides encoding one or more of the components of the system. In certain cases, the vector may be a lentiviral vector, a retroviral vector, or In certain cases, the vector is either a viral vector or a non-viral vector. The vector is a non-viral vector, and the non-viral vector is a Sleeping Beauty transposon.

[0045] As used herein, a polynucleotide encoding a first gene switch polypeptide, a polynucleotide encoding a gene switch polypeptide of 2, and a gene of interest ( A polynucleotide construct comprising a polynucleotide encoding a GOI (a group of GOIs), a polynucleotide encoding a first gene switch polypeptide; a polynucleotide encoding a second gene switch polypeptide; a polynucleotide comprising a continuous open reading frame (ORF) located between the The construct further comprises a polynucleotide encoding a linker, wherein said GOI is The first gene switch polypeptide and the second gene switch polypeptide are linked by a linker. A polynucleotide construct is presented that is linked to each of the peptides.

[0046] As used herein, (i) a DNA fragment fused to a first nuclear receptor ligand-binding domain a first sequence encoding a first gene switch polypeptide comprising a binding domain; and (ii) transactivation fused to a second nuclear receptor ligand-binding domain. a second sequence encoding a second gene switch polypeptide comprising a domain at least one of said first sequence and said second sequence; Polynucleotides whose expression is modulated by one or more NFAT response elements A punchline is presented.

[0047] Provided herein are methods for stimulating the proliferation and / or survival of engineered cells, comprising: (a) inducing a cell (b) obtaining a cell sample from a subject; and (b) subjecting cells of said cell sample to one or more transfections. transfecting one or more polynucleotides, including a nucleotide sequence encoding ... wherein the one or more transposons are chimeric antigen receptors (CARs) or TCs. R, cytokine, one or more cell tags, ligand-induced regulation of said cytokine a gene switch polypeptide for the gene transfer and said one or more polynucleotides A transposase effective to integrate into the genome of a cell, resulting in a population of engineered cells. wherein the gene switch polypeptide is encoded by: i) a first nuclear A first genetic construct comprising a DNA binding domain fused to a receptor ligand binding domain. and ii) a nuclear receptor ligand-binding domain fused to a second nuclear receptor ligand-binding domain. a second gene switch polypeptide comprising a transactivation domain linked to the first gene switch polypeptide; The first gene switch polypeptide and the second gene switch polypeptide are A method is presented in which the nucleotides are linked by a linker.

[0048] Provided herein are methods for increasing the persistence of engineered cells in vivo in a subject in need thereof. (a) obtaining a cell sample from a subject; (b) detecting the cell sample; One or more polynucleotides containing one or more transposons are transfected into the cells. transfecting the one or more transposons into a chimeric antibody; a CAR or TCR, a cytokine, one or more cell tags, A gene switch polypeptide for ligand-induced regulation of kinetin, and DNA, are prepared as follows: A transposase effective to integrate into the genome of a cell, resulting in a population of engineered cells. wherein the gene switch polypeptide is encoded by: i) a first nuclear A first genetic construct comprising a DNA binding domain fused to a receptor ligand binding domain. and ii) a nuclear receptor ligand-binding domain fused to a second nuclear receptor ligand-binding domain. a second gene switch polypeptide comprising a transactivation domain linked to the first gene switch polypeptide; The first gene switch polypeptide and the second gene switch polypeptide are A method is presented in which the nucleotides are linked by a linker.

[0049] In some embodiments, the method of transfecting cells provides In certain embodiments, the step of electroporating the cells comprises electroporating the cells. At least one of the polynucleotides is a gene switch polypeptide. At least one polynucleotide encodes a polypeptide, and is modulated by a promoter. The promoter is a tissue-specific promoter or the EF1A promoter. or a functional variant thereof. In certain cases, the promoter is EF1A promoter having a sequence shown in any one of Nos. 58 to 60, and is this functional variant. In certain cases, the promoter is a tissue-specific promoter. The tissue-specific promoter contains a T cell-specific response element. In certain cases, the promoter is a tissue-specific promoter. The NFAT receptor comprises one or more NFAT response elements. The answer element has a sequence shown in any one of SEQ ID NOs: 50 to 57. In certain cases, the cytokines are IL-1, IL-2, IL-15, IL-12, 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, fusion of at least one of IL-21, IL-15, IL-15Rα, or an IL-15 mutant In other cases, the cytokine is in a secreted form. In yet another embodiment, the cytokine is in a membrane-bound form. 3-209. , NK cells, NKT cells, T cells, or T cell precursors.

[0050] In other cases, the methods provided herein may involve administering an effective amount of engineered cells to a subject requiring such administration. In certain instances, the administering step further comprises administering to the subject In certain instances, the method includes rapidly infusing the engineered cells into the animal. The method further comprises administering a ligand of the present invention to induce cytokine expression. In the case of , CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, R OR1, CEA, EGP-2, EGP-40, HER2, HER3, folate binding protein Quality of life, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22 , EGFR, MUC-1, MAGE-A1, MUC-16, h5T4, PSMA, TAG -72, EGFRvIII, CD123, and at least one of VEGF-R2 In certain instances, the CAR is capable of binding to CD19. In certain cases, the CAR is capable of binding to CD33. In certain cases, the transposase is a salmonid-type Tc1-like transposase. In certain cases, the transposase is SB11 or SB100x transposase. In certain instances, the one or more cell tags are HER1 truncated mutants and and a CD20 truncation mutant. In certain instances, one or The plurality of cell tags have a sequence shown in any one of SEQ ID NOs: 189 to 202. do.

[0051] Provided herein is a method of treating a subject with a solid tumor, comprising: (a) administering to a subject a cell sample; (b) introducing one or more transposons into cells of said cell sample. transfecting one or more polynucleotides comprising One or more transposons may be used to encode a chimeric antigen receptor (CAR) or TCR, cytokine In one or more cell tags, a gene for ligand-induced regulation of said cytokine The switch polypeptide and DNA are integrated into the genome of the cell, a gene switch polypeptide encoding a transposase effective to generate a population of The domain comprises: i) a DNA-binding domain fused to a first nuclear receptor ligand-binding domain; and ii) a first gene switch polypeptide comprising a second nuclear receptor ligand. A second gene switch promoter comprising a transactivation domain fused to a binding domain. a first gene switch polypeptide and a second gene switch polypeptide, (c) the step of preparing the engineered cell. and administering the population to the subject.

[0052] In some embodiments, the cytokines in the methods of treatment 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-19, IL- L-15, IL-12, IL-21, IL-15, IL-15Rα, or IL-15 variant In some embodiments, the cytokine comprises at least one of a fusion of a heterologous cytokine, a fusion of ... In some embodiments, at least one of the one or more polynucleotides comprises L-12. At least one polynucleotide encodes a gene switch polypeptide and One polynucleotide is modulated by a promoter, and the promoter is a tissue-specific promoter or the EF1A promoter, or a functional variant thereof In some embodiments, the promoter is any one of SEQ ID NOs: 58-60. or a functional variant thereof. In certain embodiments, the promoter is a tissue-specific promoter, In some embodiments, the promoter comprises a T cell specific response element. , a tissue-specific promoter, wherein the tissue-specific promoter is a promoter that is capable of expressing one or more NFAT In some embodiments, the NFAT response element comprises SEQ ID NO: 50. 57.

[0053] Also disclosed are engineered effector cells, wherein the engineered effector cells are a) engineered receptor constructs that selectively bind to specific cell surface proteins expressed by target cells; (b) a first polynucleotide encoding the construct; and (b) one or more engineered gene switches. One or more polynucleotides encoding a polypeptide, The switch polypeptide comprises a transactivation domain, a DNA-binding domain, and a ligase. one or more polynucleotides comprising one or more of the nucleotide-binding domains; and (c) a ligand that is modulated by the engineered gene switch polypeptide. a heterologous gene under the control of an inducible promoter, encoding a cytokine; Contains genes.

[0054] In some embodiments, the target cell is a mammalian cell. In some embodiments, the cell surface protein is a protein on the surface of said tumor cell. In some embodiments, the cell surface protein is expressed on the surface of an artificial antigen-presenting cell (aA In some embodiments, the engineered receptor construct is a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is selected from the group consisting of CD19, CD33, BCMA, CD4 4, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-4 0, HER2, HER3, folate-binding protein, GD2, GD3, IL-13R-α2 , KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1 , h5T4, PSMA, TAG-72, EGFRvIII, CD123, or VEGF In some embodiments, the chimeric antigen receptor binds to EGFRvIII. do.

[0055] In some embodiments, the engineered receptor construct is an engineered T cell receptor. In this study, cytokines were IL-2, IL-15, IL-12, IL-21, or IL- In some embodiments, the cytokine is a fusion of IL-15 with IL-15Rα. In some embodiments, the effector cells encode proteins comprising a cell tag. In some embodiments, the cell tag further comprises a polynucleotide that encodes a truncated HE In some embodiments, the effector cells are R1 mutants or CD20 truncated mutants. are immune effector cells. In some embodiments, the effector cells are T cells, In some embodiments, the one or more engineered genes are selected from the group consisting of NK cells, NK cells, and tumor-infiltrating lymphocytes. The gene switch polypeptide comprises a transactivation domain, a DNA-binding domain, and In some embodiments, the polypeptide further comprises one or more engineered gene switches. The polypeptide comprises a DNA-binding domain fused to a nuclear receptor ligand-binding domain. A first gene switch polypeptide comprising a domain and a nuclear receptor ligand binding property a second gene switch polypeptide comprising the transactivation domain fused to a domain; Contains peptides.

[0056] In some embodiments, the first gene switch polypeptide and the second gene switch polypeptide In some embodiments, the polypeptide is linked to the ribozyme by a polypeptide linker. , the polypeptide linker may be a cleavable linker sequence or a ribosome-skipping linker In some embodiments, the polypeptide linker is a sequence of 2A, F / T2A, GSG -2A, GSG linker, SGSG linker, furin linker, and their mutations In some embodiments, the transactivation domain comprises one of: In some embodiments, one or more engineered genes comprise a VP16 transactivation domain. The switch polypeptides are the ecdysone receptor (EcR), the ubiquitous receptor, and the orphan steroid hormone receptor 1, NER-1, steroid hormone nuclear receptor 1, retinoid X receptor interaction Functional protein 15, liver X receptor β, steroid hormone receptor-like protein, liver X receptor, liver X receptor α, farnesoid X receptor, receptor-interacting protein 14, and the farnesol receptor. In some embodiments, the DN The A-binding domain (DBD) is a GAL4 (GAL4 DBD), LexADBD, transcription factor Factor DBD, steroid / thyroid hormone nuclear receptor superfamily member DBD , a bacterial LacZ DBD, and a yeast DBD.

[0057] Provided herein is a method for modulating the expression of a heterologous gene, comprising: injecting a target cell with a gene encoding a heterologous gene according to the present invention. Further disclosed are methods comprising contacting an effector cell disclosed herein. Optionally, modulating means increasing or decreasing expression of the heterologous gene. Optionally, expression comprises expression of said ligand in the absence of said ligand. In some cases, expression is reduced or eliminated compared to expression in the presence of The patient is resuscitated by administering a further amount of the ligand.

[0058] Furthermore, there is provided a method for regulating expression of a heterologous gene in an effector cell, comprising: and administering to a target cell one or more engineered gene switch polypeptides, an engineered receptor construct, and introducing a polynucleotide encoding said heterologous gene, the heterologous gene is under the control of an inducible promoter; and activating the effector cells via After the step of activating, the one or more engineered gene switch polypeptides and delivering a ligand to the effector cells to induce expression of the heterologous gene via the and presenting the step of:

[0059] Optionally, the activating step comprises contacting the effector cell with an antigen. Optionally, the antigen is a tumor antigen. Optionally, the ligand is Effector cells are incubated with tumor antigens for at least 7 days before presentation to target cells. Optionally, the step of activating the effector cells comprises exposing the effector cells to Optionally, co-culturing the cells with artificial antigen-presenting cells (aAPCs) The feeding period is for at least 7, 14, 21, or 28 days. In some cases, the aAPCs are transgenic K562 cells. The receptor construct comprises a chimeric antigen receptor (CAR). Optionally, the CAR is a CD19 , CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, C EA, EGP-2, EGP-40, HER2, HER3, folate-binding protein, GD2 , GD3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EGFR , MUC-1, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvIII , CD123, or VEGF-R2. Optionally, the effector cell comprises an engineered T cell receptor (TCR). contacting the cell with a TCR binding polypeptide. The polypeptide comprises a TCR-binding antibody or fragment thereof. , effector cells for at least 7 days before presentation to effector cells. , to the TCR binding polypeptide. In some cases, the TCR binding polypeptide , expressed via the aAPC. Optionally, the heterologous gene encodes a cytokine. In some cases, the cytokines are IL-2, IL-15, IL-12, IL-21, or or a fusion of IL-15 and IL-15Rα. It is L-12.

[0060] Optionally, the heterologous gene encodes at least one cell tag. Optionally, the cell tag is a truncated HER1 mutant or a truncated CD20 mutant. The one or more gene expression cassettes further comprise a nucleotide sequence encoding a cell tag. Optionally, the effector cell is an immune effector cell. In this case, the immune effector cells are T cells, NK cells, or tumor-infiltrating lymphocytes. wherein the one or more engineered gene switch polypeptides comprise a transactivation domain. , a DNA-binding domain, and a ligand-binding domain. The dopamine-binding domain is involved in the ecdysone receptor (EcR), a ubiquitous receptor, and an orphan receptor. NER-1, steroid hormone nuclear receptor 1, retinoid X receptor interacting protein Protein 15, liver X receptor β, steroid hormone receptor-like protein, liver X receptor , liver X receptor α, farnesoid X receptor, receptor-interacting protein 14, and The farnesol receptor may optionally contain a DNA-binding domain. In is GAL4 (GAL4 DBD), LexADBD, transcription factor DBD, steroid / Thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DBD and at least one of a yeast DBD. The in contains the VP16 transactivation domain.

[0061] In some cases, the one or more engineered gene switch polypeptides comprise the DNA-binding Optionally, one or more of the following may be present: Several engineered gene switch polypeptides are known, including ultraspiracle proteins (USPs), Retinoid receptor X (RXR), or at least some of their functional fragments and variants and further comprising at least one of the functional fragments and variants of the present invention, wherein the functional fragments and variants bind to EcR. Optionally, the one or more engineered gene switch polypeptides can be Optionally, the one or more polypeptide linkers Peptide linkers include 2A, GSG-2A, GSG linker, SGSG linker, and furosemide. and at least one of the following: In some cases, the ligand is (2S,3R,5R,9R,10R,13R,14S,1 7R)-17-[(2S,3R)-3,6-dihydroxy-6-methylheptan-2-yl

[00100] -2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,1 1,12,15,16,17-Decahydro-1H-cyclopenta[a]phenanthrene 6-one;N'-(3,5-dimethylbenzoyl)-N'-[(3R)-2,2-dimethyl 5-Methyl-2, 3-Dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethyl) -benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide;5 -Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3 ,5-Dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl -propyl)-hydrazide;5-methyl-2,3-dihydro-benzo[1,4]dioxy N'-(1-tert-butyl-butyl)-N'-(3,5-dimethyl- 5-Methyl-2,3-dihydro-benzo[1,4]dihydrobenzo[1,4]dihydrazide N'-(1-tert-butyl-butyl)-N'-(3,5-dioxin-6-carboxylic acid) Methoxy-4-methyl-benzoyl)-hydrazide; 5-Ethyl-2,3-dihydro-benzo Benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethyl-benzoyl)- N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5-ethyl-2,3 -Dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy -4-methyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)- 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carvone Acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl) -hydrazide; 5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-chlor N'-(1-tert-butyl-butyl)-N'-(3,5-dimethoxy-4-methylbenzoate N-(1-ethyl-2, 3,5-dimethylbenzoic acid N-(1-ethyl-2, 2-Dimethyl-propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydra 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl) ester -propyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide;3,5 -dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3-methoxy- 2-Methyl-benzoyl)-hydrazide;3,5-Dimethoxy-4-methyl-benzoic acid N -(1-tert-butyl-butyl)-N'-(3-methoxy-2-methyl-benzoyl )-hydrazide;3,5-dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propionyl) 3,5-Di(2-methyl-3-methoxybenzoyl)hydrazide; Methoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N '-(2-Ethyl-3-methoxy-benzoyl)-hydrazide;3,5-Dimethyl-benzo N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)benzoate 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert -butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 2-Methoxy-nicotinic acid N-(1-tert-butyl-pentyl)-N'-(4-ethyl) N-(2,2-dimethyl-3,5-dimethylbenzoic acid)hydrazide; 1-Phenyl-propyl)-N'-(4-ethyl-benzoyl)-hydrazide;3,5- Dimethylbenzoate N-(1-tert-butyl-pentyl)-N'-(3-methoxy- 2-Methyl-benzoyl)-hydrazide; and 3,5-dimethoxy-4-methyl-benzo N-(1-tert-butyl-pentyl)-N'-(3-methoxy-2-methyl-benzyl)benzoate The compound contains at least one of the following:

[0062] Described herein are polynucleotides for ligand-induced control of heterologous gene expression in engineered cells. (a) binding to a specific cell surface protein expressed by a target cell; and (b) a first sequence encoding an engineered receptor construct capable of: by modulating a promoter linked to said heterologous gene in response to the presence of and one or more engineered gene sequences for ligand-induced control of the expression of said heterologous gene. a second sequence encoding a switch polypeptide, Alternatively, a plurality of engineered gene switch polypeptides are linked by a polypeptide linker. Further disclosed are polynucleotides comprising:

[0063] In some embodiments, the polypeptide linker comprises a truncated linker sequence. In some embodiments, the cleavable linker sequence is an F2A linker. The engineered gene switch polypeptide or polypeptides are linked to the nuclear receptor ligand binding domain. a first gene switch polypeptide comprising a fused DNA-binding domain; and a second domain comprising a transactivation domain fused to an endoreceptor ligand binding domain; In some embodiments, the first gene switch polypeptide The first and second gene switch polypeptides are linked by a linker. In some embodiments, the linker is a cleavable linker sequence or a ribosomal skipping linker. In some embodiments, the linker is a 2A linker, a GSG-2A linker, or a GSG linker, SGSG linker, furin linker, and their mutants and and derivatives thereof. In some embodiments, the 2A linker is a F2A linker. In some embodiments, the heterologous gene encodes a cytokine. The cytokines are IL-2, IL-15, IL-12, IL-21, or IL-15 and IL-21. In some embodiments, the cytokine is IL-12. In some embodiments, the heterologous gene encodes at least one cell tag. In embodiments, the cell tag is a HER1 truncated mutant or a CD20 truncated mutant. In some embodiments, the polynucleotide is incorporated into an engineered cell.

[0064] As used herein, a system for expressing cytokines in host cells adjacent to target cells is described. wherein, in the presence of a ligand, the engineered receptor construct binds to a cell surface protein, (a)(i) in the target cell, so that host cells in the vicinity of the target cell express a cytokine; an engineered receptor construct that selectively binds to a predetermined cell surface protein, expressed in ) a transactivation domain, a DNA-binding domain, and a ligand-binding domain (iii) an engineered gene switch polypeptide comprising one or more of the following: a heterologous gene linked to a promoter modulated by a gene switch polypeptide; Further, a system comprising (a) a host cell encoding a cytokine expressed from a gene; and (b) a ligand. will be disclosed.

[0065] Optionally, the engineered receptor construct comprises a chimeric antigen receptor. Antigen receptors include CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folate Synthetic protein, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelial CD22, EGFR, MUC-1, MAGE-A1, h5T4, PSMA, TAG- 72, EGFRvIII, CD123, or VEGF-R2. , the chimeric antigen receptor binds to EGFRvIII. Optionally, the engineered receptor construct The antibody comprises an engineered T cell receptor. Optionally, the cytokine is IL-2, IL-15, It is a fusion of IL-12, IL-21, or IL-15 with IL-15Rα. Thus, the cytokine is IL-12. Optionally, the host cell is Optionally, the cell tag further encodes a HER1 truncated mutant or or a truncated CD20 mutant.

[0066] Further provided herein are methods for expressing cytokines from host cells in the vicinity of target cells. contacting the target cells with the system disclosed herein in the presence of a ligand. Also provided herein are methods comprising the steps of: A method of modulating the expression of a cytokine of the present invention, comprising administering to a subject a cytokine comprising the steps of: The method comprises the steps of contacting the system disclosed in the document with target cells and adjusting the amount of ligand. Optionally, expression is in the absence of said ligand, and is reduced or abolished compared to expression in the presence of a ligand. Expression is then restored by administering additional amounts of the ligand. The cells are animal or mammalian cells. Optionally, the mammalian cells are human cells. Optionally, the mammalian cell is a T cell, an NK cell, or a tumor-infiltrating lymphocyte. Optionally, the target cell is a tumor cell. Optionally, the system comprises one or more Contained within a vector. Optionally, each vector comprises a plasmid. Optionally, Optionally, each vector comprises an expression plasmid. The vector may be a viral vector, a retroviral vector, or a non-viral vector. , a non-viral vector, Sleeping Beauty transposase and SB Contains transposons.

[0067] Provided herein is a polypeptide for binding to EGFRvIII, comprising SEQ ID NO:2 33, 234, 235, 236, 237, 238, 239, 240, 241, 242, 2 43, or 244. %, 90%, 95%, 99%, or 99.5% identity to a polypeptide sequence Further disclosed are polypeptides comprising:

[0068] Optionally, the polypeptide is expressed in an engineered effector cell. The effector cell is an immune effector cell. The cell is a T cell, a NK cell, or a tumor-infiltrating lymphocyte. Optionally, the polypeptide comprises a chimeric antigen receptor (C Optionally, the EGFRvIII comprises the antibody or fragment thereof or a Optionally, the polypeptide binds to the scFv antigen-binding domain of the CAR. Does not cross-react with live EGFR.

[0069] As used herein, a) an engineered receptor construct capable of binding to EGFRvIII; a first polypeptide comprising a transactivation domain and a nuclear receptor ligand binding domain; and c) a DNA-binding domain and a nuclear receptor ligand-binding domain. a polynucleotide encoding a second polypeptide comprising a F / T2A linker at least one of an engineered receptor construct, a first polypeptide, and a second polypeptide; Further disclosed are polynucleotides that link the two together.

[0070] Optionally, an F / T2A linker connects the engineered receptor construct to the first polypeptide. and said second polypeptide. , the F / T2A linker connects the first polypeptide to the second polypeptide Optionally, the polynucleotide further encodes an IRES linker. Thus, the IRES linker connects the first polypeptide to the second polypeptide.

[0071] As used herein, effector cells comprising the polynucleotides disclosed herein are further Optionally, the effector cell is a cell that expresses said first polypeptide or said Linked to an inducible promoter that can be modulated by a second polypeptide. The ligated heterologous gene encodes the ligated heterologous gene.

[0072] Herein, a chimeric antigen receptor (CAR) capable of binding to EGFRvIII is (a) an EGFRvIII binding region; (b) a transmembrane region; and (c) a transmembrane a spacer region connecting the EGFRvIII binding region to the EGFRvIII binding region, a stalk region comprising at least one dimerization site, and The CAR further comprises a stalk extension region comprising a sequence with at least 75% sequence identity. will be disclosed.

[0073] As used herein, a compound or antibody that is effective to bind to an EGFRvIII molecule or a variant thereof includes: (b) an EGFRvIII-binding moiety on the surface of engineered effector cells; one or more of a activating domain, a DNA-binding domain, and a ligand-binding domain; (c) a polypeptide encoding a cytokine; a heterologous gene that is modulated by an engineered gene switch polypeptide; Further disclosed are engineered effector cells containing a heterologous gene controlled by a promoter. do.

[0074] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the present invention and its advantages may be obtained by reference to exemplary embodiments in which the principles of the present disclosure are employed. This can be had by reference to the following detailed description and accompanying drawings, which set forth: [Brief explanation of the drawings]

[0075] [Figure 1] Figure 1 shows a schematic depiction of the Sleeping Beauty (SB) system adapted for genetically modifying T cells. DNA plasmids expressing the SB transposon system, i.e., SB11, membrane-bound IL-15 (mbIL-15), and chimeric antigen receptor (CAR), are transfected into peripheral blood mononuclear cells (PBMCs) to redirect T cell specificity. Upon contact with designer activating and propagating cells (AaPCs), T cells stably expressing the integrated sequences are propagated and expanded. [Figure 2A]

[0023] Figure 10 is a schematic illustrating various structural components of various ligand-inducible gene switch vector systems. Depicted exemplary CARs can include CD19 CARs. The EF1A promoter is an exemplary constitutive promoter. [Figure 2B] See the description of FIG. 2A. [Figure 2C] See the description of FIG. 2A. [Figure 2D] See the description of FIG. 2A. [Figure 3] FIG. 1 shows a schematic illustration of a multiple vector system for ligand-inducible gene switch polypeptides. [Figure 4]

[0023] Figure 2 depicts quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector system described herein at day 1 post-nucleofection in the presence / absence of berezimex ligand (solvent: DMSO). Constructs 1, 4, 5, 6, 7, and 9 correspond to the constructs depicted schematically in Figures 2A-2D. [Figure 5]

[0023] Figure 2 depicts quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector system described herein and gated for the CD19-specific CAR-positive population at day 21 post-nucleofection in the presence / absence of berezimex ligand (solvent: DMSO). Ligand was added 48 hours prior to flow cytometry analysis. Constructs 1, 4, 5, 6, and 9 correspond to the constructs depicted schematically in Figures 2A-2D. [Figure 6]

[0023] Figure 2 depicts quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector system described herein and gated for the CD19-specific CAR-positive population at day 29 post-nucleofection in the presence / absence of berezimex ligand (solvent: DMSO). Expression of mbIL-15 is turned on after ligand addition. Constructs 1, 4, 5, 6, 7, and 9 correspond to the constructs depicted schematically in Figures 2A-2D. [Figure 7]Figure 2 shows quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector system described herein and gated for the CD19-specific CAR-positive population at day 35 after nucleofection in the presence / absence of berezimex ligand (solvent: DMSO). Expression of mbIL-15 is turned off after ligand removal. Constructs 1, 4, 5, 6, and 9 correspond to the constructs depicted schematically in Figures 2A-2D. [Figure 8] Figure 2 shows quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector system described herein and gated for the CD19-specific CAR-positive population at day 40 after nucleofection in the presence / absence of berezimex ligand (solvent: DMSO). Expression of mbIL-15 is maintained after reintroduction of the ligand. Constructs 1, 4, 5, 6, and 9 correspond to the constructs depicted schematically in Figures 2A-2D. [Figure 9] Figure 2 shows quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector system described herein and gated for the CD19-specific CAR-positive population at day 48 after nucleofection in the presence / absence of berezimex ligand (solvent: DMSO). Expression of mbIL-15 is continuously maintained after reintroduction of the ligand. Constructs 1, 4, 5, 6, and 9 correspond to the constructs depicted schematically in Figures 2A-2D. [Figure 10] Figure 2 shows quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector system described herein and gated for the CD19-specific CAR-positive population at day 50 after nucleofection in the presence / absence of berezimex ligand (solvent: DMSO). Expression of mbIL-15 is continuously maintained after reintroduction of the ligand. Constructs 1, 4, 5, 6, and 9 correspond to the constructs depicted schematically in Figures 2A-2D. [Figure 11]FIG. 1 shows quantitative RT-qPCR analysis of mbIL-15 expression in cells transfected with the ligand-inducible gene switch vector system described herein at days 29, 33, 35, and 40 post-nucleofection in the presence and absence of the ligands described herein, confirming ligand-dependent regulation of mbIL-15 expression by the gene switch. [Figure 12] FIG. 1 shows results from a cell survival assay illustrating that ligand-regulated mbIL-15 expression promotes preferential CAR-T cell survival in the absence of IL-2 and IL-21 cytokines. [Figure 13A] Figure 13D shows quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector system described herein, which supports the dose response of berezimex ligand. Figure 13D shows quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector system described herein, which supports the dose response of berezimex ligand. Cells were gated for FSC / SSC / LIVE / CD3+. Samples were taken after 2 days of incubation with berezimex at 100 nM, 25 nM, 6.25 nM, 1.56 nM, 0.4 nM, and 0 nM (DMSO control). [Figure 13B] See the description of Figure 13A. [Figure 13C] See the description of Figure 13A. [Figure 13D] See the description of Figure 13A. [Figure 14] FIG. 1 shows a Western blot analysis of mb-IL-15 expression in cells transfected with the ligand-inducible gene switch vector system described herein in the presence / absence of berezimex ligand. [Figure 15A]

[0023] Figure 1 is a schematic depiction of the varying structural components of the general, diverse ligand-inducible gene switch vector system described herein. In the schematic, "PR" represents promoter; "IP" is the ligand-inducible promoter of the gene switch for gene transcription; "5'UTR" is the 5' untranslated region (for transcription into mRNA); "GOI," "GOI-1," and "GOI-2" are the gene of interest or the first GOI (GOI-1) and second GOI (GOI-2) transcribed and expressed as mRNA alone (e.g., siRNA), or as mRNA and polypeptide; "linker" is a cleavable linker sequence or a ribosomal skipping linker sequence; and "TAD" is a transcriptional transactivation domain (e.g., VP16 from herpesvirus). "LBD" refers to a nuclear receptor ligand binding domain (e.g., but not limited to, a USP domain, an RXR domain, or chimeras (e.g., USP / RXR chimeras), and substitution mutant LBD domains, from vertebrate and non-vertebrate species); "DBD" refers to a DNA binding domain (e.g., but not limited to, a Gal4 DBD); and "EcR" refers to a truncated EcR domain and substitution mutant EcR domains (in vitro and in vitro). an ecdysone receptor ligand binding domain, including an EcR domain, that confers the ability to form an active transcriptional activation complex in vivo in the presence of steroidal and / or non-steroidal ligands (agonists), including steroidal ligands (such as ponasterone A and muristerone A) and non-steroidal ligands (such as diacylhydrazine, tebufenozide, and methoxyfenozide); "optional GOI" is any, optional, gene of interest that is transcribed and expressed as mRNA or a polypeptide; and "BD polyA" is a bidirectional polyadenosine tail sequence. [Figure 15B] See the description of Figure 15A. [Figure 16A]FIG. 1 is a schematic depiction of the varying structural components of the various ligand-inducible gene switch vector systems described herein. [Figure 16B] See the description of Figure 16A. [Figure 17]

[0023] Figure 1 shows a schematic representation of an exemplary ligand-inducible gene switch vector system with recombinase junction sites. Such a system can use a recombinase (such as a serine recombinase) to integrate a single vector into immune cells (e.g., T cells). [Figure 18A] Figure 18A is a table depicting the % CAR-positive T cells over days 1, 8, and 15. These T cells were engineered to express a CAR and gene-switch-controlled mbIL-15 (RTS-mbIL15) from a single vector using serine recombinase (SF370)-mediated recombination. Figure 18B depicts the induction of mbIL-15 expression in the presence and absence of berezimex on day 15. The figure also confirms the loss of mbIL15 expression upon removal of berezimex on day 22. Figure 18C is an exemplary depiction of the RTS-mbIL15 constitutive CAR construct. [Figure 18B] See the description of Figure 18A. [Figure 18C] See the description of Figure 18A. [Figure 19A] FIG. 1A is a schematic illustrating the various structural components of various ligand-inducible gene switch vector systems under the control of constitutive or T cell-specific promoters. [Figure 19B] See the description of Figure 19A. [Figure 20A]Figure 20A is a diagram illustrating the various structural components of various ligand-inducible gene switch vector systems under the control of constitutive or T cell-specific promoters. Figures 20B-20E show quantitative flow cytometry analysis of cells transfected with the ligand-inducible gene switch vector systems described herein, confirming that expression of mbIL-15 is dependent on T cell activation (by addition of ConA) and the presence of berezimex. Constructs 1-6 correspond to the constructs depicted schematically in Figure 20A. [Figure 20B] See the description of Figure 20A. [Figure 20C] See the description of Figure 20A. [Figure 20D] See the description of Figure 20A. [Figure 20E] See the description of Figure 20A. [Figure 21A] FIG. 1 depicts the diverse composition of IL-12. [Figure 21B] See the description of Figure 21A. [Figure 22] FIG. 1A-C schematically illustrate various structural components of various ligand-inducible gene switch vector systems under the control of constitutive or inducible promoters. [Figure 23]

[0023] Figure 1 shows schematic diagrams illustrating the various structural components of various ligand-inducible gene switch vector systems that express different forms of IL-12 under the control of constitutive, inducible, or tissue-specific promoters (PRs). IL-12 can be expressed as single-chain IL-12, membrane-bound IL-12, or by separate expression of the p35 and p40 domains. [Figure 24A] FIG. 1A-C schematically illustrate various structural components of various ligand-inducible gene switch vector systems under the control of constitutive or inducible promoters. [Figure 24B] See the description of Figure 24A. [Figure 24C]See the description of Figure 24A. [Figure 24D] See the description of Figure 24A. [Figure 25A] FIG. 1A is a diagram illustrating the various structural components of various ligand-inducible gene switch vector systems under the control of constitutive, inducible, or T-cell specific promoters. [Figure 25B] See the description of Figure 25A. [Figure 26] Figure 10 shows quantitative flow cytometry analysis depicting CAR expression in EGFRvIII CAR T cells expanded ex vivo by sequential stimulation via co-culture with AaPCs using multiple donors. [Figure 27] FIG. 1 shows the production of IL-12 by cells transfected with the ligand-inducible gene switch vector system described herein in the presence / absence of berezimex ligand (solvent: DMSO). [Figure 28A]

[0033] Figures 28A and 28B show analyses of IL-12 expression levels. Figure 28A shows the expression levels of IL-12 by cells transfected with the ligand-inducible gene switch vector system described herein in the presence / absence of berezimex ligand (solvent: DMSO) in the absence of cell activation. Figure 28B shows the expression levels of IL-12 by cells transfected with the ligand-inducible gene switch vector system described herein in the presence / absence of berezimex ligand (solvent: DMSO) following antigen-specific activation. [Figure 28B] See the description of Figure 28A. [Figure 29] FIG. 1 shows an analysis of IL-12 expression levels in cells transfected with the ligand-inducible gene switch vector system described herein and co-cultured with aAPCs and berezimex ligand (solvent: DMSO) in the presence / absence. [Figure 30A]Figures 30A and 30B show time course analyses of IL-12 expression levels. Figure 30A shows the expression levels of IL-12 by cells transfected with the ligand-inducible gene switch vector system described herein and co-cultured in the absence of AaPCs with / without berezimex ligand (solvent: DMSO). The ligand was added 24 hours later and removed 48 hours later. Figure 30B shows the expression levels of IL-12 by cells transfected with the ligand-inducible gene switch vector system described herein and co-cultured in the presence of AaPCs with / without berezimex ligand (solvent: DMSO). The ligand was added 24 hours later and removed 48 hours later. IL-12 expression was quantified 24, 48, and 120 hours after ligand addition. [Figure 30B] See the description of Figure 30A. [Figure 31A]

[0033] Figures 31A and 31B show analysis of IL-12 expression levels. Figure 31A shows IL-12 expression levels in the presence / absence of berezimex and AaPC ​​by cells transfected with the ligand-inducible gene switch vector system described herein and previously expanded in co-culture with AaPC ​​in medium without berezimex (cycle 1). Figure 31B shows IL-12 production in the presence / absence of berezimex and AaPC ​​by cells transfected with the ligand-inducible gene switch vector system described herein and previously expanded in co-culture with AaPC ​​in medium without berezimex (cycle 1). [Figure 31B] See the description of Figure 31A. [Figure 32]Figures 32A and 32B show analysis of IL-12 expression levels. Figure 32A shows IL-12 production by cells transfected with the ligand-inducible gene switch vector system described herein. Berezimex ligand was added at time 0, removed at 48 hours, added again at 168 hours, and removed at 216 hours. Figure 32B shows CAR expression in cells transfected with the ligand-inducible gene switch vector system described herein by flow cytometry analysis. Cells were gated for the CD3-positive population. [Figure 33] FIG. 1 shows the expression levels of IL-12 by cells transfected with the ligand-inducible gene switch vector system described herein in the presence of berezimex and in the presence (cycle 1) / absence (cycle 2) of AaPC. [Figure 34] FIG. 1 shows berezimex dose-dependent expression of IL-12 by cells transfected with the ligand-inducible gene switch vector system described herein. [Figure 35] Figure 1 shows the production of IFNγ by CAR-T cells generated via transfection of the ligand-inducible gene switch vector system described herein and co-cultured with target cells at an effector to target cell ratio of 1:1. IFNγ production was measured using co-culture of CAR-T cells with EL4 target cells, EL4 EGFRvIII target cells, and a control without target cells (effector alone). [Figure 36A]

[0049] Figure 36 shows the specific cytotoxicity of CAR-T cells. Figure 36A shows the production of IFNγ by CAR-T cells generated via transfection of the ligand-inducible gene switch vector system described herein. Effector CAR-T cells were co-cultured with U87MG, U87MG-FLuc-GFP, U87MG-EGFRvIII-FLuc-GFP, U251MG, U251MG-FLuc-GFP, and U251MG-EGFRvIII-FLuc-GFP target cells at a 1:1 effector:target cell ratio for 24 hours before analyzing IFNγ expression in the culture supernatant. Figure 36B shows the percentage lysis of EGFRvIII-expressing glioblastoma cells by CAR-T cells generated via transfection of the ligand-inducible gene switch vector system described herein. Effector CAR-T cells were co-incubated with EL4, EGFRvIII-expressing EL4, K562, and EGFRvIII-expressing K562 target cell lines at varying effector:target cell ratios. [Figure 36B] See the description of Figure 36A. DETAILED DESCRIPTION OF THE INVENTION

[0076] Detailed Description of the Invention The following description and examples further illustrate embodiments of the present disclosure. It is not limited to the specific embodiments described herein and, as such, may vary. It will be appreciated that numerous variations and modifications to this disclosure will occur to those skilled in the art. will recognize that the scope of

[0077] All terms are intended to be understood as they would be understood by one of ordinary skill in the art. Unless otherwise specified, the technical and scientific terms used herein Terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. do.

[0078] The section headings used herein are for organizational purposes only and do not limit the scope of the subject matter described. It should not be considered as limiting the subject matter.

[0079] While various features of the disclosure may be described in the context of a single embodiment, the features may also be provided separately. Conversely, the terms "common" and "commonly used" may be used interchangeably herein, and may be presented in any suitable combination. Although, for clarity, the present disclosure may be described in the context of individual embodiments, the present disclosure also may be described in the context of individual embodiments. It can be implemented in one embodiment.

[0080] definition The following definitions supplement those in the art and are intended to orient the present application and any Related or unrelated matters, e.g., commonly owned, belonging to any patent or application Any methods and materials similar or equivalent to those described herein are not intended to be limiting. Although various materials and methods may be used in carrying out the tests of this disclosure, preferred materials and methods are described herein. Accordingly, the terminology used herein refers to specific embodiments and methods. are for descriptive purposes only and are not intended to be limiting. There is no.

[0081] In this application, unless otherwise stated, the use of the singular refers to the plural. As used herein, unless the context clearly dictates otherwise, Unless otherwise specified, the singular forms "a", "an" and "the" are used in conjunction with the plural It should be noted that the referent includes the Unless otherwise stated, the use of "or" means "and / or." In addition to the term "including," "include," "includes" The use of other forms, such as "included" and "included" is not limiting.

[0082] As used herein, "some embodiments," "an embodiment," "one embodiment," or References to "other embodiments" may include modifications to the particular features, structures, or aspects described in connection with the embodiments. The features are included in at least some embodiments of the present disclosure, but all embodiments include: It means that it is not necessarily included.

[0083] As used in this specification and claims, "comprising" means "comprise" and "comprises" are examples of "comprising" g)), "having" ("have" and "having any form of "has," "including," ("include" and "includes" are examples of "including" any form of "containing") or "containing" ("contain " and "contains," or any form of "containing" The term "list" is inclusive or open-ended and may include additional, unlisted elements or methods. Any embodiment discussed herein does not exclude any method steps of the present disclosure. Alternatively, it is contemplated that the present invention may be practiced with respect to compositions, and vice versa. Additionally, the compositions of the present disclosure can be used to achieve the methods of the present disclosure.

[0084] The terms "about" or "approximately" refer to an approximate value for a particular value, as determined by one of ordinary skill in the art. , within an acceptable error range, which in part depends on how the value is measured or determined. That is, within the error range that depends on the limits of the measurement system. For example, "about" means that According to practice in the art, this can mean within one standard deviation or more than one standard deviation. Alternatively, "about" can mean up to 20%, e.g., up to 10%, or up to 5% of a given value. In another example, an amount of "about 10" can mean between 10 and 9. In yet another example, the use of "about" in relation to a reference numerical value includes any amount between 1 and 11. The word also means a value ± 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or Alternatively, particularly with respect to a biological system or process, The term "about" also means within an order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold. Where specific values ​​are recited in this application and claims, they are not necessarily the same. Unless otherwise stated, "within acceptable error range" is used for a particular value. The term "about" should be assumed.

[0085] As used herein, the term "isolated" and its grammatical equivalents means to isolate an organism from its natural environment. As used herein, the term "purified" and its grammatical meanings are Equivalents are those in which the molecule or composition is isolated from nature (e.g., genomic DNA and mRNA). whether derived from DNA (including DNA fragments), synthesized (including cDNA), and / or grown under laboratory conditions Whether the product is amplified by a specific factor, the purity is increased, and in this case "purity" is a relative term. On the other hand, nucleic acids and proteins are free from diluents or Although it may be formulated with an adjuvant, for practical purposes it is best to use it in isolation. For example, when used for introduction into a cell, the nucleic acid may be Typically, it is mixed with an acceptable carrier or diluent. The term "qualitatively purified" and its grammatical equivalents refer to nucleic acids, polypeptides, proteins, Polynucleotides, proteins, polypeptides, or other compounds with which they are naturally associated. essentially free, i.e., greater than about 50%, of peptides and other molecules, More than 70% free, more than about 90% free, nucleic acid sequences, polypeptides, proteins It refers to a substance or other compound.

[0086] As used herein, a "polynucleotide" or "oligonucleotide" refers to a ribonucleotide. nucleotides or deoxyribonucleotides of any length Refers to the polymeric form of nucleic acid. This term refers only to the primary structure of the molecule. The term refers to double-stranded and single-stranded DNA, triplex DNA, as well as double-stranded and single-stranded RNA. The term also includes, for example, methylation and / or duplexing of polynucleotides. The polynucleotide may include modified forms by capping, as well as unmodified forms of the polynucleotide. The term also refers to non-naturally occurring or synthetic nucleotides, as well as nucleotides. It is also intended to include molecules containing analogs thereof.

[0087] As used herein, the terms "polypeptide," "peptide," "protein," and the like are used interchangeably. These grammatical equivalents refer to a polymer of amino acid residues. Proteins, optionally glycosylated or otherwise modified with amino acids typical of proteins within a given intracellular environment. The polypeptides and proteins disclosed herein are proteins containing other typical modifications. Proteins (including functional portions and functional variants thereof) are derived from one or more naturally occurring proteins. Instead of the amino acids listed above, synthetic amino acids may be included. Amino acids are known, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-Decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- Hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine alanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine phenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine Synthon, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, Dolin-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid , aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclo Pentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptane Carboxylic acid, α-(2-amino-2-norbornane)carboxylic acid, α,γ-diaminobutyric acid , α,β-diaminopropionic acid, homophenylalanine, and α-tert-butyl The present disclosure provides methods for producing engineered cells in which the expression of the polypeptides described herein is It is further understood that the present invention may be associated with post-translational modification of one or more amino acids of a polypeptide construct. Non-limiting examples of post-translational modifications include acetylation, phosphorylation, and formylation. Sylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, Alkylation, including methylation and ethylation, ubiquitination, and pyrrolidone carboxylic acid addition , disulfide bridge formation, sulfation, myristoylation, palmitoylation, isoprenylation , farnesylation, geranylation, glypionation, lipoylation, and iodination.

[0088] Nucleic acids and / or nucleic acid sequences may be derived from natural or artificial common ancestral nucleic acids or ancestral nucleic acid sequences. A protein and / or protein sequence is "homologous" if it is derived from that sequence. their coding DNA is derived, natural or artificial, from a common ancestral nucleic acid or ancestral nucleic acid sequence Homologous molecules are sometimes referred to as homologs. For example, Any naturally occurring protein may be modified by any available mutagenesis method. When expressed, this mutant nucleic acid produces the protein encoded by the original nucleic acid. Homology generally refers to the sequence of two or more nucleic acids or is deduced from the sequence identity between proteins (or their sequences). The exact percentage of identity between sequences useful for identifying nucleic acids and proteins of interest is Although varying, at least 25% sequence identity is used to establish homology. Sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% %, or 99% or greater sequence identity may also be used to establish homology. Methods for determining percent sequence identity (e.g., data This section describes the default parameters for BLASTP and BLASTN. , which are publicly available.

[0089] As used herein in the context of two nucleic acid or amino acid sequences of a polypeptide The terms "identical" or "sequence identity" and their grammatical equivalents are used interchangeably. Two words that are the same when aligned with respect to the maximal anaphoric relation across the comparison range As used herein, a "comparison window" refers to a range of at least about 20, typically , about 50 to about 200, more usually about 100 to about 150 consecutive positions. After optimally aligning the two sequences, the sequences are divided into the same number of It refers to a segment that can be compared to a reference sequence by consecutive positions. The optimal sequence alignment for comparison is based on the Smith method. and Waterman, Adv. Appl. Math., 2:482 (1981) local homology algorithm; Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); Pearson and Lipman, Proc. Nat. Acad. Sci USA, 85:2444 (1988); Computerized implementation of algorithms (Intelligentics, Mountai n View Calif.'s PC / Gene program, CLUSTAL, Wi sconsin Genetics Software Package, Geneti cs Computer Group (GCG), 575 Science Dr., M GAP, BESTFIT, BLAST in Washington, Wis., USA This can be done by a variety of methods, including but not limited to FASTA, FASTA, and TFASTA. For more information on the CLUSTAL program, see Higgins and Sharp, Gene, 73:237-244 (198 8) and Higgins and Sharp, CABIOS, 5:151-153 (1989), Corpet et al., Nucleic Acid s Res., 16:10881-10890 (1988), Huang et al., Computer Applications in the Biosci. ences, 8:155-165 (1992), and Pearson et al., Methods in Molecular Biology, 24: 307-331 (1994). Alignment can also be performed by visual and manual inspection. In one class of embodiments, the present invention provides a method for detecting a position of a target object. The polypeptides can be searched using, for example, BLASTP (or CL) with default parameters. USTAL, or any other available alignment software) and a sequence similar to the reference polypeptide or fragment thereof, but at least 80%, 85%, 90%, 98% identical. 99%, or 100% identical. Similarly, nucleic acids are also described in reference to a starting nucleic acid. They can also be used, for example, as in BLAST, using default parameters. N (or CLUSTAL, or any other available alignment software) 50%, 60%, 70%, 75%, 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%, 35%, can be 0%, 85%, 90%, 98%, 99%, or 100% identical. , when we say that a molecule has a certain percentage of sequence identity to a larger molecule, this means that If two molecules are optimally aligned, this percentage of residues in the small molecule will be The sequence in which the molecules are optimally aligned is used to find matching residues within the larger molecule. means.

[0090] The term "substantially identical" as applied to nucleic acid or amino acid sequences and The grammatical equivalent thereof is that a nucleic acid sequence or an amino acid sequence is encoded by a program described above, e.g. For example, at least 100% of the total number of matches was obtained by comparing the matched sequences with a reference sequence using BLAST with standard parameters. 90% or more sequence identity, at least 95%, at least 98%, and at least 99% sequence identity. For example, the BLASTN program (for nucleotide sequences) uses 11 as the default. Using a word length (W) of 10, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses a default search of 3 Code length (W), expectation value (E) of 10, and BLOSUM62 scoring matrix (See Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992) Percent sequence identity is calculated by dividing two optimally aligned sequences by the number of sequences in the comparison window. The portion of the polynucleotide sequence within the comparison window is determined by comparing the two sequences. For optimal alignment of the sequences, compare with the reference sequence (without additions or deletions) It may contain additions or deletions (i.e., gaps). The percentage is the number of identical nucleobases or amino acids. Determine the number of positions where the amino acid residue occurs in both sequences to determine the number of matched positions. The number of matched positions is divided by the total number of positions in the comparison region, and the result is multiplied by 100 to obtain the matched position. In some embodiments, substantial identity is calculated by determining the percentage of sequence identity. over a region of the sequence at least about 50 residues in length, and over a region of at least about 100 residues In some embodiments, the sequence spans at least about 150 residues and is substantially In some embodiments, the sequences are substantially identical over the entire length of the coding region. be.

[0091] A "transposon" or "transposable element" (TE) is a gene that determines its location in the genome. altering, possibly creating or storing mutations and altering the genome size of the cell , the vector DNA sequence. Transposition often results in duplication of the TE. Class I TEs are copied in two steps: first, class I TEs are copied from DNA to RNA; The resulting RNA is then reverse transcribed into DNA. The reverse transcription step is carried out by the TE itself. It is catalyzed by reverse transcriptase, which can be encoded by the host. , similar to retroviruses such as HIV. Cut-and-paste by class II TEs The transposition mechanism does not involve an RNA intermediate. Transposition is mediated by several transposase enzymes. Some transposases bind nonspecifically to any target site in DNA. whereas other transposases bind to specific DNA sequence targets. The enzymes produce staggered cleavages at the target site, resulting in 5 This results in single-stranded DNA overhangs (sticky ends) at the 1' or 3' ends. The first step is to excise the DNA transposon, and then the DNA transposon is inserted into the new The new target site is ligated to the target site, a process that involves DNA polymerase It involves the enzyme activity and the DNA ligase activity that joins the sugar-phosphate backbone. The insertion site of a DNA transposon is located at the target DNA site. A can be created by staggered cuts in A and filling in the gaps with DNA polymerase. short direct repeats, followed by excision of the TE by transposase. During the S phase of the cell cycle, the donor site Transposition occurs when the target site has not yet replicated, but the target site has already replicated. The metastasis is classified into two types: Class I TE and Class II TE. Both can be classified as "autonomous" or "non-autonomous." While E can move on its own, non-autonomous TEs require the presence of another TE to move. This means that non-autonomous TEs require a transposase (in the case of class II) or This is often due to a lack of reverse transcriptase (in the case of class I).

[0092] "Transposase" refers to a gene that binds to the ends of transposons and functions as a cut-and-paste enzyme. It catalyzes the movement of transposons to other parts of the genome by either the mitotic or replicative transfer machinery. In some embodiments, the catalytic activity of a transposase is used to transduce genes In some embodiments, the transposer can be used to move the vector into the genome. The catalytic activity of the enzyme is used to transfer genes from vectors (e.g., transposons) into the genome. In certain embodiments, the Sleeping Beauty The transposase is provided as mRNA. In some embodiments, the mRNA is capped. and a polyA tail.

[0093] The nucleic acid sequences and vectors disclosed or contemplated herein can be "transfected" into cells. introduced by "transfection," "transformation," "nucleofection," or "transduction" As used herein, "transfection," "transformation," or or "transduction" means the transfer of one or more genes to a cell by using physical or chemical methods. The term "transfection" refers to the introduction of multiple exogenous polynucleotides into a host cell. Infection methods are known, for example, calcium phosphate DNA co-precipitation (e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expr. See Session Protocols, Humana Press (1991); DEAE-dextran method ;Electroporation;Cationic liposome-mediated transfection;Tungsten particles Enhanced gene gun method (Johnston, Nature, 346: 776-777 (1990)); strontium phosphate co-transfection precipitation method (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)); and nucleofection method (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). (Trompeter et al., J. Immunol. Methods 274:245-256 (2003)). The vectors or viral vectors are available in suitable packaging, many of which are commercially available. The infectious particles can be propagated in the growing cells and then introduced into host cells.

[0094] As used herein, a "tumor antigen" refers to an antigen that is produced or overexpressed in tumor cells. Tumor antigens refer to any antigenic substance that can elicit an immune response in a host, for example. Alternatively, for purposes of this disclosure, a tumor antigen is an antigen expressed by both healthy and tumor cells. However, to identify certain tumor types, proteins may be suitable therapeutic targets. In embodiments, the tumor antigen is CD19, CD33, BCMA, CD44, alpha-leaflet Acid receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER 2, HER3, folate-binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1, MUC1 6, h5T4, PSMA, TAG-72, EGFRvIII, CD123, or VEG In one embodiment, the tumor antigen is a myeloid malignancy, e.g., glioblastoma or EGF, a target of CAR T cell therapy for treating glioblastoma multiforme (GBM), In another embodiment, the tumor antigen is CD19. In some embodiments, the tumor antigen is CD33.

[0095] As used herein, the term "enhancer" refers to, for example, a gene that is operably linked to An enhancer refers to a DNA sequence that increases the transcription of a nucleic acid sequence. They can be located many kilobases away from the target region, allowing for regulatory factor binding, DNA These may mediate changes in the methylation patterns of the target gene or in the DNA structure. Numerous enhancers are known and have been cloned from various sources. available as a peptide or within a cloned polynucleotide (e.g., (e.g., from depositories such as ATCC, as well as other commercial or private sources). Many polynucleotides, including promoters (such as the commonly used CMV promoter), It also includes enhancer sequences. Enhancers may be located upstream of a coding sequence. It may be located within this or downstream of it. The term "enhancer" refers to an enhancer region that maps within the immunoglobulin (Ig) locus. Enhancer elements derived from (such enhancers include, for example, heavy chain (mu) 5' enhancer, light chain (kappa) 5' enhancer, kappa intron enhancer This refers to enhancers that are intronic and mu intronic enhancers, as well as 3' enhancers. In general, see Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New Y See Work (1993), pages 353-363, and U.S. Pat. No. 5,885,827. sea ​​bream).

[0096] As used herein, a "coding sequence" refers to a polynucleotide that encodes a polypeptide. A region or sequence is a sequence that begins near the 5' end and ends with an initiation codon. Near the 3' end, a stop codon is bounded. It can also be called a gframe.

[0097] As used herein, "operably linked" refers to the ability of a DNA segment to interact with another DNA segment. a physical and / or functional linkage to the A segment, wherein the segment is It refers to a linkage that allows the gene products to function in their intended manner. The DNA sequence to be loaded may be, for example, a promoter, enhancer, and / or Regulatory sequences, such as silencers, can modulate the transcription of DNA sequences either directly or operably linked to a regulatory sequence when linked in an indirectly enabling manner For example, a DNA sequence may be selected if it is located downstream of the transcription start site of a promoter. ligated to the promoter in the correct reading frame relative to the transcription start site. When a DNA sequence is inserted into a target region, allowing transcription elongation to proceed through the DNA sequence, An enhancer or silencer is operably linked to a promoter. are ligated to a DNA sequence to increase or decrease transcription of the DNA sequence, respectively. When inserted, it is operably linked to a DNA sequence encoding a gene product. Enhancers and silencers are often located upstream of the coding region of a DNA sequence. It may be located downstream of this or embedded within it. Null sequences are signal transduction pathways when expressed as a preprotein that participates in the secretion of the polypeptide. The DNA for the polypeptide sequence is operably linked to the DNA encoding the polypeptide. Linkage of the A sequence to the regulatory sequence is typically by ligation at appropriate restriction sites. or by using restriction endonucleases known to those skilled in the art to insert This is achieved via an adapter or linker.

[0098] The term "transcriptional regulator" refers to a promoter-driven DNA Biochemical elements that act to prevent or inhibit transcription of a sequence (e.g., repressors) promoter proteins or nuclear inhibitory proteins), or under certain environmental conditions, A biosynthetic molecule that acts to enable or stimulate the transcription of a motor-driven DNA sequence. "Enhanced expression" refers to a genetic element (e.g., inducer or enhancer) that mediates a specific target gene expression.

[0099] As used herein, the terms "induce" and "induce" and their grammatical equivalents The substance is a compound that controls the transcription of a nucleic acid sequence, the activity of a promoter, and and / or refers to an increase in the expression of a promoter relative to some basal level of transcription.

[0100] A "target" gene or "heterologous" gene or "gene of interest (GOI)" is a gene refers to a gene that is introduced into a host cell by transduction. Exemplary GOIs are described herein. antigen-binding polypeptides, chimeric receptors, CARs, TCRs, cytokines, and It may be an antigen-binding polypeptide, which may / may comprise a cell tag.

[0101] As used herein, a "recombinase" refers to a gene that induces site-specific recombination between defined sites. A group of enzymes that can facilitate the synthesis of DNA fragments, where the sites are physically separated on a single DNA molecule. Defined recombination refers to a group of enzymes in which the target gene is a nucleotide or site on a separate DNA molecule. The DNA sequences at the sites are not necessarily identical. Recombination is initiated by protein-DNA interaction. Within the group of enzymes, there are enzymes involved in phage integration and excision (e.g., λ Integra). enzyme, ΦC31), resolution of circular plasmids (e.g., Tn3, gamma delta, Cre, Flp), DNA inversion for expression of alternative genes (e.g. Hin, Gin, Pin), genes during development (e.g., in the Anabaena genus) assembly of nitrogen fixation genes) and transposition (e.g., IS607 transposon) There are many proteins that catalyze the recombination of DNA. Most site-specific recombinases have evolved over time. Based on affinity and mechanistic similarity, they fall into one of two families: , λ integrase family or tyrosine recombinases (e.g., Cre, Flp , XerD), and the resolvase / integrase family, or serine recombinase enzyme family (e.g., ΦC31, TP901-1, Tn3, gamma delta) .

[0102] "Recombination junction sites" refer to sites recognized by the recombinase enzymes described herein. Typically, one site is a specific polynucleotide sequence. a site within the target region (e.g., a chromosome or episome in a prokaryotic or nucleotidic organism) that allows targeted recombination Two different sites (called "complementary sites") present on the nucleic acid to be integrated at the site ) are involved. Herein, the terms "conjugation" and "conjugation" refer to the bacterial target and phage donor, respectively. The terms "attB" and "attP" are used to refer to the fusion (or recombination) sites. However, recombination sites for specific enzymes may have different names. The core region includes a left arm and a right arm separated by a core region or spacer region. Therefore, the attB recombination site is BOB' [where B and B' are , the left arm and the right arm, and O is the core region. tP is POP' [in the sequence, P and P' are arms, and O is again a codon] The attB site is the target region. The recombination sites flanking the DNA to be integrated during simultaneous integration of nucleic acids are designated as "attL" and and "attR." Thus, using the terminology above, the attL site and The attR site consists of BOP' and POB', respectively. In the designation, the "O" is omitted and attB and attP are designated, for example, as BB' and BB', respectively. It's called PP'.

[0103] Modulation of gene expression In the field of genetic engineering, precise control of gene expression is crucial for development and other physiological processes. Gene expression is a valuable tool for studying, manipulating, and controlling many It is a complex biological process that involves specific protein-protein interactions. Inducible gene expression systems or "gene switches" are useful for gene therapy, intracellular, and large-scale targeting. Protein generation, cell-based high-throughput screening assays, functional and traits in transgenic plants and animals However, for therapeutic purposes, regulated and localized The required expression is important to prevent off-target effects. In some cases, and in specific proximity or at specific locations of target cells, such as cytokines, A means for regulating the expression of heterologous genes is presented. A method for regulating the expression of a heterologous gene, such as a cytokine, by ligand-induced Optionally, the method comprises the step of: In its presence, it results in a reduced or absent basal level of heterologous gene expression. In certain embodiments, the ligand-inducible promoter is a ligand-inducible promoter of the gene switch. Further, the present invention relates to the use of heterologous genes, such as cytokines, in engineered cells. In this specification, engineered cells are those that are in their natural state, and methods for regulating expression from these cells are presented. or cells that have been modified from their endogenous state. Examples of engineered cells are cells that have been modified (e.g., poly the cells described herein), In some cases, the engineered cells can be engineered immune effector cells. In some cases, the engineered cells are T cells. In other cases, the engineered cells are NK cells. In some cases, the engineered cells are activated before inducing expression of the heterologous gene with a ligand. In some cases, the engineered cells are activated by exposing the cells to an antigen. The antigen may be an antigen recognized by an antigen-binding polypeptide expressed by a tumor cell. In one case, the engineered cells are designed to respond to such antigens. The polypeptide may comprise an antigen-binding polypeptide that binds to the polypeptide.

[0104] In a further case, the gene switch construct of the ligand-inducible promoter of the gene switch The component can be further regulated by a tissue-specific promoter. In this case, the gene switch component may be a tissue-specific promoter, e.g., in activated T cells or It is expressed only upon activation in activated NK cells.

[0105] vector

[0106] An "expression vector" or "vector" is an autonomous vector that mediates replication of a polynucleotide within a cell. behaves as a unit (i.e., is capable of replicating under its own control), or is any genetic element that can replicate upon insertion into a host cell chromosome, e.g. , plasmids, chromosomes, viruses, transposons, and the like, which are complexes of joined segments. and / or by incorporating another polynucleotide segment into it to effect its production and / or expression. Suitable vectors include plasmids, transposons, Vectors include, but are not limited to, bacteriophages and cosmids. Ligation or insertion of the vector into a host cell of the joined segments is carried out. The host may contain polynucleotide sequences necessary to effect expression of the host. Varies depending on the host organism: promoter sequences that effect transcription, enhancers that increase transcription nucleotide sequence, a ribosome binding site sequence, and a transcription / translation termination sequence. The vector involves ligation or integration of the vector into the DNA sequence of the host cell. It may be possible to directly express the product of a nucleic acid sequence encoded therein without the need for transfection.

[0107] A vector may also contain a "selectable marker gene." The term "selectable marker gene" refers to a gene that identifies cells expressing a nucleic acid sequence in the presence of a corresponding selectable agent. It refers to a nucleic acid sequence that allows specific selection in the forward or reverse direction of the nucleic acid sequence in the presence of the nucleic acid sequence. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Application Publication No. Publication No. 1992 / 08796 and No. 1994 / 28143 Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980), O'Hare et al. , Proc. Natl. Acad. Sci. USA, 78: 1527 (1981), Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981), Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981 ), Santerre et al., Gene, 30: 147 (1984), Kent et al., Science, 237: 901-903 (19 87), Wigler et al., Cell, 11: 223 (1977), Szybalska & Szybalski, Proc. Natl. Aca d. Sci. USA, 48: 2026 (1962), Lowy et al., Cell, 22: 817 (1980), and U.S. Pat. The invention described in U.S. Pat. Nos. 5,122,464 and 5,770,359 It is being done.

[0108] In some embodiments, the vector is capable of replicating in a host cell and can be selected appropriately. In the presence of selective pressure, "episodes" persist as extrachromosomal segments of DNA within the host cell. These vectors are "episomes" or "episomes" (see, e.g., Conese et al., Gene Therapy (See, for example, J. Immunol., 11:1735-1742 (2004)). Representative, commercially available episomal expression vectors include: , Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EB V) Episomal plasmids, including but not limited to, those that use an origin of replication (oriP) The vectors pREP4, pCEP4, and pREP7, as well as the Invitrogen pcDNA3.1 from Stratagen (Carlsbad, Calif.) pBK-CMV from e (LaJolla, Calif.) contains EBNA1 and oriP Non-limiting examples of episomal vectors include those that use the T antigen and SV40 origin of replication instead of the This shows a typical example.

[0109] As used herein, a "gene expression cassette" refers to a portion of a vector, such as EF1a Constitutive promoters such as 5'UTR, 3'UTR, and and polyA elements, as well as one or more genes of interest (GOI). In an embodiment, the polyA element is SV40e polyA (SEQ ID NO: 65). In an embodiment, the polyA element is a bidirectional aCA polyA (SEQ ID NO: 66). In another embodiment, the polyA is PA2 polyA (SEQ ID NO: 67). The gene expression cassette contains a DNA-binding domain fused to a nuclear receptor ligand-binding domain. transactivating domains and / or nuclear receptor ligand binding domains fused to The vector may further comprise a gene switch component, such as an activation domain. It may contain multiple gene expression cassettes.

[0110] Vector modification Polynucleotide vectors useful in the methods and compositions described herein are referred to as "pharmaceutical vectors." The vector complies with the Good Manufacturing Practice (GMP) for pharmaceuticals and quasi-drugs. For example, GMP vectors may be more pure than non-GMP vectors. Therefore, purity can be measured by bioburden. For example, bioburden is In the vector composition, aerobic bacteria, anaerobic bacteria, spore-forming bacteria, fungi, or a combination thereof Optionally, the pure vector may be low in endotoxin or Purity is confirmed by double-stranded primer walking sequencing. Plasmid identity can also be determined by the source of the vector to determine its purity. The GMP vectors of the present disclosure may be 10% to 99% purer than non-GMP vectors. GMP vectors are not bioburden, endotoxin, sequencing, or any of these. than non-GMP vectors, as measured by the presence of the combination , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% pure It can be stylish.

[0111] Optionally, a terminator sequence at the end of the first gene program is used. Terminator sequences ensure that transcripts terminate before initiating a second gene program. For example, expression vectors may contain the sequences required for the termination of transcription and stabilization of mRNA. Such sequences may be derived from eukaryotic or viral DNA or cDNA. A is available from the 5' untranslated region and sometimes from the 3' untranslated region. These regions are transcribed as polyadenylated fragments in the untranslated portion of the mRNA. The cells containing the expression vector may be transformed in vivo or in vivo. are grown in vitro under conditions that result in expression of the desired polypeptide.

[0112] In some cases, the spacer sequence is encoded by a polynucleotide in the vector. , can be used at the end of the first polypeptide. The sequence can be used at the end of the second gene in the vector. The sequence may also be used in a vector following a first gene and a second gene. can.

[0113] These vectors are used to insert vectors encoding the gene of interest, or a portion of the gene of interest. The gene or portion of the gene can be expressed in a viral vector. The insertion can be performed using any method, viral or non-viral. For example, the method can be It can be a non-viral based technique.

[0114] IRES elements Also provided herein are methods for the expression of the polynucleotides and polypeptides described herein. Also disclosed herein are constructs containing IRES elements that facilitate the expression and functionality of the The term "internal ribosome entry site (IRES)" used refers to an internal ribosome entry site (IRES). In a vector containing an IRES sequence, the first gene However, by a mechanism involving its unique 5'-UTR, cap-dependent ribosome scanning The IRES is the site of translation, whereas subsequent gene translation is achieved by direct ribosome movement to the IRES. IRES sequences are accessible to eukaryotic ribosomes in a cap-independent manner. The IRES sequence may be capable of initiating translation without binding to the 5' cap end. , which may allow the expression of multiple genes from a single transcript (Mountford and Smith 1995 ).

[0115] As used herein, the term "CAP" or "cap" The term generally refers to a 7-methylglucan residue linked 3'-5' to the 5' end of a eukaryotic mRNA. A modified nucleotide that is an anosine (7meG-ppp-G) from this mRNA It is used as a required element in the normal translation initiation pathway during the expression of the protein It refers to a modified nucleotide that is

[0116] In certain cases, the IRES region may be a promoter region for a picornavirus, an encephalomyocarditis virus, a hepatitis C virus, or a hepatitis B virus. In other cases, the IRES region may be derived from a virus, such as the IRES sequence of the inflammatory virus. As used herein, "EMCV" or "encephalomyocarditis" may be derived from an encephalomyocarditis virus. The term "virus" refers to the encephalomyocarditis virus, a virus of the genus Picornaviridae. An isolate or strain that is any member of the Ruccella genus. Examples include EMCV-R (Ruccella genus). The strains of the virus are Colombian SK virus and eukaryotic initiation factor 4G. Immunoglobulin heavy chain binding protein, c-myc proto-oncogene, vascular endothelial growth factor, fibronectin cellular I, such as fibroblast growth factor 1 IRES, or any combination or modification thereof; RES elements can be used. In some cases, cellular IRESs can be used in viral It may have an increased gene expression when compared to an IRES.

[0117] A viral IRES sequence, a cellular IRES sequence, or a combination of these can be inserted into the vector. The IRES can be used in the encephalomyocarditis virus (EMCV) or poliovirus. In some cases, the IRES element can be derived from poliovirus (PV) ), encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), porcine schizophrenia virus rus 1 (PTV-1), Aichi virus (AiV), Seneca Valley virus (SVV), C Hepatitis C virus (HCV), Classical Swine Fever Virus (CSFV), Human Immunodeficiency Virus 2 ( HIV-2), human immunodeficiency virus 1 (HIV-1), Moloney murine leukemia virus (MoMLV), feline immunodeficiency virus (FIV), mouse mammary tumor virus (MMTV) , latent human cytomegalovirus (pUL138), Epstein-Barr virus (E BNA-1), herpesvirus Marek's disease (MDVRLORF9), SV40 polycystic Tronic 19S (SV4019S), Rhopalosiphum padi (wheat aphid) RhPV, Cricket paralysis virus (CrPV), Ectr opis obliqua picorna-like virus (EoPV), Plautia stal i) PSIV, Triatoma virus (TrV), and bee hemp Infection with paralytic sissist virus (IAPV, KBV), Blackcurrant revertant virus (BRV), Pelargonium variegation virus (PFBV), Hibiscus chlorotic variegation virus Virus (HCRSV), Crucifer-infecting Tobamovirus (CrTMV), Potato leaf cigar Virus (PLRV), Tobacco etch virus (TEV), Giardia virus (GLV) ), Leishmania RNA virus 1 (LRV-1), and these Optionally, the IRES is selected from the group consisting of Apaf-1, Apaf-2, Apaf-3, Apaf-4, Apaf-5, Apaf-6, Apaf-7, Apaf-8, Apaf-9, Apaf-11, Apaf-12, Apaf-13, Apaf-14, Apaf-15, Apaf-16, Apaf-17, Apaf-18, Apaf , XIAP, HIAP2 / c-IAP1, DAP5, Bcl-2, c-myc, CAT- 1, INR, Differentiation LEF-1, PDGF2, HIF-1a, VEGF, FGF2, BiP , BAG-1, CIRP, p53, SHMT1, PITSLREp58, CDK1, Rp r, hid, hsp70, grim, skl, Antennapedia, dFoxO, dInR, Adh-Adhr, HSP101, ADH, URE-2, GPR1, NCE1 02, YMR181a, MSN1, BOI1, FLO8, GIC1, and any of these The IRES element is selected from the group consisting of two opening When inserted between open reading frames (ORFs), translation initiation occurs within the first ORF. canonical 5'-m7GpppN cap-dependent mechanism and IRES element This can occur by a cap-independent mechanism within a second ORF downstream of the first.

[0118] In some cases, the genes may be linked by an internal ribosome entry site (IRES). IRES can allow simultaneous expression of multiple genes. For example, The sequence may allow for the production of multiple proteins from a single mRNA transcript. The IRES is capable of binding to the 5' cap-independently and initiating translation.

[0119] In some cases, the IRES sequence may be 500 base pairs or approximately this length. The IRES sequence can be 300 to 1000 base pairs. For example, 300, 350, 400, 450, 500, 550, 600, 650, 700, 75 It can be 0, 800, 850, 900, 950, or 1000 base pairs in length.

[0120] Reducing expression of downstream genes in vectors that optionally contain IRES sequences For example, the gene following the IRES sequence can be linked to the gene preceding the IRES sequence. The expression can be reduced by 1% to 99% of the preceding gene. It may be a reduction.

[0121] In certain embodiments, the IRES has at least one nucleotide sequence with the nucleotide sequence of SEQ ID NO:18. 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 1 The EMCV IRES contains a nucleotide sequence with 0.000% identity.

[0122] Linker Also, the expression and functionality of the polynucleotides and polypeptides described herein Also disclosed are constructs comprising a linker that facilitates Linkers can be used within the polynucleotides described herein. The nucleotide linker is compatible with the vectors containing the polynucleotides described herein. Two strands of DNA containing desired restriction sites that can be added to create certain end structures. Optionally, the polynucleotide linker may be a chain segment, as described herein. It may be useful to modify vectors containing polynucleotides to be transfected. For example, Modifications of the vector, including nucleotide linkers, can include changes in the multiple cloning site, and The polynucleotide linker may also be a blunt-ended polynucleotide linker. The ends of the insert DNA are cut with restriction enzymes and cloned into a vector with sticky ends. The use of polynucleotide linkers can also be used to accommodate This may be more efficient than blunt-end ligation to a vector, and may be useful for downstream applications. Optionally, the insert may be released from the vector. It may be a polynucleotide sequence that encodes a polypeptide useful for therapeutic applications. Thus, the linker can be a cleavable linker.

[0123] The polynucleotide linker can be an oligomer. It can be a double-stranded DNA, a single-stranded DNA, or a combination thereof. The polynucleotide linker can be RNA. Optionally, the polynucleotide linker can be ligated by T4 ligase. and ligating the vector containing the polynucleotide described herein. To facilitate ligation, an excess amount of polynucleotide linker can be used. The vector may be added to a composition containing the insert and vector. Before introducing the anchor, the insert and vector are pretreated, e.g., with methylase. Pretreatment with HCl can prevent undesired cleavage of the insert DNA.

[0124] In certain embodiments, the polypeptides encoded by the polynucleotides described herein are The two or more polypeptides may include an intervening linker polypeptide encoding an intervening linker polypeptide. As used herein, a polynucleotide may be encoded by a sequence "Intervening linker polypeptide" refers to an amino acid sequence that separates two or more polypeptides. The term "peptide" refers to a transmembrane domain that is a cell surface polypeptide (e.g., a naturally occurring polypeptide). The polypeptides disclosed herein may be linked to a polypeptide of the present invention (including truncated variants of the polypeptide). "Peptide linker" refers to a sequence of amino acids that is optionally incorporated into a peptide construct. In certain cases, the intervening linker may be a cleavable intervening linker. In some embodiments, the linker is a truncated linker or a ribonucleotide. In some embodiments, the linker is a cleavable linker or a ribosomal linker. The skipping linker sequences are 2A, GSG-2A, GSG linker, SGSG linker, Carr, Furinlink, and variants and derivatives thereof. In some embodiments, the 2A linker is a p2A linker, a T2A linker, an F2A linker, or a In some embodiments, the polypeptide of interest is a readily available E2A linker. They are expressed as fusion proteins linked by a cleavable intervening linker polypeptide. In certain embodiments, the cleavable intervening linker polypeptide is F / T2A, T2A, p of 2A, 2A, GSG-p2A, GSG linker, and furin link mutants In certain embodiments, the linker polypeptide can be any one or more of: These include the linker polypeptides disclosed in the table below.

[0125] [Table 1-1]

[0126] [Table 1-2]

[0127] In some embodiments, the intervening linker polypeptide is a Whitlow linker (SEQ ID NO: 146), GSG linker (SEQ ID NO: 148), SGSG linker (SEQ ID NO: 149), (G4S)3 linker (SEQ ID NO: 150), furin cleavage site / furin link 1 ( Furlink 1) (SEQ ID NO: 151), Fmdv linker (SEQ ID NO: 152), Tosea aci Thosea asigna virus region 2A (T2A) (SEQ ID NO: 153), furin- GSG-T2A (SEQ ID NO: 154), furin-SGSG-T2A (SEQ ID NO: 155) , Porcine teschovirus 12A region (P2A) (SEQ ID NO: 156), GSG-P2A ( Equine rhinitis virus type A region 2A (E2A) (SEQ ID NO: 158), at least one amino acid sequence identical to that of the foot-and-mouth disease virus 2A region (F2A) (SEQ ID NO: 159). Also 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 10 Optionally, the intervening linker polypeptide comprises an amino acid sequence having 0% identity to the amino acid sequence of the present invention. , and the amino acid sequence of the linker (SEQ ID NO: 147, 161, or 162) 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 100 % identity. In some cases, viral 2A sequences may be used. The 2A element can be shorter than the IRES, with 5 to 100 base pairs. In some cases, the 2A sequence may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 5 0, 55, 60, 65, 70, 75, 80, 85, 90, or 100 nucleotides in length The 2A-linked genes may be expressed in a single open reading frame. The "self-cleavage" can be achieved by cleaving the last two amino acids at the C-terminus of the 2A polypeptide. It is possible for co-translational interactions between GPs, which are acids, to occur co-translationally, resulting in equal amounts of co-expressed proteins. Drop.

[0128] The viral 2A sequence can be about 20 amino acids. Optionally, the viral 2A sequence is , the consensus motif, Asp-Val / Ile-Glu-X-Asn-Pro -Gly-Pro. The consensus motif sequence may act co-translationally. For example, it can prevent normal peptide bond formation between glycine and proline residues. This results in ribosome skipping and cleavage of the nascent polypeptide. This effect can result in multiple genes at equimolar levels.

[0129] 2A peptides are the sequences of multiple proteins within a single open reading frame. Afterwards, the polypeptides can be cleaved into individual polypeptides via the ribosomal skipping mechanism. This may allow translation into a polypeptide (Funston, Kallioinen et al. 2008). In the form, the 2A sequence is F / T2A, T2A, p2A, 2A, T2A, E2A, F2A, and BmCPV2A, BmIFV2A, and any combination thereof.

[0130] In some cases, the vector may include an IRES sequence and a 2A linker sequence. In this case, the expression of multiple genes linked to the 2A peptide is controlled by the This can be facilitated by a spacer sequence (GSG). A pacer, a linker, an adapter, a promoter, or a combination thereof may be combined. For example, the linker may be a spacer (SGSG linker or or GSG linker or Whitlow linker) and furin linker (RA- The spacer may be I-CeuI. The linker can be engineered. For example, the linker can contain chemical features such as hydrophobicity. Optionally, the at least two linker sequences can be designed to include: In other cases, multiple linkers may be used within a vector, resulting in the same protein. For example, the gene of interest can be at least They may be separated by two linkers.

[0131] In certain embodiments, the polypeptides encoded by the polynucleotides described herein are The two or more polypeptides may be separated by an intervening sequence encoding a linker polypeptide. In certain cases, the linker is a cleavable linker. In some embodiments, the polypeptide of interest is linked to the cleavable intervening linker polypeptide. In certain embodiments, the protein is expressed as a fusion protein in which the cleavable phosphorylase is linked to the cleavable phosphorylase. Car polypeptides include furin-link, fmdv, p2a, GSG- p2a, and / or fp2a. Thus, the linker is APVKQGSG (SEQ ID NO: 161).

[0132] In certain instances, the linker polypeptide has the amino acid sequence "RAKR" (SEQ ID NO: 151). In certain instances, the furin linker polypeptide may comprise a "CG TGCAAAGCGT" (SEQ ID NO: 6) or "AGAGCTAAGAGG" (SEQ ID NO: 9) can be encoded by a polynucleotide sequence comprising

[0133] In some embodiments, the linker is a The linker may be a flexible linker, a non-flexible linker, an in vivo cleavable linker, or a In some cases, the linker may be a functional The domains are linked together (as in the case of flexible and inflexible linkers) In some cases, such as in the case of in vivo cleavable linkers, functional domains can be It may also be released in vivo.

[0134] The linker improves biological activity and increases expression yield, thereby achieving desirable pharmacokinetics. The linker may also be a hydrazone, peptide, disulfide, or thioether. It may also include thioesther.

[0135] In some cases, the linker sequences described herein may include flexible linkers. Flexible linkers allow for some degree of movement or interaction between the joined domains. Flexible linkers can be small, non-polar (e.g., Gly), or can be composed of polar amino acids (e.g., Ser or Thr). , and may have a sequence consisting primarily of a string of Gly and Ser residues ("GS" linker). An example of a flexible linker is a linker having the sequence (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number "n", this exemplary G The length of the S linker can also be optimized to achieve proper separation of functional domains, if necessary. In addition to the GS linker, other flexible linkers can also be used to maintain the essential interdomain interactions. Linkers can also be used in recombinant fusion proteins. may also be rich in small or polar amino acids, such as Gly and Ser. , containing additional amino acids such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu are used to improve solubility. It can be improved.

[0136] The flexible linkers incorporated into the linker sequences described herein provide good flexibility. Small or polar amino acids, such as Gly and Ser, are used to confer stability and solubility. The flexible linker may be rich in amino acids. In addition, flexible linkers may be suitable for non-rigid protein domains. Passive linkers that may not have flexible structures but maintain the distance between functional domains The length of the flexible linker can be adjusted to ensure proper folding. This may allow for better binding and optimal biological activity of the fusion protein. Cut.

[0137] The linkers described herein can optionally further comprise a non-flexible linker. Using a non-flexible linker to maintain a constant distance between domains of a polypeptide Examples of non-flexible linkers include alpha-helix-forming linkers, to name a few. , Pro-rich sequence, (XP)n, X-Pro backbone, A(EAAAK)nA (n = 2-5 The rigid linker may optionally be an α-helical linker. , or may contain multiple Pro residues, thereby exhibiting a relatively rigid structure.

[0138] The linkers described herein may, in some cases, be cleavable. Non-cleavable linkers are used in in vivo or ex vivo processes. Functional domains are covalently linked together so that they act as one molecule throughout the process. The linker may also be cleaved in vivo. Carriers can be introduced in vivo to release free functional domains. Cleavable linkers can be degraded in the presence of reducing agents and proteases, to name a few. For example, reduction of disulfide bonds can be used to create cleavable linkers. In the case of disulfide linkers, the linker can be made by disulfide bonding with thiols such as glutathione. A cleavage event via sulfide exchange could result in cleavage. Cleavage of the linker in the recombinant fusion protein in vivo has also been demonstrated. vo, which occurs in specific cells or tissues under pathological conditions (e.g., cancer or inflammation). by proteases that may be expressed or may be restricted to specific cellular compartments. In some cases, a cleavable linker may allow for targeting of cleavage. For example, the specificity of many proteases results in slow cleavage of the linker within the constraint compartment. Cleavable linkers may also be hydrazones, peptides, disulfides, or thioesters. For example, hydrazones may confer serum stability. The hydrazone may be capable of cleavage within an acidic compartment. The acidic compartment has a pH of up to 7. The linker may also include a thioether. The thioether may be non-reducing. Thioethers can be designed for intracellular proteolysis.

[0139] In certain embodiments, the fmdv linker polypeptide comprises SEQ ID NO: 152 and at least At most about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, It includes sequences that may be 90%, 95%, 97%, 98%, 99%, or 100% identical. In certain embodiments, the fmdv linker polypeptide comprises two or more fusion One or more of the linkers encoded in a single vector linking the fusion proteins In certain instances, the fmdv linker polypeptide is a polynucleotide It may be encoded by a nucleic acid sequence of an open reading frame (ORF). Thus, the ORF encoding fmdv comprises or consists of the sequence of SEQ ID NO:7. In certain embodiments, the polynucleotide encoding fmdv comprises SEQ ID NO: 7 and at least one At least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical.

[0140] In certain cases, the linker polypeptide may be a "p2a" linker. In certain embodiments, the p2a polypeptide has at least about 45% identity with SEQ ID NO: 156; 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, The sequences may be 97%, 98%, 99%, or 100% identical. In embodiments, the p2a linker polypeptide links two or more fusion proteins. The linker may be one or more of the linked sequences encoded within a single vector. In some cases, the p2a linker polypeptide is a polynucleotide having an open reading frame. In certain embodiments, the p2 The ORF encoding a comprises or consists of the sequence of SEQ ID NO: 11. In this case, the polynucleotide encoding p2a is a sequence identical to SEQ ID NO: 11 and at least 45 %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 %, 97%, 98%, 99%, or 100% identical, or at least nearly so The ratios of these may be the same.

[0141] Optionally, the linker polypeptide can be a "GSG-p2a" linker. In certain embodiments, the GSG-p2a linker polypeptide comprises SEQ ID NO: 157 and at least one At least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , including sequences that may be 90%, 95%, 97%, 98%, 99%, or 100% identical In certain embodiments, the GSG-p2a linker polypeptide may comprise two or more Linkers encoded within a single vector linking more than 10 fusion proteins Optionally, the GSG-p2a linker polypeptide may be one or more of: The open reading frame (ORF) of a nucleic acid sequence encoded by the The ORF encoding GSG p2a may comprise the sequence of SEQ ID NO: 12. Therefore, the polynucleotide encoding GSG-p2a has a sequence similar to SEQ ID NO: 12 and at least 4 5%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9 5%, 97%, 98%, 99%, or 100% identical, or at least nearly identical These ratios may be the same.

[0142] The linker polypeptide can be the "fp2a" linker provided herein. In certain embodiments, the fp2a linker polypeptide comprises at least one of SEQ ID NO: 160 and SEQ ID NO: 161. Also about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% The sequences may be 95%, 97%, 98%, 99%, or 100% identical. In certain instances, the fp2a linker polypeptide may comprise two or more fusion proteins. One or more linkers encoded within a single vector link the proteins. Optionally, the fp2a linker polypeptide is The nucleic acid sequence may be encoded by an open reading frame (ORF). In one embodiment, the polynucleotide encoding the fp2a linker has at least one sequence similar to SEQ ID NO: 15. Also 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 97%, 98%, 99%, or 100% identical, or at least nearly identical These ratios may be approximately the same.

[0143] In some cases, the linker can be engineered. For example, the linker can be hydrophobic, etc. In some cases, at least two The linker sequence may result in the same protein. or 162 polypeptide sequences and 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 The sequences may be 0.001% identical, or may be approximately these percentages. A number of linkers can be used in the vector, for example, The gene of interest and one or more gene switch polypeptide sequences are shown in Figure 15 and Figure 16. 16, can be separated by at least two linkers. The genes can be 2, 3, 4, 5, 6, 7, 8, 9, or up to 10. They may be separated by a linker.

[0144] The linker can be an engineered linker. Methods for designing linkers include computer-aided In some cases, the computational method may be a graphical method. Three-dimensional peptides derived from a database using computational methods. A library of structures can be searched for suitable peptides. Haven Protein Data Bank (PDB) was used to select linkers. The distance between selected amino acids can be measured.

[0145] In some embodiments, a polypeptide comprising a furin polypeptide and a 2A polypeptide A polynucleotide encoding a peptide construct is provided, in this case a furin polypeptide. and 2A polypeptides containing at least three hydrophobic amino acids. Optionally, the at least three hydrophobic amino acids are linked by a linker. Lysine (Gly) (G), Alanine (Ala) (A), Valine (Val) (V), Leucine Leucine (Leu) (L), isoleucine (Ile) (I), proline (Pro) (P), phenylalanine (P) Phe (F), Methionine (Met) (M), Tryptophan (Trp )(W). Optionally, the polypeptide linker is also selected from the list consisting of: One or more GS linker sequences, e.g., (GS)n, (SG)n, (GSG)n (sequence Sequence No. 78), and (SGSG)n (SEQ ID NO: 79) [wherein n is any number from 0 to 15] [may be a number of .times. ...

[0146] A method for obtaining improved expression of a polypeptide construct, comprising: and a polynucleotide encoding said polypeptide construct comprising a second functional polypeptide. providing a polypeptide comprising the first functional polypeptide and a second functional polypeptide; and a peptide with at least 60% identity with the sequence APVKQ (SEQ ID NO: 162). and wherein the polypeptides are linked by a linker polypeptide comprising the sequence; Expressing the polynucleotide, wherein the sequence of the polypeptide construct is A linker polypeptide having a sequence with at least 60% identity to APVKQ. and a step of: A method is provided that includes:

[0147] promoter

[0148] "Promoter" refers to a region of a polynucleotide that induces transcription of a coding sequence A promoter is a gene that is located near the transcription start site of a gene, on the same strand of DNA, and A promoter is located upstream (towards the 5' region of the sense strand). Constitutive promoters are active under all conditions, whereas other promoters, e.g. Inducible promoters are regulated and active in response to specific stimuli. The promoter may be a tissue-specific promoter, including but not limited to a T cell-specific promoter. It is a regulator or an activated promoter.

[0149] As used herein, the term "promoter activity" and its grammatical equivalents refer to The degree of expression of a nucleotide sequence operably linked to a promoter whose activity is measured. The promoter activity can be measured by, for example, Northern blot analysis, to determine the activity of the RNA produced. It can also be measured directly by determining the amount of transcript and promoter-linked The amount of a product encoded by a linked nucleic acid sequence, such as a linked reporter nucleic acid sequence, is determined. It can also be measured indirectly by

[0150] As used herein, an "inducible promoter" refers to a promoter that is regulated by a transcriptional regulator, e.g., a biological factor. Or refers to a promoter that is induced to activity by the presence or absence of an abiotic factor. Inducible promoters are promoters that allow expression of genes operably linked to them to be expressed in an organism or in a particular They are useful because they can be turned on or off at certain stages of tissue development. Examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, and promoter, steroid-regulated promoter, metal-regulated promoter, pathogenesis-regulated promoter promoters, temperature-regulated promoters, and light-regulated promoters. In this embodiment, the inducible promoter is part of a gene switch. It can be a ligand-inducible promoter of the gene switch. The switch is a small molecule ligand-induced A potential ecdysone receptor-based gene switch with two polypeptides In some cases, the gene switches are incorporated herein by reference in their entirety. No. 2001 / 009050 (International Publication No. PCT / US2001 / 009050) No. 070816; U.S. Pat. No. 7,091,038; U.S. Pat. No. 7,777 Specification No. 6,587; Specification No. 7,807,417; Specification No. 8,202,718 PCT / US Application No. 2001 / 030608 (International Publication No. 2002 / 029 No. 075; U.S. Patent No. 8,105,825; U.S. Patent No. 8,168,4 26; PCT / US Application No. 2002 / 005235 (International Publication No. WO 200 2 / 066613); U.S. Patent Application Publication No. 10 / 468,200 (U.S. Publication No. 20120167239); PCT / U.S. Application No. 2002 / 005 706 (International Publication No. 2002 / 066614); U.S. Pat. Specification No. 531,326; Specification No. 8,236,556; Specification No. 8,598,409 Specification; PCT / US Application No. 2002 / 005090 Specification (International Publication No. 2002 / 0 No. 66612; U.S. Pat. No. 8,715,959 (U.S. Publication No. 2005-010012) 060100416); PCT / US Application No. 2002 / 005234 ( WO 2003 / 027266; ​​U.S. Pat. No. 7,601,508 Specification; Specification No. 7,829,676; Specification No. 7,919,269; No. 8, No. 030,067; PCT / US Application No. 2002 / 005708 (International Publication U.S. Patent Application Publication No. 2002 / 066615; U.S. Patent Application Publication No. 10 / 468,19 2 (U.S. Publication No. 20110212528); PCT / U.S. Application No. 200 2 / 005026 (International Publication No. 2003 / 027289); U.S. Patent No. 7,563,879; Patent No. 8,021,878; Patent No. 8,497 ,093; PCT / US Application No. 2005 / 015089 (International Publication No. No. 005 / 108617; U.S. Pat. No. 7,935,510; No. 8,076,454; PCT / US Application No. 2008 / 011270 (National International Publication No. 2009 / 045370; U.S. Patent Application Publication No. 12 / 241, 018 (U.S. Publication No. 20090136465); PCT / U.S. Application No. 008 / 011563 (International Publication No. WO 2009 / 048560); U.S. Patent Application Publication No. 12 / 247,738 (U.S. Publication No. 20090123441 PCT / US Application No. 2009 / 005510 (International Publication No. 2010 / 042189 pamphlet); U.S. Patent Application Publication No. 13 / 123,129 specification ( U.S. Publication No. 20110268766; PCT / U.S. Application No. 2011 / 0296 82 (International Publication No. 2011 / 119773); U.S. Patent Application Publication No. 13 / 636,473 (U.S. Publication No. 20130195800); PC T / US Application No. 2012 / 027515 (International Publication No. WO 2012 / 122025 brochure); and U.S. Pat. No. 9,402,919, However, without limitation, any of the systems described therein may be used. The component may be selected from any of the ecdysone-based receptors.

[0151] As used herein, a subject may be provided with at least two functional proteins or portions thereof; at least one promoter; and at least one engineered recombination site. At least one polynucleotide encoding the polypeptide sequence described in administering a non-viral vector comprising: In one embodiment, the expression of at least two functional proteins is driven by a method comprising: Thus, at least one promoter may be a constitutive promoter. Thus, at least one promoter may be a tissue-specific promoter. At least one promoter may be an inducible promoter. The inducible promoter is a small molecule ligand-induced, two-polypeptide ecdysone promoter. In other cases, at least one promoter can be inducible, and at least one promoter is activation-specific Possibly, a combination of promoters can be used.

[0152] The inducible promoter is for dose-regulatory control of the expression of the at least two genes. In some cases, the ligand is an ecdy steroid, 9-cis- Retinoic acid, synthetic analogues of retinoic acid, N,N'-diacylhydrazines, oxazolidinyl Dibenzoyl alkyl cyanohydrazine, N-alkyl-N,N'-diaroyl hydrazine hydrazine, N-acyl-N-alkylcarbonylhydrazine, N-aroyl-N-alkyl -N'-Aroylhydrazine, Alnidoketone, 3,5-di-tert-butyl-4-hydrazine Hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, oxysterol 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 2 5-Epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxy Cholesterol-3-sulfate (ECHS), 7-ketocholesterol 3-sulfate, Flamezol, bile acids, 1,1-biphosphonate (b iphosphonate) ester, juvenile hormone III, RG-115819 (3,5-dimethyl N-(1-ethyl-2,2-dimethylpropyl)-N'-(2-methyl- 3-Methoxy-benzoyl)-hydrazide-), RG-115932 ((R)-3,5- Dimethylbenzoate N-(1-tert-butyl-butyl)-N'-(2-ethyl-3- methoxy-benzoyl)-hydrazide), and RG-115830 (3,5-dimethyl -Benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxybenzoate -benzoyl)-hydrazide), and any combination thereof. It is possible.

[0153] In some embodiments, the promoter is an inducible promoter. In some cases, the promoter is a non-inducible promoter. As used herein, "tissue specific" refers to a promoter that is specific to a tissue or cell type. In some cases, a tissue-specific promoter is a promoter that regulates gene expression within a subset of the target gene. promoters that drive expression only in certain tissues or cell types of an organism. In other cases, tissue-specific promoters can be spatially regulated. The target differentially tracks expression in cell types or tissues over time, including during the development of an organism. In some cases, a tissue-specific promoter can be used to control the timing of the activation of the promoter. The promoter is spatially and temporally regulated. In certain embodiments, tissue-specific promoters are Motors can be expressed constitutively within a particular cell type or in specific regions at a particular time or cell type. For example, tissue-specific promoters may be activated in T cells or It may be a promoter that is activated when specific cells, such as NK cells, are activated. The cells are presented in various forms, for example, by MHC class II molecules, together with peptide antigens. or engineered T cells containing the antigen-binding polypeptide are activated when engaged with the antigen. In one case, such engineered T cells or engineered NK cells may be engineered to be chimeric antigen receptors. They express a receptor (CAR) or a T cell receptor (TCR).

[0154] In one case, at least one promoter is an engineered promoter or a mutation thereof. As described herein, the promoter is a minimal promoter derived from IL-2. and a nuclear factor of activated T cells (NFAT) response element, such as SEQ ID NO: 51. NFIL2D response element, NFkB / TCF response element, NF_AT / N FIL2B response element, or one of the NFIL2A / OCT response elements Examples of response elements are described herein in their entirety. Incorporated, Mattila et al., EMBO J. 1990 December; 9(13): 4425-4433 It is listed.

[0155] In some embodiments, the at least one promoter is the IL-2 core promoter ( In one embodiment, the at least one promoter comprises an IL-2 promoter. In another embodiment, at least one promoter The target includes an IL-2 enhancer / promoter mutant (SEQ ID NOs: 42 to 43). In yet another embodiment, at least one promoter comprises an NF-κB binding site (SEQ ID NO: In some embodiments, at least one promoter comprises (NF In some embodiments, the promoter comprises a variant of the IL-κB1-IL2 promoter (SEQ ID NO: 47). At least one promoter is a (NF-κB)3-IL2 promoter variant (sequence In some embodiments, the at least one promoter comprises (NF-κB). B) 6-IL2 promoter variant (SEQ ID NO: 49). The other promoter is a 1×NFAT response element-IL2 promoter mutant (sequence In another embodiment, the at least one promoter comprises an activated T cell promoter. In yet another embodiment, the nucleic acid sequence of the present invention comprises a nuclear factor A (NFAT) response element (SEQ ID NO: 51). At least one promoter is a 6×NFAT response element-IL2 promoter In yet another embodiment, at least one promoter is selected from the group consisting of nucleotides 1 to 5, nucleotides 6 to 7, and nucleotides 8 to 9. The promoter was a 3xNFAT response element-IL2 promoter mutant (SEQ ID NO: 56 In some embodiments, the at least one promoter comprises a human EF1A promoter. In some embodiments, the promoter comprises at least one of the following variants: One promoter is the human EF1A1 promoter / enhancer (SEQ ID NO: 60). In some embodiments, the at least one promoter is a human UBC promoter. (SEQ ID NO: 61). In some embodiments, at least one promoter comprises a hexamer. It contains the GAL4-inducible proximal factor binding element (PFB) (SEQ ID NO: 62). In an embodiment, at least one promoter is Synthetic Minimal Promoter 1 (an inducible promoter). The human IL-2 gene and 5' flanking region are , comprising the nucleotide sequence of SEQ ID NO:95.

[0156] As described herein, a gene switch for ligand-induced control of IL-12 expression is The use of IL-1 receptors may be beneficial, for example, by allowing for modulation of expression and improving the therapeutic index. However, using gene switches, ligands, The conditions for dose-dependent IL-12 expression are determined by the administration of an activating ligand (e.g., berezimex). In certain embodiments, the presence or absence of: Further conditional control is also envisioned. A gene switch component is provided, which is a transgene under the control of the gene switch. Conditioning of gene switch components required for berezimex-regulated expression of genes (e.g. In some embodiments, this results in the expression of T cells. In the presence of tumors and activated T cells, I This results in the preferential expression of cytokines such as IL-12 or IL-15. This results in increased localized levels of transgene expression controlled by a gene switch. It is possible.

[0157] For example, T cell activation-specific expression of gene switch components may be mediated by activated T cell nuclear factors. It can be controlled by a promoter containing a NFAT response element. T transcription factors are key modulators of the effector T cell state. NFAT NFAT is an early transcriptional checkpoint that progressively drives TCR stimulation and subsequent depletion. After stimulation, they are rapidly activated in T cells and, induced by appropriate costimulatory signaling, form APs. Together with IL-1, they form a protein complex that regulates effector genes and T cell function. The NFAT response element is expressed in other minimal promoter sequences (e.g., the IL2 minimal promoter). to drive transgene expression in response to T cell activation. This can be done.

[0158] Other examples of activation-specific promoters are the interleukin 2 (IL2) promoter and and programmed cell death (PD) 1 (CD279) promoter, but are not limited to Gene switch components also regulate the pro-inflammatory signaling pathways, such as NF-κB. Fusion of binding sites for other nuclear factors into the minimal promoter sequence (e.g., IL2) By using this method, it is possible to conditionally express the gene even upon activation of immune cells.

[0159] In some embodiments, the promoter comprises an NF-κB binding site (SEQ ID NOs: 44-46), Nuclear factor of activated T cells (NFAT) response element (SEQ ID NO: 51), 6-site GAL4-induced Conductive proximal factor binding element (PFB) (SEQ ID NO: 62), or synthetic based on RPL6 In certain embodiments, the promoter comprises the IL- 2 core promoter, IL-2 minimal promoter, IL-2 enhancer / promoter Mutant, (NF-κB)1-IL2 promoter mutant, (NF-κB)3-IL2 promoter Motor mutant, (NF-κB)6-IL2 promoter mutant, 1×NFAT response element ment-IL2 promoter mutant, 3xNFAT response element-IL2 promoter mutant, 6xNFAT response element-IL2 promoter mutant, human EEF1A1 promoter promoter mutant, human EEF1A1 promoter / enhancer, human UBC promoter The promoter may be any one or more of: a promoter, a synthetic minimal promoter, and a synthetic minimal promoter 1. In certain embodiments, the promoter nucleotide is a linker polypeptide disclosed in the table below. Includes petite.

[0160] [Table 2-1]

[0161] [Table 2-2]

[0162] [Table 2-3]

[0163] [Table 2-4]

[0164] [Table 2-5]

[0165] Genetic switches As used herein, gene switch polypeptides, ligand-inducible gene switch polypeptides, Polynucleotides encoding these polypeptides and / or polypeptides Methods and systems for incorporating nucleotides are presented.

[0166] "Gene switch" or "genetic switch" The term refers to a combination of a promoter and associated response elements, e.g., one or In the presence of multiple ligands, expression of genes incorporating response elements and promoters This refers to an EcR-based system that modulates gene expression. Or gene switches, gene therapy, intracellular, large-scale protein production, cell Based high-throughput screening assays, functional genomics, and It has a wide range of applications, including the regulation of traits in transgenic plants and animals. Such an inducible gene expression system is useful for the production of a ligand-inducible heterologous gene expression system. It may include.

[0167] An early form of EcR-based gene switch was discovered in Drosophila melanogaster ( anogaster) EcR (DmEcR) polypeptide and mouse (Mus musculus) RXR ( MmRXR polypeptides, and these receptors bind to the steroid ponasterone. In the presence of A, reporter genes were expressed in mammalian cell lines and transgenic mice. It has been shown to transactivate genes (Christopherson et al., 1992; No et al., 1996). Subsequently, Suhr et al., 1998, investigated the nonsteroidal ecdysone agonist Tebufenozide inhibits the growth of Bombyx mori in the absence of exogenous heterodimer partners. ) EcR (BmEcR) for high-level transcription of reporter genes in mammalian cells. It was shown to induce lance activation.

[0168] PCT International Patent Application / US Application No. 97 / 05330 (International Publication No. 97 / 381 17) and PCT International Patent Application / U.S. Application No. 99 / 08381 (WO 99 / 58155) modulates the expression of exogenous genes a DNA construct comprising an exogenous gene and an ecdysone response element; The construct reacts with the ligand for the construct and, optionally, with a silent partner. In the presence of a receptor that can act as a Methods of activation by a second DNA construct containing an ecdysone receptor that induces gene expression In this example, the ecdysone receptor was expressed in Drosophila melanogaster (Dr. Typically, such systems have been isolated from Escherichia coli (Bombyx mori) for optimal activation. To achieve this, the presence of a silent partner, preferably the retinoid X receptor (RXR), is required. In mammalian cells, the insect ecdysone receptor (EcR) is required for the mammalian ecdysone receptor. Heterodimerizes with rhodopsin X receptor (RXR), thereby targeting target genes or heterologous genes. It can be used to regulate gene expression in a ligand-dependent manner. Patent application / US application No. 98 / 14215 specification (International Publication No. 99 / 02683 pamphlet The ecdysone receptor isolated from the silkworm moth, Bombyx mori, It has been shown to be functional in mammalian systems without the need for an exogenous dimer partner. In some embodiments, RXR is mutated to SEQ ID NO: 69 by at least 45%, 50%, or 60% of the RXR signaling sequence. %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97 %, 98%, 99%, or 100% identical nucleotides, or SEQ ID NO: 182; At least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, 97%, 98%, 99%, or 100% identical polypeptides. In some embodiments, the gene switch comprises VP16-linker-RxR, 16-Linker-RXR is a nucleotide sequence of SEQ ID NO: 70 and at least 45%, 50%, 55%, 60%, or %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99 %, or 100% identical nucleotides, or at least 45% identical to SEQ ID NO: 183 , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , 97%, 98%, 99%, or 100% identical polypeptides.

[0169] U.S. Patent No. 6,265,173 describes a steroid / thyroid superfamily of receptors. Various members of the Millipore Standards are incorporated herein by reference in their entirety, at least in accordance with the U.S.P. Drosophila melanogaster ultraconjugates containing dimerization domains It is disclosed that the spirulina receptor (USP) or a fragment thereof can be combined with the spirulina receptor (USP) or a fragment thereof. U.S. Patent No. 5,880,333 describes the use of cinnamon in plants. Drosophila melanogaster EcR and Ultraspiracle (USP) A heterodimer system according to the present invention, wherein the transactivation domain and the DNA binding domain are The present invention discloses a heterodimeric system, which is located on two different hybrid proteins. In each of these cases, a transactivation domain and a DNA-binding domain (P Native EcR as described in CT International Patent Application / US Application No. 98 / 14215. or as in PCT International Patent Application / US Application No. 97 / 05330 and (as modified EcR) into a single molecule, and other The heterodimer partners were used in their native state. In some embodiments, The gene switch comprises an EcR ligand-binding domain-VY mutant, and The binding domain-VY variant has at least 45%, 50%, or 60% affinity to SEQ ID NO: 72 or 73. %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97 %, 98%, 99%, or 100% identical nucleotides, or SEQ ID NO: 185 or 186 and at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical In another embodiment, the gene switch comprises a GAL4-linker-EcR polypeptide. and GAL4-linker-EcR comprises SEQ ID NO: 74 or 75 and at least 45 %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 %, 97%, 98%, 99%, or 100% identical nucleotides to SEQ ID NO: 1 87 or 188 and at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% Contains identical polypeptides.

[0170] PCT International Patent Application / US Application No. 01 / 0905 discloses a transactivation domain The DNA-binding domain and the ATP-binding domain are separated from each other by placing them on two different proteins. In the absence of ligand, an ecdysone receptor-based inducible gene expression system was shown to produce background This results in a significant reduction in the activity of the ligand, which is above background This results in a significant increase in activity over that of the two high The hybrid line is described in PCT Application / US Application No. 97 / 05330 and PCT Application / US In comparison with the two systems disclosed in the application of the patent application No. 98 / 14215 The two-hybrid system is a significantly improved inducible gene expression modulation system. When the DNA-binding domain binds to the DNA-binding site on the gene, it becomes a transactivation domain. The main target is to increase the number of interacting proteins to activate the promoter more efficiently. but the ability to place the transcription activation domain in a more favorable position relative to the DNA binding domain. (See, for example, U.S. Pat. No. 5,283,173. The two-hybrid gene expression system uses a DNA fragment fused to a nuclear receptor polypeptide. A first gene expression cassette encoding a binding domain and a different nuclear receptor polypeptide. a second gene expression cassette encoding a transactivation domain fused to the peptide In the presence of a ligand, the first polypeptide This induces a conformational change in the polypeptide that facilitates its interaction with a second polypeptide, This results in dimerization of the DNA-binding domain with the transactivation domain. The DNA binding domain and the transactivation domain are thought to be Because the ligands are present on different molecules, in the absence of ligand, the background activity is is significantly reduced.

[0171] Another surprising finding was that certain modifications of the two-hybrid system also inhibited the production of steroidal ligands. and ponasterone A ("PonA") or muristerone A ("MurA") In comparison, sensitivity to nonsteroidal ligands, e.g., diacylhydrazines The results also showed that nonsteroidal anti-inflammatory drugs, when compared with steroids, improved The ligand resulted in greater gene transcription activity at lower ligand concentrations. Hybrid systems are characterized by the R It also avoids some of the side effects caused by overexpression of XR. In a preferred two-hybrid system, E The native DNA-binding and transactivation domains of cR or RXR are removed As a result, these hybrid molecules interact with other steroid hormones present in the cell. Reduces the likelihood of interaction with steroid receptors, resulting in fewer side effects bring about.

[0172] The ecdysone receptor (EcR) is a member of the nuclear receptor superfamily. Classified into subfamily 1, group H (referred to herein as "group H nuclear receptors") Members of each group share 40–60% amino acid identity within the E (ligand-binding) domain. (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor r Subfamily, 1999; Cell 97: 161-163). In addition to the ecdysone receptor, this nuclear receptor Other members of subfamily 1, group H, are ubiquitous receptors (URs), orphan receptors, and steroid hormone nuclear receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1), RXR interacting Liver protein 15 (RIP-15), liver x receptor beta (LXRβ), steroid hormone receptor receptor-like protein (RLD-1), liver x receptor (LXR), liver x receptor α (LXRα ), farnesoid x receptor (FXR), receptor-interacting protein 14 (RIP-1 4), and the farnesol receptor (HRR-1).

[0173] In some cases, the inducible promoter is manufactured by Intrexon Corporation. RHEOSWITCH® gene switches, such as small molecule ligand-inducible It may be an ecdysone receptor-based gene switch with a single polypeptide. Thus, the gene switches are incorporated herein by reference in their entirety. , PCT / US Application No. 2001 / 009050 (International Publication No. 2001 / 0708 No. 16; U.S. Patent Nos. 7,091,038; 7,776,58 Specification No. 7; Specification No. 7,807,417; Specification No. 8,202,718; PC T / US Application No. 2001 / 030608 (International Publication No. WO 2002 / 029075 Pamphlet); U.S. Patent No. 8,105,825; U.S. Patent No. 8,168,426 Details: PCT / US Application No. 2002 / 005235 (International Publication No. 2002 / 06 No. 6613); U.S. Patent Application Publication No. 10 / 468,200 (U.S. Patent Application Publication No. PCT / US Application No. 2002 / 005706 Specification (International Publication No. 2002 / 066614); U.S. Patent No. 7,531, Specification No. 326; Specification No. 8,236,556; Specification No. 8,598,409; PCT / US Application No. 2002 / 005090 (International Publication No. WO 2002 / 06661 No. 2 pamphlet); U.S. Patent No. 8,715,959 (U.S. Publication No. 200601 No. 00416; PCT / US Application No. 2002 / 005234 (International Publication No. 2003 / 027266; ​​U.S. Patent No. 7,601,508; Specification No. 7,829,676; Specification No. 7,919,269; Specification No. 8,030, 067; PCT / US Application No. 2002 / 005708 (International Publication No. WO 2002 / 005708) 02 / 066615 Brochure); U.S. Patent Application Publication No. 10 / 468,192 Specification Publication No. 20110212528; PCT / US Application No. 2002 / 00 5026 (International Publication No. 2003 / 027289); U.S. Pat. , 563,879; 8,021,878; 8,497,093 PCT / US Application No. 2005 / 015089 (International Publication No. 2005 / No. 108617; U.S. Patent No. 7,935,510; U.S. Patent No. 8,07 No. 6,454; PCT / US Application No. 2008 / 011270 (International Publication No. 2009 / 045370); U.S. Patent Application Publication No. 12 / 241,018 Specification (U.S. Publication No. 20090136465); PCT / U.S. Application No. 2008 / No. 011563 (International Publication No. WO 2009 / 048560); U.S. Pat. Application Publication No. 12 / 247,738 (U.S. Publication No. 20090123441 PCT / US Application No. 2009 / 005510 (International Publication No. 2010 / 042 No. 189); U.S. Patent Application Publication No. 13 / 123,129 (U.S. Patent Application Publication No. PCT / US Application No. 2011 / 029682 Detailed description (International Publication No. 2011 / 119773); U.S. Patent Application Publication No. 13 / 636,473 (U.S. Publication No. 20130195800); PCT / U.S. Application No. 2012 / 027515 specification (International Publication No. 2012 / 122025 pamphlet and among the systems described in U.S. Pat. No. 9,402,919. The ecdysone-based receptor component may be selected from, but is not limited to, any of the following: Not determined.

[0174] System for modulating expression of heterologous genes and interleukins in host cells a polynucleotide that expresses a gene switch polypeptide disclosed herein; Systems containing the gene are provided that are under the control of a constitutive or inducible promoter. Various structural components of non-limiting exemplary ligand-inducible gene switch vector systems are shown. The element is shown in Figure 22.

[0175] In some embodiments, the expression cassette of the gene switch vector system is XON-64. and has the sequence set forth in SEQ ID NO: 131. In some embodiments, the gene switch vector The expression cassette of the TA system is XON-30 and has the sequence shown in SEQ ID NO: 132. In some embodiments, the expression cassette of the gene switch vector system is XON-59. and has the sequence set forth in SEQ ID NO: 133. In some embodiments, the gene switch vector The expression cassette of the TA system is XON-60 and has the sequence shown in SEQ ID NO: 134. In some embodiments, the expression cassette of the gene switch vector system is XON-61. and has the sequence set forth in SEQ ID NO: 135. In some embodiments, the gene switch vector The expression cassette of the TA system is XON-62 and has the sequence shown in SEQ ID NO: 136. .

[0176] In some embodiments, the expression of heterologous genes and cytokines in the host cell is modulated. a first polynucleotide encoding a first polypeptide; a first gene expression cassette comprising: a second polynucleotide encoding a second polypeptide; a second gene expression cassette comprising a ligand; and a ligand, wherein the host cell is In the presence of the ligand, the first gene expression cassette and the second gene expression cassette Upon contact with the set, the heterologous gene and the cytokine are expressed in the host cell. In order to achieve this, the first polypeptide and the second polypeptide are (i) transactivatable. (ii) a DNA-binding domain; and (iii) a ligand-binding domain. (iv) said heterologous gene; and (vi) one or more of said cytokines. Optionally, the heterologous gene comprises an antigen-binding gene as described herein. Optionally, the antigen-binding polypeptide comprises a polypeptide as described herein. CARs, e.g., CD19, CD33, BCMA, CD44, α-folate receptor, CAIX , CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folic acid Binding proteins, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelial cell membrane protein Lin, CD22, EGFR, MUC-1, MUC-16, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123, and VEGF- R2.

[0177] cytokines As used herein, gene switch polypeptides and cytokines, or variants thereof, Polynucleotides encoding the antibody or derivatives, and methods of incorporating the same Cytokines are molecules with a molecular weight of approximately 5 to 20 kDa that are involved in cell signaling. Cytokines are a class of small proteins between Includes interferons, interleukins, colony-stimulating factors, or tumor necrosis factors. In some embodiments, chemokines act as chemoattractants, directing cell migration. However, they are classified into four subfamilies: CXC, CC, CX3C, and XC. Representative chemokines are the CC subfamily: CCL1, CCL2 (MCP-1), CCL 3, CCL4, CCL5(RANTES), CCL6, CCL7, CCL8, CCL9( or CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL2 2, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28 ;CXC subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL 5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CX CL17; XC subfamily: XCL1 and XCL2; and CX3C subfamily These include chemokines derived from the CX3CL1 receptor.

[0178] Interferons (IFNs) are type I interferons (e.g., IFN-α, IFN -β, IFN-ε, IFN-κ, and IFN-ω), type II interferons (e.g. , IFN-γ), and type III interferons. In some embodiments, IFN -α is IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17 IFNA21 and IFNA22.

[0179] Interleukins are produced by leukocytes or white blood cells It is expressed and promotes the development and differentiation of T and B lymphocytes and hematopoietic cells. Exemplary 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, L-6, IL-7, IL-8(CXCL8), 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-2 5, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL IL-32, IL-33, IL-35, and IL-36.

[0180] In some embodiments, the interleukin comprises mbIL-15. mbIL-15 is co-expressed with the engineered effector cells described herein. In some embodiments, mbIL-1 is a membrane-bound chimeric IL-15 that can 5 is in-frame with full-length IL-15Rα, or a functional fragment or variant thereof full-length IL-15 (e.g., a native IL-15 polypeptide), or Optionally, IL-15 is phospho-transferred to IL-15Rα. In some cases, mbIL-15 is indirectly linked via a marker. “Tethered IL-15 augments antitumor activity and promotes a stem-cell memory sub As described in “Public Healthcare’s Immunoglobulin Gene Expression and Antigens,” PNAS 2016.

[0181] Tumor necrosis factors (TNFs) are a group of cytokines that modulate apoptosis. In some cases, within the TNF family, there are TNFα, lymphotoxin alpha (L T-alpha), lymphotoxin beta (LT-beta), T cell antigen gp39 ( CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L, and including, but not limited to, TNF-releasing apoptosis-inducing ligand (TRAIL); There are about 19 members.

[0182] Colony-stimulating factors (CSFs) interact with receptor proteins on the surface of hematopoietic stem cells. It is a secreted glycoprotein that acts as a signaling pathway that subsequently regulates cell proliferation and differentiation into specific types of blood cells. CSFs are secreted glycoproteins that modulate differentiation. phage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (G-CSF), or promegapoietin.

[0183] In some embodiments, the cytokine is administered in combination with a chimeric antigen receptor described herein. It is a co-expressed membrane-bound cytokine.

[0184] In some embodiments, one or more of the methods described herein comprises administering a cytokine. In some cases, the cytokine may be a chemokine, an interferon, an interferon, or a Interleukins, colony-stimulating factors, or tumor necrosis factors. The methods described herein may involve the use of one or more of the following: chemokines, interferons, interleukins, and further administering a cytokine selected from a cytokine, a colony-stimulating factor, or a tumor necrosis factor. In some instances, one or more of the methods described herein may include administering IL-2, IL-3, or IL-7, IL-12, IL-15, IL-21, IFNγ, or TNF-α The method further includes administering a cytokine that

[0185] Optionally, the cytokine is at least one of a chemokine, an interferon, Interleukins, lymphokines, tumor necrosis factors, or variants or combinations thereof Optionally, the cytokine is an interleukin. Optionally, the interleukin Turleukin inhibits IL-12, IL-2, IL-15, IL-21, and their functions. In some embodiments, the cytokine is at least one of a functional variant and a fragment. IL-1, IL-2, IL-15, IL-12, IL-21, IL-15, IL In one embodiment, the IL-15Rα is a fusion of IL-15Rα or a fusion of an IL-15 variant. In some embodiments, the cytokine is a variant of IL-15 (SEQ ID NO: 90; SEQ ID NO: 203). In one embodiment, the cytokine is IL-15 receptor alpha (SEQ ID NO: 91; In some embodiments, the cytokine may be membrane-bound or secreted. In other embodiments, the cytokine may be an intracellular cytokine. Interleukins include membrane-bound IL-15 (mbIL-15), IL-15, and IL-15 fusion with Ra (SEQ ID NO: 91; SEQ ID NO: 204), or IL-15 mutant (SEQ ID NO: 90; SEQ ID NO: 203). In some embodiments, mbIL-15 may comprise the polypeptides described herein. Membrane-bound chimeric IL-15 that can be co-expressed using engineered effector cells described in In some embodiments, mbIL-15 is full-length IL-15Rα, or a variant of this function. full-length IL-15 (e.g., native IL-15) fused in-frame with a representative fragment or variant thereof Optionally, the IL-15 polypeptide or a fragment or variant thereof. is indirectly linked to IL-15Rα via a linker. mbIL-15 contains a signal peptide (SEQ ID NO: 92; SEQ ID NO: 205). Therefore, mbIL-15 is a potential antitumor agent, as described by Hurton et al., "Tethered IL-15 augments antitumor activity." activity and promotes a stem-cell memory subset in tumor-specific T cells,” PNAS 2 In another embodiment, the interleukin is IL-1 2. In some embodiments, the IL-12 may comprise single-chain IL-12 (scIL-12). , protease-sensitive IL-12, destabilized IL-12, membrane-bound IL-12, or inserted IL-12. In some embodiments, the IL-12 may be murine IL-12 subunit. In some embodiments, IL-12 is nit beta (p40) (SEQ ID NO: 206). , mouse IL-12 subunit alpha (p35) (SEQ ID NO: 207). In this embodiment, the IL-12 is a murine single chain IL-12 (p40-linker-p35) ( In some embodiments, the IL-12 is a human single chain IL-12(p40-linker-p35) (SEQ ID NO: 94; SEQ ID NO: 209). In certain embodiments, the IL-12 is a single-chain IL-12. 2 variants are disclosed in International Publication No. WO 02 / 04999, all of which are incorporated by reference in their entirety. Pamphlet No. 2015 / 095249, Pamphlet No. 2016 / 048903 and the method described in WO 2017 / 062953. This is true.

[0186] Provided herein is a polynucleotide encoding a gene switch polypeptide, comprising: The gene switch polypeptide is a) fused to a nuclear receptor ligand binding domain. a) a first gene switch polypeptide comprising a DNA-binding domain associated with the first gene switch polypeptide; and b) a nuclear A second gene comprising a transactivation domain fused to a receptor ligand binding domain. a gene switch polypeptide, a first gene switch polypeptide, and a second gene switch polypeptide; and a switch polypeptide are linked by a linker. Optionally, the linker is a linker described herein, e.g., a GSG linker. 2A links such as Carr, Furin Link, F / T2A, T2A, p2A, GSG-p2A In other cases, the linker may be a 1, 2, 3, 4, 5, 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, 39, 3 In some embodiments, the IRES can be an EMCV IRES or a 2xR bm3 IRES. Exemplary IRES sequences can be found in SEQ ID NOs: 18 and 19. In certain cases, a polynucleotide encoding the 2xRbm3 IRES can be The codes are SEQ ID NO: 19 and 45%, 50%, 55%, 60%, 65%, 70%, 75%, 8 Can be 0%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical In certain cases, the EM The polynucleotide encoding the CV IRES is SEQ ID NO: 18 and SEQ ID NO: 45, 50, 5 5%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 9 They may be 8%, 99%, or 100% identical, or at least approximately these percentages. They may be identical.

[0187] In some cases, the DNA binding domain (DBD) may be any DBD described herein, e.g., For example, GAL4 (GAL4 DBD) (SEQ ID NO: 71; SEQ ID NO: 184), LexADB D, transcription factor DBD, a member of the steroid / thyroid hormone nuclear receptor superfamily The protein comprises at least one of a soluble nucleotide sequence (DBD), a bacterial LacZ DBD, and a yeast DBD. The transactivation domain may be any of the transactivation domains described herein, e.g., VP 16 transactivation domain (SEQ ID NO: 68; SEQ ID NO: 181), p53 transactivation domain, and the B42 acidic activator transactivation domain The nuclear receptor ligand-binding domain is involved in the ecdysone receptor (EcR), a ubiquitous receptor body, orphan receptor 1, NER-1, steroid hormone nuclear receptor 1, retinoid X Receptor-interacting protein 15, liver X receptor β, steroid hormone receptor-like protein Protein, liver X receptor, liver X receptor α, farnesoid X receptor, receptor-interacting protein The protein may comprise at least one of protein 14, and farnesol receptor.

[0188] Optionally, linked by a polypeptide linker or ribosome skipping sequence. The gene switch polypeptide induces dose-dependent ligand induction of gene expression. These present an improvement in sexual control compared to ligand-induced gene switches, where the gene The switch polypeptides are linked by non-coding sequences, such as an IRES. Thus, gene switch polypeptides linked by 2A linkers are effective in heterologous gene expression. Although it offers dose-dependent, ligand-inducible control, it offers an improvement over gene switches. In this case, the gene switch polypeptides are separated by an IRES.

[0189] The polypeptides and polynucleotides described herein can be expressed in engineered cells. As used herein, engineered cells are cells that have been modified from their native or endogenous state. Examples of engineered cells include cells that have been engineered to express, for example, a gene switch polypeptide, a gene of interest, The gene encoding the target gene (GOI), cell tag, heterologous gene, and any other polynucleotides described herein may be used. modified to encode peptides and polynucleotides (e.g., polynucleotides The cells are those described herein (by transfection of the vector into the cells).

[0190] Ligand In some embodiments, the ligand used to regulate the inducible gene switch is Bottom: N-[(1R)-1-(1,1-dimethylethyl)butyl]-N'-(2-ethyl- 3-Methoxybenzoyl)-3,5-dimethylbenzohydrazide (also known as berezimex) (2S, 3R, 5R, 9R, 10R, 13R, 14S, 17R)-17-[( 2S,3R)-3,6-dihydroxy-6-methylheptan-2-yl]-2,3,14 -trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,1 6,17-Decahydro-1H-cyclopenta[a]phenanthren-6-one; N'-( 3,5-dimethylbenzoyl)-N'-[(3R)-2,2-dimethyl-3-hexanyl ]-2-Ethyl-3-methoxybenzohydrazide;5-Methyl-2,3-dihydrobenzohydrazide Zo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethyl-benzoyl)-N '-(1-Ethyl-2,2-dimethyl-propyl)-hydrazide;5-Methyl-2,3- Dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy- 4-Methyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydroxide 5-Methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1-tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl)- Hydrazide; 5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbohydrazide N'-(1-tert-butyl-butyl)-N'-(3,5-dimethoxy-4-methyl)phosphoric acid 5-Ethyl-2,3-dihydro-benzo[1,4]dihydrobenzo[1,4]dihydrazide Xine-6-carboxylic acid N'-(3,5-dimethyl-benzoyl)-N'-(1-ethyl) -2,2-dimethyl-propyl)-hydrazide;5-ethyl-2,3-dihydro-benzo [1,4]dioxine-6-carboxylic acid N'-(3,5-dimethoxy-4-methyl-benzoyl) 5-Ethyl)-N'-(1-ethyl-2,2-dimethyl-propyl)-hydrazide 1-(2,3-dihydrobenzo[1,4]dioxine-6-carboxylic acid N'-(1-te rt-Butyl-butyl)-N'-(3,5-dimethyl-benzoyl)-hydrazide;5- Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-(1- tert-Butyl-butyl)-N'-(3,5-dimethoxy-4-methyl-benzoyl) -hydrazide;3,5-dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propionyl) (3-Methoxy-2-methyl-benzoyl)hydrazide;3,5-Dimethylbenzoyl N-(1-ethyl-2,2-dimethylpropyl)-4-methylbenzoic acid N' -(3-Methoxy-2-methyl-benzoyl)-hydrazide;3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3-methoxy-2-methyl-benzoic acid 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-yl)-hydrazide Butyl-butyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide;3 ,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-( 2-Ethyl-3-methoxy-benzoyl)-hydrazide;3,5-dimethoxy-4-methyl N-(1-ethyl-2,2-dimethylpropyl)-N'-(2-ethyl-)benzoic acid 3-Methoxy-benzoyl)-hydrazide;3,5-Dimethyl-benzoic acid N-(1-te rt-Butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazine N-(1-tert-butyl-3,5-dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-butyl) ester -N'-(2-ethyl-3-methoxybenzoyl)hydrazide;2-Methoxy-nicotinic acid N-(1-tert-butyl-pentyl)-N'-(4-ethyl-benzoyl)- Hydrazide; 3,5-Dimethyl-benzoic acid N-(2,2-dimethyl-1-phenyl-propionyl) Pyr)-N'-(4-ethyl-benzoyl)-hydrazide;3,5-dimethyl-benzoic acid N-(1-tert-butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl) yl)-hydrazide; and 3,5-dimethoxy-4-methyl-benzoic acid N-(1-te rt-Butyl-pentyl)-N'-(3-methoxy-2-methyl-benzoyl)-hydra The compound may be selected from, but is not limited to, any of the following:

[0191] Possibly, dose-regulated control of ecdysone receptor-based inducible gene switches. The ligands used for this purpose are ecdysone, 20-hydroxyecdysone, and ponasterone. A, ecdy steroids such as muristerone A, 9-cis-retinoic acid, retinoin Synthetic analogs of the acid, each of which is incorporated herein by reference, are disclosed in U.S. Pat. Specification No. 13,836; Specification No. 5,117,057; Specification No. 5,530,028 and US Patent No. 5,378,726, and US Patent Application Publication No. 2005 / 02 09283 and 2006 / 0020146 N,N'-diacylhydrazines such as N,N'-diacylhydrazines containing N,N'-diacylhydrazines; U.S. Patent Application Publication No. Oxazolines as described in European Patent Application No. 2004 / 0171651; Dibenzoylalkylcyanohydrazines such as those disclosed in US Pat. No. 61,809 Which dibenzoylalkylcyanohydrazine; U.S. Pat. No. 5,225,443 N-alkyl-N,N'-diaroylhydrazines and other N-alkyl-N,N'-diaroylhydrazines disclosed in N,N'-diaroylhydrazine; as disclosed in European Application No. 234,994 N-acyl-N-alkylcarbonylhydrazines, such as N-acyl-N-alkylcarbonylhydrazines, carbonylhydrazine; as described in U.S. Pat. No. 4,985,461 N-aroyl-N-alkyl-N'-aroylhydrazines Alkyl-N'-aroylhydrazines; U.S. Patent Application Publication No. 2004 / 0049037 Alnidoketones such as those described in the document; 3,5-di-tert -butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpage hydroxysteroids, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol Cholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6- Alpha-epoxycholesterol-3-sulfate (ECHS), 7- Ketocholesterol-3-sulfate, Flamezol, bile acid, 1,1- Other classes include biphosphonate esters, juvenile hormone III, etc. The material may be selected from, but is not limited to, any of a variety of similar materials. An example of a suitable diacylhydrazine ligand is RG-115819 (3,5-dimethyl-benzoyl) N-(1-ethyl-2,2-dimethylpropyl)-N'-(2-methyl-3-methoxy)- (R)-3,5-dimethyl-benzoyl)-hydrazide), RG-115932 ((R)-3,5-dimethyl- N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzyl)benzoate N-benzoyl-hydrazide), and RG-115830 (3,5-dimethyl-benzoic acid N -(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl )-hydrazides), any of which are incorporated by reference in their entirety. No. 12 / 155,111 and PCT application Ser. No. 2004 / 0109994, which are incorporated herein by reference. See U.S. Application No. 2008 / 006757.

[0192] antigen-binding polypeptide The polypeptides and proteins disclosed herein (including functional portions and functional The amino acid sequence (including synthetic variants) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane. carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetyl aminomethyl-cysteine, trans-3-hydroxyproline and trans-4- Hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4- Chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hybrid Hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexyl Alanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetracarboxylic acid Trihydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide , N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxy- Roxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexyl α-aminocycloheptanecarboxylic acid, α-(2-amino-2-nor Bornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homo phenylalanine, and α-tert-butylglycine.

[0193] As used herein, "antibody" refers to a monoclonal or polyclonal antibody. As used herein, the term "monoclonal antibody" refers to a single clone of B cells. It refers to an antibody produced by a single clone and binding to the same epitope. "Monotypic antibodies" are antibodies produced by different B cells that bind to different epitopes of the same antigen. Refers to a population of antibodies. Whole antibodies typically consist of four polypeptides: heavy (H) chain polypeptides and two identical copies of the light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, CH3, CH4, CH5, CH6, CH7, CH8, CH9, CH10, CH11, CH12, CH13, CH14, CH15, CH16, CH17, CH18, CH19, CH20, CH21, CH22, CH23, CH24, CH25, CH26, CH27, CH28, CH29 Each light chain contains an N-terminal variable (VL) region and one C-terminal variable (CH2, CH3) region. The variable regions of each pair of light and heavy chains comprise the antibody's constant (CL) region and two C-terminal constant (CL) regions. The VH region and the VL region form an antigen-binding site. They share a common general structure, including four framework regions that are relatively conserved. The framework regions are linked by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, are responsible for binding to the antigen. These form the "hypervariable region" of the antibody.

[0194] An "antigen recognition portion" or "antibody recognition domain" refers to a molecule or molecule that specifically binds to an antigen. refers to a portion of a molecule. In one embodiment, the antigen recognition moiety is an antibody, an antibody-like molecule, or and the antigen is a tumor antigen or an infectious disease antigen.

[0195] "Antibody-like molecules" are, for example, Ig superunits that are capable of selectively binding to a partner. MHC molecules and T cell receptors can be proteins that are members of the IL-1 family. In one embodiment, the antibody-like molecule is a TCR. In this study, the TCR has been modified to increase its binding affinity to MHC.

[0196] As used herein, the terms "antibody fragment," "antibody fragment," "functional fragment of an antibody," and "antigen The term "binding moiety," or grammatical equivalents thereof, refers to a molecule capable of specifically binding to an antigen. and the like. (See generally Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)) An antibody fragment may comprise, for example, one or more CDRs, a variable region (or a portion thereof), a constant region, or both. It is desirable for the antibody fragment to contain a region (or portion thereof), or a combination thereof. Examples of antibody fragments include (i) a single antibody consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) a Fab fragment, which is a covalent fragment; (iii) a Fab fragment, which is linked by a disulfide bridge in the stalk region; (iii) an F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments separated by a single bond; (iv) an Fv fragment consisting of a VL domain and a VH domain of a single arm; The main chains are joined by a synthetic linker that allows them to be synthesized as a single polypeptide chain. It is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) combined together. Some single-chain Fvs (scFvs) (e.g., Bird et al., Science, 242: 423-426 (1988); H uston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et a l., Nat. Biotechnol., 16: 778 (1998); and (v) each polypeptide. The peptide chains are too short to allow pairing between VH and VL on the same polypeptide chain. It contains a VH linked to a VL by a tid linker, thereby forming two functional antigens. on different VH-VL polypeptide chains to create a dimeric molecule having a binding site. Diabodies, dimers of polypeptide chains that drive pairing between complementary domains Antibody fragments are known in the art and include, but are not limited to, those described in, for example, U.S. Pat. These are described in more detail in Patent No. 8,603,950. may comprise a variable fragment of a heavy chain antibody (VHH).

[0197] The term "functional portion" when used with respect to a CAR refers to any portion of a CAR of the present disclosure. an arbitrary portion or fragment of the biology of the CAR of which the functional portion is a part (parent CAR) With respect to the nucleic acid sequence encoding the parent CAR, the term CA refers to a portion or fragment that retains the parent CAR's specific activity. The nucleic acid sequence encoding the functional portion of R is, for example, about 10%, 25%, or , 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR It can encode a protein containing

[0198] As used herein, the term "functional variant" refers to a functional variant of a reference polypeptide. Polypeptides or proteins with qualitative or significant sequence identity or sequence similarity It refers to a variant that retains the biological activity of the reference polypeptide of which it is a variant. In embodiments, the functional variant may be, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 locations A reference protein with conservative amino acid substitutions, or approximately these number of conservative amino acid substitutions Functional variants include, for example, the amino acid sequence of the CAR (parent CA R) mutants that target cells to a similar extent as, or to a similar extent as, the parent CAR The nucleic acid sequence encoding the parent CAR includes variants that retain the ability to recognize to a higher degree. In accordance with the sequence, the nucleic acid sequence encoding the functional variant of CAR is the same as the nucleic acid sequence encoding the parent CAR. For example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 70% identical, about 80% identical, about 90% identical, about 100% identical, about 120% identical, about 140% identical, about 160% identical, about 180% identical, about 200% identical, about 220% identical, about 240% identical, about 25 % identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical.

[0199] The term "conservative amino acid substitution" or "conservative mutation" refers to a substitution of one amino acid. Refers to the replacement of an amino acid with another amino acid with common characteristics. The functional mechanism that defines sex is the amino acid variation between corresponding proteins of homologous organisms. The aim is to analyze the standardized frequency of phenotypes (Schulz, GE and Schirmer, RH, Principle s of Protein Structure, Springer-Verlag, New York (1979)). , amino acids within a group are preferentially exchanged with each other, thus affecting the overall protein structure. It is possible to define groups of amino acids that are most similar to each other in their effect on (Schulz, GE and Schirmer, RH, supra). Examples of conservative mutations are Amino acid substitutions of amino acids, e.g., lysine, aryl, etc., which may maintain a positive charge. substitution of glutamic acid for glutamic acid, and vice versa, so that the negative charge can be maintained. , aspartic acid substitutions, and vice versa; serine substitutions that maintain a free -OH. and substitution of glutamine with threonine, which may maintain free -NH2. Alternatively, or in addition, the functional variant may contain at least one asparagine substitution. It may comprise the amino acid sequence of a reference protein with one non-conservative amino acid substitution.

[0200] The term "non-conservative mutation" refers to an amino acid substitution between different groups, e.g., a lysine These include the substitution of tryptophan with phenylalanine, or the substitution of serine with phenylalanine. In this case, the non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions preferably do not impair or disrupt the biological activity of functional variants. may enhance the biological activity of the functional variant such that is increased compared to the parent CAR.

[0201] In some embodiments, the antigen-binding portion of the CAR described herein binds to CD19, C D33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA , EGP-2, EGP-40, HER2, HER3, folate-binding protein, GD2, G D3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EGFR, M UC-1, MUC-16, MAGE-A1, MUC16, h5T4, PSMA, TAG- 72, specific for EGFRvIII, CD123, and VEGF-R2, or In some embodiments, the antigen-binding domain binds to a target antigen-specific monoclonal antibody. The variable domain light chain (VL) and variable domain heavy chain (VH) of a clonal antibody , linked by a flexible linker such as a glycine-serine linker or a Whitlow linker. In some embodiments, the antibody fragments include single-chain antibody fragments (scFv) comprising an fused VL and VH. The scFv is a humanized scFv. In some embodiments, the antigen-binding portion comprises, for example, From the N-terminus to the C-terminus, the VH-linker-VL or VL-linker-VH can be used in a variety of directions. Optionally, the antigen-binding domain may comprise a VH and a VL linked by a target In some embodiments herein, the antigen-binding domain recognizes a target epitope. CAR or CAR-(ab')2, Fab', Fab, Fv, or scFv T cells are described.

[0202] In one embodiment, the antigen-binding portion of the CAR described herein is specific for CD19. In one embodiment, the antigen-binding portion of the CAR described herein binds to CD33. In another embodiment, the antigen-binding portion of the CAR described herein is specific for B In yet another embodiment, the antigen binding of the CAR described herein is specific for CMA. In some embodiments, the synthetic moiety is specific for CD44. The antigen-binding portion of R is specific for the alpha-folate receptor. The antigen-binding portion of the CAR described in is specific for CAIX. The antigen-binding portion of the CAR described herein is specific for CD30. In one embodiment, the antigen-binding portion of the CAR described herein is specific for ROR1. In embodiments, the antigen-binding portion of the CAR described herein is specific for CEA In some embodiments, the antigen-binding portion of a CAR described herein binds to EGP-2. In one embodiment, the antigen-binding portion of the CAR described herein is specific for EG In another embodiment, the antigen-binding ability of the CAR described herein is specific for P-40. In yet another embodiment, the CA moiety is specific for HER2. The antigen-binding portion of R is specific for HER3. The antigen-binding portion of the described CAR is specific for folate binding protein. In embodiments, the antigen-binding portion of the CAR described herein is specific for GD2. In some embodiments, the antigen-binding portion of the CAR described herein is specific for GD3. In one embodiment, the antigen-binding portion of the CAR described herein is IL-13 In one embodiment, the antigen-binding portion of the CAR described herein is specific for R-a2. In one embodiment, the antigen-binding domain of the CAR described herein is specific for KDR. In another embodiment, the CAR fragment described herein is specific for EDB-F. In yet another embodiment, the antigen-binding portion of the antibody described herein is specific for mesothelin. The antigen-binding portion of the described CAR is specific for CD22. The antigen-binding portion of the CAR described herein is specific for EGFR. In one embodiment, the antigen-binding portion of the CAR described herein is specific for MUC-1. In one embodiment, the antigen-binding portion of the CAR described herein is specific for MUC-16. In one embodiment, the antigen-binding portion of the CAR described herein is MAGE-A1 In some embodiments, the antigen-binding portion of a CAR described herein is specific for In some embodiments, the antigen binding of the CAR described herein is specific for h5T4. In another embodiment, the CAR antibody described herein is specific for PSMA. In a further embodiment, the antigen-binding portion of the The antigen-binding portion of the CAR described in is specific for EGFRvIII. In some embodiments, the antigen-binding portion of the CAR described herein is specific for CD123. In a further embodiment, the antigen-binding portion of the CAR described herein binds to VEGF-R Specific to 2.

[0203] Chimeric antigen receptor (CAR) In some embodiments, the chimeric receptors described herein encode chimeric receptors expressed on the cell surface. In some cases, the chimeric receptor may be a polypeptide that binds to an antigen, e.g., a tumor enable recognition and binding to tumor antigens, such as tumor-associated antigens or tumor-specific antigens In some cases, the antigen-binding region comprises an antibody or binding fragment, e.g., For example, Fab, Fab', F(ab')2, F(ab')3, scFv, sc(Fv)2 , dsFv, diabodies, minibodies, and nanobodies or their binding Optionally, the antigen-binding region comprises an scFv. Optionally, the antigen-binding region comprises a chimeric The receptor comprises an scFv (e.g., a chimeric antigen receptor (CAR)). Chimeric antigen receptors include pattern recognition receptors. In other cases, chimeric receptors are engineered Contains T cell receptors (TCRs).

[0204] As used herein, "chimeric antigen receptor (CAR)", "artificial T cell receptor", " The term "chimeric T cell receptor," or "chimeric immune receptor," refers to a chimeric T cell receptor that expresses an exogenous specificity in a CARs refer to engineered receptors that are grafted onto immune effector cells. In some cases, CARs are , antigen-binding domain, stalk region, transmembrane domain, and intracellular domain (endo They contain an extracellular domain (ectodomain) that contains an intracellular domain. The domain further comprises one or more intracellular signaling domains. The CARs described herein contain an antigen-binding domain, a stalk, and a marker for T cell activation. a transmembrane domain, one or more costimulatory domains, and a signaling domain. include.

[0205] In embodiments, the CAR of the present disclosure comprises an antigen-binding portion, otherwise referred to as an antigen-binding moiety: In some embodiments, the target-specific binding element specifically binds to an antigen on a tumor cell. Targeting tumor antigens of interest by engineering desired antigen-binding moieties that bind to the target In the context of this disclosure, "tumor antigen" or "hyper- "Hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen that is associated with a specific hyperproliferative disorder, such as cancer. Refers to an antigen common to hyperproliferative disorders.

[0206] The antigen-binding domain is the complementarity-determining region of a monoclonal antibody. The complementarity determining regions (CDRs) may comprise a variable region, a complementarity determining region (CDR), ... ) is complementary to the antigen and therefore transmits to the receptor its specificity for this particular antigen. of antigen receptor (e.g., immunoglobulin and T cell receptor) proteins that mediate A short sequence of amino acids found within the variable domain of each polypeptide chain of an antigen receptor. may contain three CDRs (CDR1, CDR2, and CDR3). The antigen-binding domain is F(ab')2, Fab', Fab, Fv, or scFv. Optionally, the antigen-binding domain is an scFv. Optionally, the antigen-binding domain is an scFv. Optionally, the antigen-binding domain is a Fab. Optionally, the antigen-binding domain is a Fab'. Optionally, the antigen-binding domain is F(ab')2. The synthetic domain is Fv.

[0207] In some embodiments, the CARs described herein are capable of targeting CD19, CD33, BCMA, , CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, E GP-40, HER2, HER3, folate-binding protein, GD2, GD3, IL-13 R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC -16, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, EGFRv III, CD123, and VEGF-R2. In some embodiments, the CARs described herein comprise a CD19, CD3 3, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, E GP-2, EGP-40, HER2, HER3, folate-binding protein, GD2, GD3 , IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC -1, MUC-16, MAGE-A1, MUC16, h5T4, PSMA, TAG-72 , an antigen that binds to epitopes on EGFRvIII, CD123, and VEGF-R2 In some embodiments, the CARs described herein comprise a CD19 binding domain. , CD33, or EGFRvIII. In some cases, the CARs described herein bind to an epitope on CD19. Optionally, the CAR described herein comprises an antigen-binding domain that binds to CD33. In a further embodiment, the antibody comprises an antigen-binding domain that binds to the above epitope. The CAR or chimeric receptor or antigen-binding polypeptide described in , myelin oligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MA Autoantigen binding, binding to epitopes on dCAM1, SDF1, or type II collagen It includes a binding region or an antigen-binding region.

[0208] In another embodiment, the CAR described herein is an EGFRvIII-specific CAR. "EGFRvIII," "EGFR variant III," "type III EGFR mutation" "EGFR.D2-7" or "de2-7EGFR" refers to a human subject and a non-human subject. Extracellular protein ligands, epidermal growth factor (EGF) family, in human subjects The epidermal growth factor receptor (EGFR), a transmembrane protein or receptor for members of the EGFRvIII is a mutant form of EGFR (EGFR; ErbB-1; HER1). A 267 amino acid, in-frame sequence within the extracellular domain of the wild-type EGFR protein by a deletion of exons 2-7 of the wild-type EGFR gene, resulting in a deletion in EGFRvIII also contains a novel glycoprotein inserted at the fusion junction. The truncated receptor, EGFRvIII, also contains EGFR residues. Although unable to bind to tyrosine kinases, they exhibit constitutive tyrosine kinase activity. is important for its pro-tumorigenic effect. Kinase-deficient EGFRvIII has a similar EGFRvIII is unable to confer an oncogenic advantage. It can be highly expressed in tumors of the ovarian and rectal nuclei (M) and can also be detected in some other solid tumor types. but cannot be detected in normal tissues.

[0209] In some embodiments, the antigen-binding portion of a CAR described herein is EGFRvI. II-specific (EGFRvIII CAR). EGFRvIII-specific CARs are When expressed on the cell surface, it redirects T cell specificity to human EGFRvIII. In some embodiments, the antigen-binding domain is a target antigen-specific, anti-EGFRvII The variable light chain (VL) and variable heavy chain (VH) of a monoclonal antibody and a flexible linker such as a glycine-serine linker or Whitlow linker. and single-chain antibody fragments (scFv) comprising a VL and a VH linked together. In some embodiments, the scFv is mouse MR1 IgG. EGFRvIII scFv clone MR1 (SEQ ID NO: 115; SEQ ID NO: 229), anti-E GFRvIII scFv clone MR1-1 (SEQ ID NO: 116; SEQ ID NO: 230), anti EGFRvIII scFv clone huMR1-1 (SEQ ID NO: 117; SEQ ID NO: 231 ), anti-EGFRvIII scFv clone huMR1-2 (SEQ ID NO: 118; SEQ ID NO: 232). In some embodiments, the antigen-binding portion is, for example, N-terminal to C-terminal and VH-linker-VL or VL-linker-VH and V linked in a direction It may include H and VL.

[0210] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 221. (VL of anti-EGFRvIII clone MR1) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In some embodiments, the antigen-binding portion comprises a VL polypeptide having the same sequence as in the first embodiment. The CAR described herein has the amino acid sequence of SEQ ID NO: 220 (anti-EGFRvIII clone). At least 90%, 91%, 92%, 93%, 94% of the loan amount of MR1 (VH) 95%, 96%, 97%, 98%, 99%, or 100% identity to a VH polypeptide. In some embodiments, the antigen-binding portion comprises a C polypeptide as described herein. AR is the amino acid sequence of SEQ ID NO: 223 (anti-EGFRvIII clone MR1-1 VL) and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Antibodies containing VL polypeptides with 97%, 98%, 99%, or 100% identity In some embodiments, the CAR described herein comprises a nucleotide sequence similar to SEQ ID NO:2. The 22 amino acid sequence (VH of anti-EGFRvIII clone MR1-1) At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9% or 100% identity to the antigen-binding portion comprising a VH polypeptide. .

[0211] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 225. (VL of anti-EGFRvIII clone humMR1-1) at least 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or The antigen-binding portion comprises a VL polypeptide having 100% identity. In one embodiment, the CAR described herein comprises the amino acid sequence of SEQ ID NO: 224 (anti-EGFR VIII clone (humMR1-1 VH) at least 90%, 91%, 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In some embodiments, the antigen-binding portion comprises a VH polypeptide having a specific identity to the VH polypeptide. The CAR described herein has the amino acid sequence of SEQ ID NO: 227 (anti-EGFRvIII clone At least 90%, 91%, 92%, 93% of the VLs of the humMR1-2 clones , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the antigen-binding portion comprises a VL polypeptide as described herein. The CAR to be used has the amino acid sequence of SEQ ID NO: 226 (anti-EGFRvIII clone h At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, VH polypeptides with 5%, 96%, 97%, 98%, 99%, or 100% identity The antigen-binding portion comprises a peptide.

[0212] In one embodiment, the CAR described herein is a CD19-specific CAR. "CD19" refers to antigen cluster 19 or B lymphocyte antigen CD19. In humans, CD19 The CD19 gene is a protein encoded by the CD19 gene, which is involved in antigen receptor-dependent stimulation. Assembled by the antigen receptor of B lymphocytes to lower the threshold for stimulation It encodes a cell surface molecule. CD19 is expressed on follicular dendritic cells and B cells. In this context, CD19 is derived from the earliest recognizable B-lineage cells during the development of B-cell blasts. It is present on B cells but is lost during maturation into plasma cells. CD19 is primarily expressed in the presence of CD21 and Together with CD19 and CD81, it acts as a B cell coreceptor. Upon activation, it mediates the cytoplasmic transcription of CD19. The agonist is phosphorylated, which promotes binding by Src family kinases and PI-3 kinases. As on T cells, several surface molecules on B lymphocytes act as antigens, resulting in the recruitment of antibodies. The CD19 phosphoglycoprotein complex forms a proto-receptor that is (almost) B cell specific. Protein is one of these molecules. Other surface molecules are CD21 and CD8 1. These surface immunoglobulin (sIg)-related molecules facilitate signal transduction. On B cells, anti-immunoglobulin antibodies that mimic exogenous antigens bind to CD19, sI The reverse process has not been demonstrated, so this The formation of the receptor complex is suggested to be antigen-induced. This molecular association is chemically This has been confirmed by research.

[0213] In yet another embodiment, the CAR described herein is a CD33-specific CAR. Siglec-3, sialic acid-binding Ig-like lectin 3, SIGLEC3, SIGLE "CD33" refers to a 67 kDa single-spanning membrane glycoprotein, which may be C-3, gp67, or p67. Proteins, sialic acid-binding immunoglobulin-like lectins (Siglecs) superfamily CD33 is a member of the V-set family, which is responsible for binding to sialic acid. It is characterized by a C2 set of Ig-like domains in its extracellular domain. Alternative splicing of CD33 mRNA leads to the V-set Ig-like domain. In addition to the Ig-like domains of the V set, there are also disulfide bonds that connect the Ig-like domains of the C2 set. In healthy subjects, C D33 is primarily expressed on normal multipotent myeloid progenitor cells, unipotent colony-forming cells, monocytes, and CD33 is expressed as a myeloid differentiation antigen found on mature granulocytes and leukocytes. , expressed on more than 80% of myeloid leukemia cells, but not on normal hematopoietic stem cells or mature granules It is not expressed on lymphocytes (Andrews, R. et al., The L4F3 antigen is expressed by unipote nt and multipotent colony-forming cells but not by their precursors, Blood, 68(5 ):1030-5 (1986). CD33 is expressed on malignant myeloid cells, activated T cells, and activated NK cells. It has been reported to be expressed on at least some of the blast cells in the majority of AML patients. is also found on a subset (Pollard, J. et al., Correlation of CD33 expression l evel with disease characteristics and response to gemtuzumab ozogamicin containi ng chemotherapy in childhood AML, Blood, 119(16):3705-11 (2012)). On AML blasts In addition to its widespread expression, CD33 can also be expressed on the stem cells underlying AML.

[0214] In embodiments, the antigen-binding portion of the CAR described herein is specific for CD33. When expressed on the cell surface, CD33-specific CARs Redirects T cell specificity to human CD33. In some embodiments, antigen binding The variable domain is the variable domain light chain of an anti-CD33 monoclonal antibody specific for the target antigen. (VL) and a variable domain heavy chain (VH) comprising a glycine-serine linker or A single-chain antibody comprising a VL and a VH linked by a flexible linker such as a lysine linker. In some embodiments, the scFv includes M195, m2H12, In some embodiments, the scFv is humanized. In some embodiments, the antigen-binding portion is, e.g., a scFv, e.g., hM195. , from the N-terminus to the C-terminus, VH-linker-VL or VL-linker-VH, The VH and VL may be linked by In some embodiments, the CD33 antigen binding domain has the amino acid sequence of SEQ ID NO: 214. (hM195 VL) with at least 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99%, or 100% identity to the polypeptide. In some embodiments, the CD33 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 215. At least 90%, 91%, 92%, 93%, 94%, Polypeptides with 95%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the CD33 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 216. At least 90%, 91%, or 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% It includes polypeptides having identity.

[0215] In some embodiments, the polynucleotides, polypeptides and methods described herein The method may be used to treat hyperproliferative diseases such as cancer, autoimmune diseases, or viral or bacterial infections. In some embodiments, the compounds can be used to treat infections, such as helminths, helminths, or parasitic infections. Antigens can be found in cancer cells, autoimmune cells, or infected cells by viruses, bacteria, or parasites. Pathogens that can be targeted include, but are not limited to: , Plasmodium, Trypanosomes, Aspergillus ergillus, Candida, hepatitis A virus, hepatitis B virus Virus, Hepatitis C virus, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, or Ebola. Autoimmune diseases include graft-versus-host disease, rheumatoid arthritis, and Machi, systemic lupus erythematosus, celiac disease, Crohn's disease, Sjogren's syndrome, Rheumatism Polymyalgia nigra, multiple sclerosis, neuromyelitis optica, ankylosing spondylitis, type 1 diabetes, alopecia areata psoriasis, vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigus vulgaris, or autoimmune bud May include uveitis.

[0216] The pathogens that are essentially recognized by CAR can be any type of pathogen, but some In embodiments, the pathogen is a fungus, bacterium, or virus. Exemplary Viral Pathogens The body is composed of Adenoviridae, Epstein-Barr virus (EBV), and CMV (cytomegalovirus), Respiratory Syncytial Virus (RSV), JC virus, BK virus, HPV, HSV, HHV family Viruses of the Hepatitis family, Picornaviridae, Hepatitis virus Herpesviridae, Hepadnaviridae, Flaviviridae Flaviviridae, Retroviridae, Orthomyxoviruses Family (Orthomyxoviridae), Family (Paramyxoviridae), Papovavirus Family (Papovaviridae), Genus (Polyomavirus), Family (Rhabdoviridae) bdoviridae, and Togaviridae pathogens. Viruses include smallpox, influenza, mumps, measles, chickenpox, Ebola, and rubella. Exemplary pathogenic fungi include Candida, Aspergillus, llus), Cryptococcus, Histoplasma, Includes the genera Pneumocystis, and Stachybotrys. Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, seudomonas, Shigella, Campylobacter, Staphylococcus aureus Staphylococcus, Helicobacter, E. coli i), Rickettsia, Bacillus, Bordetella a), Chlamydia, Spirochetes, and Salmonella In some embodiments, the pathogen receptor Dectin 1 is used. It recognizes carbohydrate structures on the cell walls of fungi, such as Aspergillus sp. In another embodiment, a CAR can be generated by targeting viral determinants (e.g., CMV and Based on antibodies that recognize glycoproteins derived from rabies and Ebola, It is possible to create a CAR that blocks the rhesus pathology.

[0217] In some embodiments, a "spacer," "spacer region," or "spacer" may be used. The terms "main" or "hinge", "hinge" region, or "hinge domain" The "stalk," "stalk region," or "stalk domain" is defined as a region that binds to an antigen. A synthetic domain is used to link the transmembrane domain. The "stalk domain" or "stalk region" is a region within the polypeptide chain that separates the transmembrane domain from the cell. Any oligonucleotide that functions to link to the exodomain or cytoplasmic domain. In some embodiments, the stalk domain comprises an antigen-binding domain or polypeptide. sufficient to allow the domains to orient in different directions to facilitate antigen recognition. In some cases, the stalk region is made up of a hinge region derived from IgG1. In an alternative case, the stalk region may be a combination of the CH2CH3 region of an immunoglobulin and optionally and optionally a portion of CD3. Optionally, the stalk region is The CD8α hinge region (SEQ ID NO: 29) described in the pamphlet of No. 73755 ; SEQ ID NO: 170), the 12 amino acid hinge region of IgG4-Fc (ESKYGPPCP PCP), or IgG4 hinge region.

[0218] In some embodiments, the stalk region is CD8 alpha 2X (SEQ ID NO: 30; SEQ ID NO: 171), CD8 alpha 3X (SEQ ID NO: 31; SEQ ID NO: 172), or CD8 alpha At least 90%, 91%, or more of the amino acid sequence of α4X (SEQ ID NO: 32; SEQ ID NO: 173) %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the polypeptides.

[0219] In other embodiments, a spacer is provided between the extracellular domain of the CAR and the transmembrane domain. The spacer may comprise a stalk region and a stalk extension region. In one embodiment, the spacer may comprise a single stalk region. The spacer may comprise a stalk region and a stalk extension region. For example, the spacer may comprise one Stokes region of, and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 stokes In a further embodiment, the stalk region may be linked to the stalk via a linker. It can be linked to an extension region.

[0220] The transmembrane domain may be derived from natural or recombinant sources. When the source is natural, the domain may be any membrane-bound or transmembrane protein. Suitable transmembrane domains can be derived from the alpha chain, beta chain, and It may also contain the transmembrane domain of the CD28, CD3 epsilon, or zeta chain. 3ζ, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD 33, CD37, CD64, CD80, CD86, CD134, CD137, or CD Alternatively, the transmembrane domain may be derived from a synthetic In some embodiments, the amino acid sequence may be a nucleotide sequence containing 2 or more amino acids, and may contain hydrophobic residues such as leucine and valine. At one or both ends of the transmembrane domain, phenylalanine, tryptophan, and valine triplets are found. Optionally, in some embodiments, 2 to 10 Short oligonucleotide or polypeptide linkers, the length of which is between amino acids This may form a link between the transmembrane domain and the cytoplasmic signaling domain of CAR. In some embodiments, the linker is a glycine-serine linker. In this state, the transmembrane domain contains the CD8α transmembrane domain or the CD3ζ transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain. In some embodiments, the transmembrane domain comprises a CD3ζ transmembrane domain. The transmembrane region is a small amino acid sequence that is identical to the amino acid sequence of the CD8 alpha transmembrane domain (SEQ ID NO: 174). At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, It contains at least one of the polypeptides having 99% or 100% identity. In some embodiments, the transmembrane region comprises an amino acid sequence of the CD28 transmembrane domain (SEQ ID NO: 175). At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Polypeptides having at least 97%, 98%, 99%, or 100% identity Also includes one.

[0221] The intracellular domain may include one or more costimulatory domains. Exemplary costimulatory domains include: The markers are CD8, CD27, CD28, 4-1BB (CD137), ICOS, and DAP10. , DAP12, OX40 (CD134), or fragments or combinations thereof. Optionally, the CARs described herein are not limited to CD8, CD27, , CD28, 4-1BB(CD137), ICOS, DAP10, DAP12, OX40 (CD134), or a fragment or combination thereof. In some cases, the present invention may include one or more of the following: The CARs described in the specification are CD27, CD28, 4-1BB (CD137), and ICOS. OX40 (CD134), or a fragment or combination thereof. It may include one or more of the main Thus, the CARs described herein are CD8, CD28, 4-1BB (CD137), or one or more of the costimulatory domains selected from fragments or combinations thereof In some cases, the term "a" refers to a group of compounds described herein, including two or more. CARs may be CD28, 4-1BB (CD137), or fragments or combinations thereof. one or more, or two or more, of the costimulatory domains selected from In some cases, the CARs described herein may contain a costimulatory domain, CD 28 and 4-1BB (CD137), or their respective fragments. Thus, the CARs described herein contain the costimulatory domains CD28 and OX40 ( CD134), or their respective fragments. The CARs used in this study are those that combine the costimulatory domains CD8 and CD28, or each of these. In some cases, the CARs described herein comprise a costimulatory domain, a CD4 fragment, or a CAR-specific fragment. 28, or a fragment thereof. Optionally, the CARs described herein comprise a co-stimulatory domain. The present invention also includes the main 4-1BB (CD137) or a fragment thereof. The CARs described herein contain the costimulatory domain OX40 (CD134), or fragments thereof. In some cases, the CARs described herein comprise a co-stimulatory domain, CD8 , or a fragment thereof.

[0222] The intracellular signaling domain, also known as the cytoplasmic domain, of the CAR of the present disclosure comprises: Contributes to the activation of at least one of the normal effector functions of CAR-loaded immune cells The term "effector function" refers to the specialized function of a cell. The target function may be, for example, cytotoxic activity or helper activity including secretion of cytokines. Therefore, the term "intracellular signaling domain" is used to describe Proteins that transmit target functional signals and direct cells to carry out specialized functions Usually, the entire intracellular signaling domain can be used, but in many cases It is not necessary to use the entire chain. A truncated portion of the intracellular signaling domain may be used. Insofar as such cleavage transduces an effector function signal, it is possible to cleave such cleavage from the intact strand. can be used alternatively. Hence the term intracellular signaling domain. of an intracellular signaling domain sufficient to transmit an effector function signal. In some embodiments, the intracellular domain is a T cell Optionally, the polypeptide further comprises a signaling domain for T cell activation. The signaling domains for TCR zeta, FcR gamma, FcR beta, and CD3 CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d-derived domains. In some cases, they act as a signal for T cell activation. The transduction domain comprises a domain derived from CD3ζ.

[0223] In some embodiments, the intracellular signaling domain is CD28 (SEQ ID NO: 176), CD3 zeta signaling domain (SEQ ID NO: 177), 4-1BB signaling domain (SEQ ID NO: 178), DNAX-activating protein 10 (DAP10) signaling domain In (SEQ ID NO: 179), or DNAX activator protein 12 (DAP12) signal At least 90%, 91%, 92%, or 100% identical to the amino acid sequence of the transduction domain (SEQ ID NO: 180). %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical The polypeptides include at least one of the polypeptides having the following properties:

[0224] CD19-specific CAR CD19 is a cell surface glycoprotein of the immunoglobulin superfamily. In some cases, CD19 is expressed within solid tumors, such as pancreatic, liver, and prostate cancers. It has been detected in.

[0225] In some embodiments, the antigen-binding portion of the CAR described herein is specific for CD19. When expressed on the cell surface, CD19-specific CARs can enhance the specificity of T cells by targeting human T cells. In some embodiments, the antigen-binding domain redirects the target antigen to CD19. The variable domain light chain (VL) and variable domain of an anti-CD19 monoclonal antibody specific for The main heavy chain (VH) is preferably a glycine-serine linker or a Whitlow linker. A single-chain antibody fragment (scFv) comprising VL and VH linked by a flexible linker In embodiments, the scFv is SJ25C1 and / or FMC63. In some embodiments, the scFv is a humanized scFv. The binding moieties can be, for example, from N-terminus to C-terminus, VH-linker-VL or VL-linker It may comprise a Kerr-VH and a directionally linked VH and VL.

[0226] In some embodiments, the antigen-binding domain is a sc antibody that binds CD19. CD19-specific CARs are described that contain an Fv. In some cases, the antigen-binding domain recognizes an epitope on CD19.

[0227] In some embodiments, the antigen-binding domain is selected from the group consisting of JCAR014, JCAR015, JC AR017, or 19-28z CAR (Juno Therapeutics) In some embodiments, the present invention recognizes an epitope on CD19 that is also recognized by In the specification, the antigen-binding domain is JCAR014, JCAR015, JCAR017, Also approved by CAR (Juno Therapeutics) on 19-28z CD19-specific CAR-T cells that recognize an epitope on CD19 have been described. In some cases, CD19-specific CAR-T cells express the CD8 alpha transmembrane domain. a transmembrane domain selected from the group consisting of CD27, CD28, 4-1BB(CD137), ICOS, DAP10, DAP12, OX40(CD134 ), or a fragment or combination thereof, and further comprising a signaling domain derived from CD3ζ.

[0228] In some embodiments, the CD19-specific CAR-T cells described herein are The antigen-binding domain comprises JCAR014, JCAR01 5, JCAR017, or 19-28z CAR (Juno Therapeutic s) recognizes an epitope on CD19 that is also recognized by CD19-specific CAR-T cells express either the CD8 alpha transmembrane domain or the CD3ζ transmembrane domain Transmembrane domain selected from the domains: CD27, CD28, 4-1BB (CD137 ), ICOS, DAP10, OX40 (CD134), or fragments or combinations thereof and a signal derived from CD3ζ; It further comprises a transduction domain.

[0229] In some embodiments, the CD19-specific CAR-T cells described herein are The anti-CD19 antibody described in Patent Application Publication No. 20160152723 include.

[0230] In some embodiments, the antigen-binding domain is KTE-C19 (Kite Pharm It recognizes an epitope on CD19 that is also recognized by some In embodiments, the antigen-binding domain is also recognized by KTE-C19. CD19-specific CAR-T cells that recognize an epitope on CD19 have been described. In some cases, CD19-specific CAR-T cells express the CD8 alpha transmembrane domain. a transmembrane domain selected from the group consisting of CD27, CD28, 4-1BB(CD137), ICOS, DAP10, DAP12, OX40(CD134 ), or a fragment or combination thereof, and further comprising a signaling domain derived from CD3ζ.

[0231] In some embodiments, the CD19-specific CAR-T cells described herein are The antigen-binding domain of V is also bound by KTE-C19. Recognizes an epitope on CD19. In some cases, CD19-specific CA RT cells are selected from CD8 alpha transmembrane domain or CD3 zeta transmembrane domain. Transmembrane domains: CD27, CD28, 4-1BB (CD137), ICOS, DA P10, DAP12, OX40 (CD134), or fragments or combinations thereof one or more costimulatory domains selected; and a signaling domain derived from CD3ζ. Also includes main.

[0232] In some embodiments, the CD19-specific CAR-T cells described herein are available from international publications. The anti-CD19 antibody described in the pamphlet of Patent Publication No. 2015187528, includes fragments or derivatives thereof.

[0233] In some embodiments, the antigen-binding domain is a polypeptide of the type designated CTL019 (Novartis). In some embodiments, the present invention recognizes an epitope on CD19 that is also recognized by The specification states that the antigen-binding domain is a region on CD19 that is also recognized by CTL019. CD19-specific CAR-T cells that recognize the epitope of , CD19-specific CAR-T cells express either the CD8 alpha transmembrane domain or the CD3ζ transmembrane domain. Transmembrane domains selected from the following: CD27, CD28, 4-1BB (CD13 7), ICOS, DAP10, DAP12, OX40 (CD134), or fragments thereof one or more costimulatory domains selected from any one or combination of: It further comprises a signal transduction domain.

[0234] In some embodiments, the CD19-specific CAR-T cells described herein are v, and the antigen-binding domain is also recognized by CTL019. In some cases, CD19-specific CARs recognize epitopes on CD19 that are recognized by the CD19-specific CARs. -T cells selected from CD8 alpha transmembrane domain or CD3 ζ transmembrane domain Transmembrane domains: CD27, CD28, 4-1BB (CD137), ICOS, DAP 10, DAP12, OX40 (CD134), or a fragment or combination thereof. one or more costimulatory domains selected from the CD3ζ-derived signaling domains; Further includes Inn.

[0235] In some embodiments, the antigen-binding domain is UCART19 (Cellectis) In some embodiments, the antibody recognizes an epitope on CD19 that is also recognized by As used herein, the antigen-binding domain is a CD4 antibody that is also recognized by UCART19. CD19-specific CAR-T cells that recognize an epitope on CD19 have been described. In this case, CD19-specific CAR-T cells are expressed as CD8 alpha transmembrane domain or CD3 A transmembrane domain selected from the ζ transmembrane domain; CD27, CD28, 4-1BB (C D137), ICOS, DAP10, DAP12, OX40 (CD134), or one or more costimulatory domains selected from fragments or combinations thereof; and CD3 It further contains a signaling domain derived from ζ.

[0236] In some embodiments, the CD19-specific CAR-T cells described herein are v, and the antigen-binding domain is also Recognizes an epitope on CD19. In some cases, CD19-specific CA RT cells are selected from CD8 alpha transmembrane domain or CD3 zeta transmembrane domain. Transmembrane domains: CD27, CD28, 4-1BB (CD137), ICOS, DA P10, OX40 (CD134), or a fragment or combination thereof; one or more costimulatory domains; and a signaling domain derived from CD3ζ. Included.

[0237] In some embodiments, the antigen-binding domain is selected from the group consisting of BPX-401 (Bellicum). In some embodiments, the antibody recognizes an epitope on CD19 that is also recognized by As used herein, the antigen-binding domain is a CD1 CD19-specific CAR-T cells that recognize the epitope on CD19 have been described. In this study, CD19-specific CAR-T cells were engineered to express either the CD8 alpha transmembrane domain or the CD3ζ A transmembrane domain selected from the group consisting of CD27, CD28, 4-1BB (CD 137), ICOS, DAP10, DAP12, OX40 (CD134), or any of these and one or more costimulatory domains selected from fragments or combinations of: It further comprises a signaling domain derived from

[0238] In some embodiments, the CD19-specific CAR-T cells described herein are v, and the antigen-binding domain is also Recognizes an epitope on CD19. In some cases, CD19-specific CA RT cells are selected from CD8 alpha transmembrane domain or CD3 zeta transmembrane domain. Transmembrane domains: CD27, CD28, 4-1BB (CD137), ICOS, DA P10, DAP12, OX40 (CD134), or fragments or combinations thereof one or more costimulatory domains selected; and a signaling domain derived from CD3ζ. Also includes main.

[0239] In some cases, the antigen-binding domain is selected from the group consisting of blinatumomab (Amgen), coltuximab (Campaigner), and gliomas with ... Bu-Rabtansine (ImmunoGen Inc. / Sanofi-aventis), MOR208 (Morphosys AG / Xencor Inc.), MEDI-55 1 (Medimmune), denintuzumab-mafodotin (Seattle Genet ics), B4 (or DI-B4) (Merck Serono), taplitumomab Paptox (National Cancer Institute), XmAb 5 871 (Amgen / Xencor, Inc.), MDX-1342 (Medarex) or an epitope on CD19 that is also recognized by AFM11 (Affimed). In some cases, CD19-specific CARs recognize the CD8 alpha transmembrane domain. a transmembrane domain selected from the group consisting of CD27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD134), or one or more costimulatory domains selected from fragments or combinations thereof; and It further contains a signaling domain derived from 3ζ.

[0240] In some embodiments, the antigen-binding domain is selected from the group consisting of F(ab'), Fab, CD19-specific CAR-T cells, including Fab, Fv, or scFv, are described. In some cases, the antigen-binding domain recognizes an epitope on CD19. Therefore, the antigen-binding domain is a marker for blinatumomab (Amgen), coltuximab (Abcam), and rabies. Tansin (ImmunoGen Inc. / Sanofi-aventis), MOR2 08 (Morphosys AG / Xencor Inc.), MEDI-551 (Me dimmune), denintuzumab mafodotin (Seattle Genetics) , B4 (or DI-B4) (Merck Serono), tapritumomab paptoc (National Cancer Institute), XmAb 5871( Amgen / Xencor, Inc.), MDX-1342 (Medarex) or A Recognizes an epitope on CD19 that is also recognized by FM11 (Affimed) In some cases, CD19-specific CAR-T cells express the CD8 alpha transmembrane domain. a transmembrane domain selected from the group consisting of CD27, CD28, 4-1BB(CD137), ICOS, DAP10, DAP12, OX40(CD134 ), or a fragment or combination thereof, and further comprising a signaling domain derived from CD3ζ.

[0241] In some cases, the CD19-specific CAR-T cells described herein contain scFv antibodies. The antigen-binding domain is a cytochrome P450 antibody, which is a cytochrome P450 antibody. Tuximab-ravtansine (ImmunoGen Inc. / Sanofi-Avent is), MOR208 (Morphosys AG / Xencor Inc.), MED I-551 (Medimmune), denintuzumab-mafodotin (Seattle G enetics), B4 (or DI-B4) (Merck Serono), Taplitz Momab-Paptox (National Cancer Institute), Xm Ab 5871 (Amgen / Xencor, Inc.), MDX-1342 (Meda rex) or AFM11 (Affimed) on CD19 In some cases, CD19-specific CAR-T cells recognize the CD8 alginate epitope. a transmembrane domain selected from the CD3ζ transmembrane domain or the CD3ζ transmembrane domain; 27, CD28, 4-1BB (CD137), ICOS, DAP10, OX40 (CD1 34), or a fragment or combination thereof, and further comprising a signaling domain derived from CD3ζ.

[0242] In embodiments, the CAR described herein has the amino acid sequence of SEQ ID NO: 210 With at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, CD19-specific CARs (CD19-) with 98%, 99%, or 100% identity In some embodiments, the C AR has at least 90%, 91%, 92%, 93%, or 100% identity with the amino acid sequence of SEQ ID NO: 211. 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity CD19-specific CAR (CD19-CD8α-CD28- with signal peptide) CD3ζ).

[0243] In some embodiments, the antigen-binding portion of the CAR described herein is specific for CD19. When expressed on the cell surface, CD19-specific CARs can enhance the specificity of T cells by targeting human T cells. In some embodiments, the antigen-binding domain redirects the target antigen to CD19. The variable domain light chain (VL) and variable domain of an anti-CD19 monoclonal antibody specific for The main heavy chain (VH) is preferably a glycine-serine linker or a Whitlow linker. A single-chain antibody fragment (scFv) comprising VL and VH linked by a flexible linker In some embodiments, the scFv comprises an anti-CD19 clone with a Whitlow linker. In some embodiments, the antibody is FMC63 scFv (SEQ ID NO: 213). The CD19 antibody is derived from the variable heavy chain of the anti-CD19 monoclonal antibody clone FMC 63. (SEQ ID NO: 212). In some embodiments, the antigen-binding portion comprises, for example, an N-terminal The VH-linker-VL or VL-linker-VH is linked directionally from the C-terminus to the C-terminus. It may comprise a linked VH and VL.

[0244] Engineered T cell receptor (TCR) In some embodiments, the chimeric antigen receptors encoded by the polynucleotides described herein are T cell receptors (TCRs) are receptors that bind to antigens on the surface of T cells. It consists of two chains (αβ or γδ) that combine to form a heterodimeric receptor In some cases, the αβ TCR is expressed on the majority of T cells in the body and specifically MHC-restricted. Each of the α and β chains is known to be involved in antigen recognition. It anchors the protein to the cell membrane and acts as an invariant subunit of the CD3 signaling apparatus. The CDRs are connected to the associated constant domain (C) via a six-loop loop. and variable domains (V) that confer antigen recognition. The main CDRs are CDR1, CDR2, CDR3, and CDR4. These CDRs comprise CDR1 and CDR2. The complex formed between the antigenic peptide and the protein (pepMHC) (e.g., , HLA-A complex, HLA-B complex, HLA-C complex, HLA-DPA1 complex, HLA-DPB1 complex, HLA-DQA1 complex, HLA-DQB1 complex, HLA- HLA-DRB1 complex, or HLA-DRB1 complex). The domains further comprise a junction region that links the constant domain to the variable domain. Thus, the beta strand further comprises a short diversity region that forms part of the joining region.

[0245] In some cases, such TCRs target specific tumor antigens, e.g., NY-ESO, Mag In other cases, such TCRs are expressed in the patient's tumor. specific neoantigens (i.e., tumor-expressed, patient-specific, somatic, In some cases, engineered TCRs have enhanced affinity. It is possible.

[0246] In some embodiments, the TCR is from International Immunogene The TCRs are described using the TCR nomenclature by tics (IMGT), and Link to public databases for sequences, e.g., their framework sequences , several types identified by CDR1 sequences, CDR2 sequences, and CDR3 sequences There are 10 types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions. Thus, the types of Vα are classified by their unique TRAV numbers in the IMGT nomenclature. For example, "TRAV21" has a unique framework sequence and CDR1 The amino acid sequences conserved from TCR to TCR are It is partially defined, but also contains amino acid sequences that vary from TCR to TCR. Similarly, "TRBV5-1" defines a region of the TCRVα having a unique CDR3 sequence. and CDR1 and CDR2 sequences, but the CDR3 sequence defines the TCRVβ region, which is only partially defined.

[0247] The beta chain diversity region is sometimes abbreviated as TRBD in the IMGT nomenclature. It will be imposed.

[0248] In some cases, the unique sequences defined by the IMGT nomenclature are widely known and These are available to those working in the field of CR. For example, these are public data from IMGT. Bass and “T cell Receptor Factsbook”, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8.

[0249] In some embodiments, the αβ heterodimeric TCR comprises, for example, a cytoplasmic domain and a transmembrane domain. In some cases, the TCR is transfected as a full-length chain containing both the TCR domain and the TCR domain. is described, for example, in WO 2006 / 000830. , containing introduced disulfide bonds between residues of each constant domain.

[0250] In some cases, the TCRs described herein are in a single chain format (e.g., (See WO 2004 / 033685). Single-stranded format are Vα-L-Vβ type, Vβ-L-Vα type, Vα-Cα-L-Vβ type, Vα-L-Vβ- αβ TCR polypeptides of the Cβ type, Vα-Cα-L-Vβ-Cβ type [in format, Vα and Vβ are the TCRα and TCRβ variable regions, respectively; Cβ and Cβ are the TCR α and TCR β constant regions, respectively, and L is a linker. In certain embodiments, the single chain TCR of the present disclosure comprises the sequence Each constant domain described in brochure 004 / 033685 The amino acid sequence may have a disulfide bond introduced between the residues of

[0251] The TCRs described herein can be associated with a detectable label, a therapeutic agent, or a PK-modifying moiety. It is possible.

[0252] For diagnostic purposes, exemplary detectable labels include fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and the like. Examples of suitable immunosuppressants include, but are not limited to, immunoglobulins, antibodies, and imaging agents.

[0253] Further genetic elements Cell therapy holds great promise for the treatment of human diseases, but the cells themselves or their Significant toxicity from the transgene products has hindered clinical exploration. In some embodiments, an immune response comprising a CAR or TCR as described herein is Vector cells transfused into a mammalian subject, e.g., a human, may be used for their If toxicity occurs, we will need to regulate the effects of these immune effector cells. Thus, in certain embodiments, the compounds described herein can be depleted. In addition to the specific chimeric antigen receptor, a second gene may also be engineered as described herein. The second gene can be introduced into the target immune effector cell. or "Ki" which allows for the depletion of cells containing TCR or antigen-binding polypeptides. In certain embodiments, a "kill switch" or a "cell tag" is used. The present invention relates to a method for treating HER1 comprising administering to a patient a therapeutically effective amount of at least one antibody-binding epitope of HER1 or a functional fragment thereof, and any Optionally, a truncated HER1 peptide (e.g., a HER1 polypeptide fragment thereof) containing a signal polypeptide sequence or a fragment thereof is also provided. Herein, this is referred to as HER1t or EGFRt).

[0254] In certain embodiments, the second gene is epidermal growth factor receptor (HER1) or In some embodiments, the second gene is a HER1 tag, which is a fragment or variant of is a truncated human epidermal growth factor receptor 1 (e.g., HER1t) (SEQ ID NO: 76; SEQ ID NO: 189), a HER1 tag. Optionally, the second gene is a human epidermal growth factor In some cases, truncated HER1 mutants are called HER1t1 ( SEQ ID NO: 77; SEQ ID NO: 190), HER1t2 (SEQ ID NO: 78; SEQ ID NO: 191), H ER1t3 (SEQ ID NO: 79; SEQ ID NO: 192), HER1t4 (SEQ ID NO: 80; SEQ ID NO: 193), HER1t5 (SEQ ID NO: 81; SEQ ID NO: 194), HER1t6 (SEQ ID NO: 8 2; SEQ ID NO: 195), HER1t7 (SEQ ID NO: 83; SEQ ID NO: 196), HER1t8 (SEQ ID NO: 84; SEQ ID NO: 197), HER1t9 (SEQ ID NO: 85; SEQ ID NO: 198), HER1t10 (SEQ ID NO: 86; SEQ ID NO: 199) or HER1t11 (SEQ ID NO: 87 ; SEQ ID NO: 200). In some embodiments, HER1, HER1t, HER1t1 , HER1t2, HER1t3, HER1t4, HER1t5, HER1t6, HER1 t7, HER1t8, HER1t9, HER1t10, and HER1t11 At least one of the following is FDA-approved: cetuximab, or HER1, HER1 t, HER1t1, HER1t2, HER1t3, HER1t4, HER1t5, HER 1t6, HER1t7, HER1t8, HER1t9, HER1t10, and / or The goal is to prevent the depletion of infused CAR-T cells through the administration of any antibody that recognizes HER1t11. Allowing thawing provides a safety mechanism.

[0255] In some embodiments, the HER1t gene comprises the nucleic acid sequence of SEQ ID NO: 76 and at least 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the HER1 gene comprises a nucleotide sequence having 100% identity to the HER1 gene. The t1 gene has at least 90%, 91%, 92%, 93% identity with the nucleic acid sequence of SEQ ID NO: 77. , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the HER1t2 gene comprises the nucleotide sequence of SEQ ID NO: 78. Nucleic acid sequences and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 nucleotide sequences with 7%, 98%, 99%, or 100% identity. In one embodiment, the HER1t3 gene has at least 90% homology with the nucleic acid sequence of SEQ ID NO: 79. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In some embodiments, the HER1t4 gene is a nucleotide sequence having 100% identity to the HER1t4 gene. The gene may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115 Nucleotides with 4%, 95%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the HER1t5 gene comprises the nucleic acid sequence of SEQ ID NO: 81. Sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% identity. In an embodiment, the HER1t6 gene is at least 90%, 90%, or 90% identical to the nucleic acid sequence of SEQ ID NO: 82. 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 In some embodiments, the HER1t7 gene comprises a nucleotide sequence having 0.0% identity to the HER1t7 gene. The child has a sequence identical to the nucleic acid sequence of SEQ ID NO: 83 and a sequence identical to the nucleic acid sequence of SEQ ID NO: 84, and the sequence identical to the nucleic acid sequence of SEQ ID NO: 85, and the sequence identical to the nucleic acid sequence of SEQ ID NO: 86, and the sequence identical to the nucleic acid sequence of SEQ ID NO: 87, and the sequence identical to the nucleic acid sequence of SEQ ID NO: 88, and the sequence identical to the nucleic acid sequence of SEQ , 95%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the HER1t8 gene comprises the nucleic acid sequence of SEQ ID NO: 84. and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 nucleotide sequences with 8%, 99%, or 100% identity. In this embodiment, the HER1t9 gene has at least 90%, 91% or more homology with the nucleic acid sequence of SEQ ID NO: 85. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In some embodiments, the HER1t10 gene comprises a nucleotide sequence having an identity to the HER1t10 gene. is at least 90%, 91%, 92%, 93%, 94%, Nucleotides with 95%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the HER1t11 gene comprises the nucleic acid sequence of SEQ ID NO: 87. and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 It includes nucleotide sequences with 8%, 99%, or 100% identity.

[0256] Truncated HER1 sequences, e.g., HER1t, HER1t1, HER1t2, HER1t 3, HER1t4, HER1t5, HER1t6, HER1t7, HER1t8, HER HER1t9, HER1t10, and / or HER1t11 are receptors for cetuximab. It inhibits EGF ligand binding, EGFR homodimerization, and hemoglobin synthesis while keeping the binding of EGFR intact. abolishes heterodimerization and potential for EGFR-mediated signaling (Ferg uson, K., 2008. A structure-based view of Epidermal Growth Factor Receptor regul ation. Annu Rev Biophys, Volume 37, pp. 353-373).

[0257] In a further embodiment, in addition to the chimeric antigen receptor specific for the therapeutic target of the present disclosure, The second gene inserted is a HER1 tag. Optionally, the HER1 tag is a full-length HER1 tag. ER1 polypeptide, or truncated HER1 polypeptide (HER1t1), HER1t 2, HER1t3, HER1t4, HER1t5, HER1t6, HER1t7, HER 1t8, HER1t9, HER1t10, or HER1t11. In some cases, the HER1 tag is a truncated HER1 mutant. , HER1, HER1t1, HER1t2, HER1t3, HER1t4, HER1t5 , HER1t6, HER1t7, HER1t8, HER1t9, HER1t10, or HER1t11 can also be treated with FDA-approved rituximab therapy. Allowing for depletion of infused CAR-T cells also allows for a safety mechanism. In certain embodiments, the HER1 tag has at least 90% identical sequence to the sequence of SEQ ID NO: 189. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 In certain embodiments, the polypeptide sequence has 0.000% identity to HER1. The tag is a HER1t1 tag and has a sequence identical to that of SEQ ID NO: 190, and is at least 90%, 91%, or , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% In certain embodiments, the HER1 tag has a polypeptide sequence having an identity of , HER1t2 tag, and the sequence of SEQ ID NO: 191 and at least 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In certain embodiments, the HER1 tag has a polypeptide sequence having the following structure: R1t3 tag, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 2 3%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity In certain embodiments, the HER1 tag has a polypeptide sequence corresponding to the HER1t 4 tags, and the sequence of SEQ ID NO: 193 and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity In certain embodiments, the HER1 tag has a polypeptide sequence. and the sequence of SEQ ID NO: 194 and at least 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or 100% identity In certain embodiments, the HER1 tag is a HER1t6 tag. , SEQ ID NO: 195 and at least 90%, 91%, 92%, 93%, 94%, 95 96%, 97%, 98%, 99%, or 100% identity to a polypeptide sequence In certain embodiments, the HER1 tag is a HER1t7 tag and has the sequence Sequence number 196 and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 having polypeptide sequences with 6%, 97%, 98%, 99%, or 100% identity In certain embodiments, the HER1 tag is a HER1t8 tag and is set forth in SEQ ID NO: 1. 97 sequences and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, having a polypeptide sequence with 97%, 98%, 99%, or 100% identity. In certain embodiments, the HER1 tag is a HER1t9 tag, and is represented by SEQ ID NO: 198 Sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% identity to a polypeptide sequence. In certain embodiments, the HER1 tag is a HER1t10 tag and has the sequence of SEQ ID NO: 199 and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 The polypeptide sequences have 8%, 99%, or 100% identity. In embodiments, the HER1 tag is a HER1t11 tag, comprising the sequence of SEQ ID NO: 200 and At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% identity to the polypeptide sequence.

[0258] In certain embodiments, the second gene encodes an activated glycosylated phosphoprotein or A fragment or variant of this is the CD20 tag (SEQ ID NO: 88; SEQ ID NO: 201). Optionally, the second gene is a truncated CD20 mutant, CD20t1 (sequence SEQ ID NO: 89; SEQ ID NO: 202). In some embodiments, the CD20 tag, (CD20t1), or fragments of the CD20 or CD20t1 tag, By administering an antibody that recognizes CAR-T cells, it is possible to deplete the infused CAR-T cells. In some embodiments, the gene encoding the CD20 tag is , SEQ ID NO: 88 and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 1 Nucleotides with 5%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the gene encoding the CD20t1 tag comprises the sequence SEQ ID NO:8 9 nucleic acid sequences and at least 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 100% identity to the nucleotide sequence.

[0259] In a further embodiment, in addition to the chimeric antigen receptor specific for the therapeutic target of the present disclosure, The second gene inserted is a CD20 tag. Optionally, the CD20 tag is a full-length C The polypeptide is a CD20 polypeptide or a truncated CD20 polypeptide (CD20t1).

[0260] In embodiments, the CAR vector comprising the CAR described herein is selected from the group consisting of SEQ ID NO: 88 nucleic acid sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 9 A full-length C containing a nucleic acid sequence with 6%, 97%, 98%, 99%, or 100% identity In some embodiments, the CAR further comprises a D20 tag. The R vector is a vector that contains at least 90%, 91%, 92%, 93%, or 100% of the nucleic acid sequence of SEQ ID NO: 89. 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity The present invention further includes a variant of the CD20 tag (CD20t1) comprising a nucleic acid sequence

[0261] In some embodiments, kill tags, e.g., HER1t, HER1t1, HER1t2, HER1t3, HER1t4, HER1t5, HER1t6, HER1t7, HER1t 8, HER1t9, HER1t10, or HER1t11 were identified as CA R or TCR or cytokine, in-frame, e.g., T. asigna ( self-cleaving peptides, including but not limited to, Thosea asigna virus (T2A) peptides In some embodiments, the T2A peptide is fused to the gene via a T2A peptide having the sequence At least 90%, 91%, 92%, 93%, 94%, or more of the amino acid sequence of No. 153 Amino acid sequences with 95%, 96%, 97%, 98%, 99%, or 100% identity In other embodiments, kill tags, e.g., HER1t, HER1t1, HER 1t2, HER1t3, HER1t4, HER1t5, HER1t6, HER1t7, H Genes encoding ER1t8, HER1t9, HER1t10, or HER1t11 to the cytokine, and at the 3' end, in-frame, e.g., (Thosea asigna) viral (T2A) peptide, but not limited to, Gene fusion is performed via a truncated peptide.

[0262] In some embodiments, both genes are cloned into a plasmid. In other embodiments, the cell tag is cloned into a separate lentiviral vector. In this state, the cell tag gene is inserted in frame with the CAR gene into the Sleeping Beacon receptor. In yet another embodiment, the vector is cloned into a H ER1t, HER1t1, HER1t2, HER1t3, HER1t4, HER1t5, HER1t6, HER1t7, HER1t8, HER1t9, HER1t10, or H Cell tags such as ER1t11 are expressed as multiple Sleeping Beauty transposons. Cloning into a vector.

[0263] In certain embodiments, the cell tag is a signal peptide, e.g., GM-CSFRα In this case, the GM-CSFRα signal peptide is SEQ ID NO: 1. At least 90%, 91%, 92%, 93%, 94%, and 95% of the 63 amino acid sequences , 96%, 97%, 98%, 99%, or 100% identity. In the form, the signal peptide has at least 90% identity with the amino acid sequence of SEQ ID NO: 164. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or In certain embodiments, the signal peptide is an IgK having 100% sequence identity. The amino acid sequence of SEQ ID NO: 165 has at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity , IgE sequence. In certain embodiments, the signal peptide is At least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence %, 97%, 98%, 99%, or 100% identity to the CD8α sequence. In certain embodiments, the signal peptide is a nucleotide sequence similar to that of SEQ ID NO: 167. At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the TVB2 (T21A) sequence. In one embodiment, the signal peptide is a nucleotide sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 168, the amino acid sequence of SEQ ID NO: 169 ... 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a CD52 sequence. The null peptide has at least 90%, 91%, 92%, or 100% identity with the amino acid sequence of SEQ ID NO: 169. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical Low-affinity nerve growth factor receptor (LNGFR, TNFRSF16) sequences with In some cases, the signal peptide is GM-CSFRα, IgK, IgE, C D8α, T21A, CD52, low-affinity nerve growth factor receptor, and their It can be selected from variants and fragments.

[0264] In some embodiments, the cell tag comprises a signal peptide, e.g., a GM-CSFRα signal peptide. In this case, the GM-CSFRα signal peptide is SEQ ID NO: 20 or or at least 90%, 91%, 92%, 93%, 94% identical to the nucleic acid sequence of SEQ ID NO: 21. , 95%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the cell tag is encoded by a signal peptide, e.g., For example, it has an IgK signal peptide, in which case the IgK signal peptide is SEQ ID NO:2 2 nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96 97%, 98%, 99%, or 100% identity to the nucleotide sequence. In some embodiments, the cell tag comprises a signal peptide, e.g., an IgE signal peptide. In this case, the IgK signal peptide has the nucleic acid sequence of SEQ ID NO: 23. of,at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 It is encoded by a nucleotide sequence with 8%, 99%, or 100% identity. In some embodiments, the cell tag comprises a signal peptide, e.g., a CD8α signal peptide. wherein the CD8α signal peptide has the sequence of SEQ ID NO: 24 or SEQ ID NO: 25 With acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 encoded from a nucleotide sequence with 7%, 98%, 99%, or 100% identity In some embodiments, the cell tag comprises a signal peptide, such as TVB2 (T21 A) It has a signal peptide, in this case the TVB2 (T21A) signal peptide. At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 96%, 97%, 98%, 99%, or 100% identity In some embodiments, the cell tag is encoded from a signal peptide, e.g., C In this case, the CD52 signal peptide is SEQ ID NO: 27 At least 90%, 91%, 92%, 93%, 94%, 95%, 96% nucleotide sequences with 97%, 98%, 99%, or 100% identity. In some embodiments, the cell tag comprises a signal peptide, e.g., a low affinity It has a signal peptide of the nerve growth factor receptor (LNGFR, TNFRSF16), In this case, low affinity nerve growth factor receptor (LNGFR, TNFRSF16) signaling The peptide has at least 90%, 91%, 92%, 93%, or 100% identity with the nucleic acid sequence of SEQ ID NO: 28. 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity It is encoded from a nucleotide sequence.

[0265] A signal peptide is located at the N-terminus of a newly synthesized protein or polypeptide. Typical amino acid sequences are those that bind to proteins or polypeptides that are targeted to the cell surface. In some embodiments, the signal peptide is an amino acid sequence that directs the signal peptide to the cell membrane. directing a polypeptide inserted into the cytoplasm (e.g., via a transmembrane domain) to the cell surface In some embodiments, the polypeptide constructs described herein include a signal peptide. The mature polypeptide construct is then synthesized with the amino acid sequence of the signal peptide. It is post-translationally processed to remove the amino acid sequence, and the signal peptide is cleaved. In this embodiment, the signal peptide sequence is not cleaved and remains in the mature polypeptide construct. It exists.

[0266] The present disclosure provides methods for directing and / or trafficking polypeptide constructs to the cell surface. A polypeptide containing any known or unknown signal peptide capable of being linked to a target gene. For example, in some embodiments, the polypeptide construct comprises GMCSFR α, Ig kappa, immunoglobulin E, CD8α, TVB2 (T21A), CD52, and is a signaling receptor for the low-affinity nerve growth factor receptor (LNGFR, TNFRSF16). It contains the signal sequence corresponding to the peptide.

[0267] In some embodiments, the signal peptide is selected from the list consisting of SEQ ID NOs: 20-28. at least 70%, 75%, 80%, 85%, 90%, Polynucleotides containing nucleotide sequences with 95%, 99%, or 100% identity In some embodiments, the signal peptide is encoded by a sequence selected from the group consisting of SEQ ID NOs: 163 to 168. 169. Amino acid sequences with 0%, 85%, 90%, 95%, 99%, or 100% identity Includes.

[0268] Engineered effector cells one or more genes regulated by the gene switch polypeptides disclosed herein Effector cells (immune effectors) engineered to express a heterologous gene or genes (also referred to as cells) are provided.

[0269] As used herein, "T cell" or "T lymphocyte" refers to a cell-mediated immune response. T cells or T lymphocytes are a type of lymphocyte that play a central role in B cells and natural killer cells due to the presence of T cell receptors (TCR) on their cell surface They can be distinguished from other lymphocytes, such as NK cells.

[0270] In some embodiments, the engineered effector cells are T cells and / or natural killer cells. T cells or T lymphocytes are modified immune cells that are involved in cell-mediated immunity. Exemplary T cells include helper T cells, cytotoxic T cells, and T H 1 7 cells, stem cell memory T cells (TSCM), naive T cells, memory T cells, These include effector T cells, regulatory T cells, or natural killer T cells.

[0271] "Helper T cells" (T H cells) regulate the development of B cells into plasma cells and memory B cells. in immune processes, including maturation, and activation of cytotoxic T cells and macrophages, Support other white blood cells. In some cases, T H The cells express the CD4 glycoprotein on their surface. Due to their expression, they are known as CD4+ T cells. Helper T cells act as antigen-presenting cells ( Peptide antigens are presented by MHC class II molecules expressed on the surface of APCs Once activated, helper T cells divide rapidly and mediate an active immune response. secrete small proteins called cytokines that inhibit or support the growth of These cells are called T H 1. T H 2. T H 3. T H 17, T H 9, or T FH Including There are several subtypes, including those that secrete different cytokines and induce different types of immune responses. Signaling from APCs facilitates differentiation into one of several subtypes. , directing T cells toward specific subtypes.

[0272] "Cytotoxic T cells" (TC cells or CTLs) are capable of targeting virus-infected cells and tumor cells. These cells also have a number of markers on their surface that destroy the nuclei of the nuclei and are involved in graft rejection. These cells express the CD8 glycoprotein and are therefore known as CD8+ T cells. These cells react with antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. Regulatory T cells recognize their targets by binding to IL-1. Through ATP, adenosine, and other molecules, CD8+ cells are inducing a state of anergy. This may activate the immune system, thereby preventing autoimmune diseases.

[0273] "Memory T cells" are antigen-specific T cells that persist for a long time after the infection has cleared. Memory T cells are a subset of T cells that respond to their cognate antigen upon re-exposure. rapidly expand into large numbers of effector T cells, which provide the immune system with a means to Memory T cells are a subtype of stem cell memory T cells. (TSCM), central memory T cells (TCM cells) and two types of effector Memory cells include CD4+ cells and T cells (TEM cells and TEMRA cells). Memory T cells can be either CD8+ or CD8+ cells. They may express CD45RO, CD45RA, and / or CCR7.

[0274] "Regulatory T cells" (Treg cells), formerly known as suppressor T cells, ) play a role in maintaining immune tolerance. The primary role of regulatory T cells is to regulate T cell-mediated immune responses. By blocking the immune response, autoreactive T cells escape the negative selection process in the thymus. The aim is to suppress cells.

[0275] "Natural killer T cells" (NKT cells) act as a bridge between the adaptive immune system and the innate immune system. Conventional antibodies recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. Unlike normal T cells, NKT cells recognize glycolipid antigens presented by a molecule called CD1d. Upon activation, these cells are attributed to both Th and Tc cells. functions (i.e., production of cytokines and release of cytolytic / cell-killing molecules) NKT cells also recognize some tumor cells and cells infected with herpes viruses. Natural killer (NK) cells are cells that naturally A type of cytotoxic lymphocyte in the immune system. In some cases, NK cells are involved in the defense against viral infections. NK cells provide the first line of defense against infection and / or tumor formation. It is possible to detect MHC presented on tumor-associated or cancerous cells and induce cytokine release. NK cells further react with antibodies and / or Alternatively, stressed cells can be detected in the absence of MHC, which allows rapid Enables a rapid immune response.

[0276] Dose of engineered effector cells In some embodiments, an amount of engineered effector cells is administered to a subject in need thereof. The amount of cytokines administered will determine their effectiveness and potential to induce the damaging effects associated with the cytokines. In some cases, the amount of modified effector cells is determined based on the amount of modified effector cells per kg of the culture medium. Approximately 10 effector cells 5 ~about 10 9 Optionally, the amount of modified effector cells Approximately 10 engineered effector cells per kg 5 ~about 10 8 Including pieces. The amount of engineered effector cells was approximately 10 engineered effector cells per kg. 5 ~about 10 7 Optionally, the amount of engineered effector cells is 1000 mg / kg of engineered effector cells. -about 10 cells 6 ~about 10 9 Optionally, the amount of engineered effector cells comprises 1 kJ / ml. Approximately 10 engineered effector cells per g 6 ~about 10 8 In some cases, modified F The amount of engineered effector cells was approximately 10 per kg. 7 ~about 10 9 Contains In some cases, the amount of engineered effector cells is about 1 kg of engineered effector cells. 10 5 ~about 10 6 In some cases, the amount of modified effector cells is Approximately 10 engineered effector cells 6 ~about 10 7 Optionally, modified effectors The cell dose was approximately 10 engineered effector cells per kg. 7 ~about 10 8 Includes pieces. Therefore, the amount of engineered effector cells is approximately 10 engineered effector cells per kg. 8 ~ about 10 9In some cases, the amount of engineered effector cells is 1000 mg / kg of engineered effector cells. Approximately 10 effector cells 9 In some cases, the amount of modified effector cells is 1 kg Approximately 10 engineered effector cells per 8 Optionally, modified effector cells The amount of engineered effector cells per kg is approximately 10 7 In some cases, modified The amount of effector cells was approximately 10 modified effector cells per kg. 6 Includes pieces. Therefore, the amount of engineered effector cells is approximately 10 engineered effector cells per kg. 5 pieces Includes.

[0277] In some embodiments, the engineered effector cells are engineered T cells. The modified T cells are CAR-T cells. In some cases, the amount of CAR-T cells is Approximately 10 CAR-T cells per 5 ~about 10 9 In some cases, the amount of CAR-T cells Approximately 10 CAR-T cells per kg 5 ~about 10 8 In some cases, CAR -T cell dose is approximately 10 CAR-T cells per kg 5 ~about 10 7 Including pieces. Therefore, the amount of CAR-T cells is approximately 10 CAR-T cells per kg. 6 ~about 10 9 Includes In some cases, the amount of CAR-T cells is approximately 10 CAR-T cells per kg. 6 ~about 10 8 In some cases, the amount of CAR-T cells may be CAR-T cells per kg. about 10 7 ~about 10 9In some cases, the amount of CAR-T cells is C per kg. AR-T cells approximately 10 5 ~about 10 6 In some cases, the amount of CAR-T cells is 1k Approximately 10 CAR-T cells per g 6 ~about 10 7 In some cases, CAR-T cells The dose is approximately 10 CAR-T cells per kg. 7 ~about 10 8 In some cases, C The amount of CAR-T cells was approximately 10 CAR-T cells per kg. 8 ~about 10 9 Includes pieces. In this case, the amount of CAR-T cells is approximately 10 CAR-T cells per kg. 9 Includes pieces. In some cases, the amount of CAR-T cells is approximately 10 CAR-T cells per kg. 8 Includes In some cases, the amount of CAR-T cells is approximately 10 CAR-T cells per kg. 7 pieces In some cases, the amount of CAR-T cells is about 10 CAR-T cells per kg. 6 In some cases, the amount of CAR-T cells is approximately 1000 CAR-T cells per kg. 10 5 Includes pieces.

[0278] In some embodiments, the CAR-T cells are CD19-specific CAR-T cells. The amount of CD19-specific CAR-T cells was approximately 10 CAR-T cells per kg. 5 ~about 10 9 In some cases, the amount of CD19-specific CAR-T cells may be Approximately 10 CAR-T cells per 5 ~about 10 8 Optionally, CD19-specific CA The amount of CAR-T cells was approximately 10 per kg. 5 ~about 10 7 Includes pieces. Therefore, the amount of CD19-specific CAR-T cells was approximately 10 CAR-T cells per kg. 6 ~about 10 9 In some cases, the amount of CD19-specific CAR-T cells may be Approximately 10 CAR-T cells 6 ~about 10 8 Optionally, a CD19-specific CAR -T cell dose is approximately 10 CAR-T cells per kg 7 ~about 10 9 Including pieces. Therefore, the amount of CD19-specific CAR-T cells is approximately 10 CAR-T cells per kg. 5 ~ about 10 6 In some cases, the amount of CD19-specific CAR-T cells may be Approximately 10 CAR-T cells 6 ~about 10 7 Optionally, CD19-specific CAR- The amount of T cells was approximately 10 CAR-T cells per kg. 7 ~about 10 8 Including pieces. The amount of CD19-specific CAR-T cells was approximately 10 CAR-T cells per kg. 8 ~about 10 9 In some cases, the amount of CD19-specific CAR-T cells may be CAR-T cells approximately 10 9 In some cases, the amount of CD19-specific CAR-T cells , approximately 10 CAR-T cells per kg 8 Optionally, CD19-specific CA The amount of CAR-T cells was approximately 10 per kg. 7 In some cases, C The amount of D19-specific CAR-T cells was approximately 10 CAR-T cells per kg. 6 Includes pieces. In some cases, the amount of CD19-specific CAR-T cells is approximately 1000 mg / kg of CAR-T cells. 10 5 Includes pieces.

[0279] In some embodiments, the modified T cells are engineered TCR T-cells. The amount of TCR T-cells produced is approximately 10 TCR cells per kg. 5 ~about 10 9 Includes pieces. Depending on the combination, the amount of engineered TCR cells is approximately 10 TCR cells per kg. 5 ~about 10 8 Pieces In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 5 ~about 1 0 7 In some cases, the amount of engineered TCR cells is about 10 TCR cells per kg. 6 ~about 10 9 In some cases, the amount of engineered TCR cells is 100 mg / kg of TCR cells. About 10 cells 6 ~about 10 8 In some cases, the amount of engineered TCR cells may be TCR cells approximately 10 7 ~about 10 9 In some cases, the amount of engineered TCR cells is 1 kg Approximately 10 TCR cells per 5 ~about 10 6 In some cases, the amount of engineered TCR cells , approximately 10 TCR cells per kg 6 ~about 10 7 In some cases, manipulated TCR cells The amount of cells is approximately 10 TCR cells per kg. 7 ~about 10 8 In some cases, operation The amount of TCR cells is approximately 10 TCR cells per kg.8 ~about 10 9 Including pieces. The amount of engineered TCR cells is approximately 10 TCR cells per kg. 9 Including pieces. The amount of engineered TCR cells is approximately 10 TCR cells per kg. 8 Including pieces. The amount of engineered TCR cells is approximately 10 TCR cells per kg. 7 Including pieces. The amount of engineered TCR cells is approximately 10 TCR cells per kg. 6 Including pieces. The amount of engineered TCR cells is approximately 10 TCR cells per kg. 5 Includes pieces.

[0280] Indications In some embodiments, the modified effector encoding the polynucleotides described herein is A method for administering human leukocytes to a subject having a disorder, such as cancer or an infectious disease, has been developed. In some cases, the cancer may be associated with CD19, CD33, BCMA, CD44, alpha-folate Receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2 , HER3, folate binding protein, GD2, GD3, IL-13R-α2, KDR, E DB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1, MUC16 , h5T4, PSMA, TAG-72, EGFRvIII, CD123, or VEGF -Cancer associated with R2 expression.

[0281] In some embodiments, the polynucleotides, polypeptides, or The modified effector cells encoding the polynucleotide are associated with overexpression of CD19. Disclosed herein are methods of administering to a subject having cancer. The modified effector cells are administered to a subject having a cancer associated with overexpression of CD33. In some embodiments, methods are disclosed herein for treating engineered effector cells by CD4+ 19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1 , CEA, EGP-2, EGP-40, HER2, HER3, folate-binding protein, G D2, GD3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EG FR, MUC-1, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, Have cancer associated with overexpression of EGFRvIII, CD123, or VEGF-R2 Methods of administering to a subject are disclosed. In some cases, the cancer is metastatic cancer. In other cases, In this case, the cancer is a recurrent or refractory cancer.

[0282] In some cases, the cancer is a solid tumor or a hematological malignancy. In some cases, the cancer is a solid tumor. In other cases, the cancer is a hematologic malignancy. The cancer is metastatic. Optionally, the cancer is recurrent or refractory.

[0283] In some cases, the cancer is a solid tumor. Exemplary solid tumors are anal cancer; appendix cancer. Cancer; bile duct cancer (i.e., cholangiocarcinoma); bladder cancer; brain cancer; breast cancer; cervical cancer; colon Cancer of unknown primary site (CUP); Esophageal cancer; Eye cancer; Fallopian tube cancer; Digestive cancer; Kidney cancer; Liver cancer liver cancer; lung cancer; medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreatic cancer; parathyroid disease; penis Cancer; Pituitary tumor; Prostate cancer; Rectal cancer; Skin cancer; Stomach cancer; Testicular cancer; Pharyngeal cancer; cancer of the uterus; vaginal cancer; vulvar cancer; or glioblastoma stomach.

[0284] "Glioblastoma" or "glioblastoma multiforme" (GBM) is an aggressive neuroepithelial brain cancer. GBM can be differentiated into glial cells, astrocytes, oligodendrocyte precursor cells, or neural stem cells. Four subtypes of glioblastoma have been identified: classical, which represents the majority of GBM; A subtype carries an additional copy of the epidermal growth factor receptor (EGFR) gene, and most This results in higher than normal expression of growth factor receptors (EGFR). In a subset of cases EGFR amplification is achieved by gene rearrangements, the most common of which is The EGFR variant III (EGFRvIII) is mutated in glioblastoma. The gene most commonly involved, TP53 (p53), is not mutated in the classical subtype. The genotype is characterized by mutations in the genes encoding TP53 (p53) and platelet-derived growth factor receptor A. PDGFRA, which is a soluble form of PDGFRA, and the gene encoding isocitrate dehydrogenase 1, which is a soluble form of PDGFRA. The mesenchymal subtype has a high proportion of alterations in IDH1, which encodes neurofibromin 1. There is a higher rate of mutations or other changes in the NF1 gene, which is responsible for the disease, than in other types, and in E It is characterized by a small number of alterations in the GFR gene and low expression of EGFR. The subtypes include neuronal markers such as NEFL, GABRA1, SYT1, and SLC12A5. Other genetic alterations in glioblastoma have also been described, Most of them cluster in two pathways, RB and PI3K / AKT. Glioblastomas express 68-78% and 8% of these pathways, respectively. 8% have changes.

[0285] In some cases, the cancer is a hematological malignancy. In some cases, the hematological malignancy is a lymphoma. Hematologic malignancies include myeloma, leukemia, myeloma, or B-cell malignancies. In some cases, exemplary hematological malignancies include: Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, Non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL) , diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma Primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B lymphoma lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma Includes B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some embodiments, the hematological malignancy comprises myeloid leukemia. The tumor comprises acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0286] In some cases, as used herein, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SMLL), lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia ( PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multifocal Myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma , Non-Burkitt's high-grade B-cell lymphoma, Primary mediastinal B-cell lymphoma (PMBL), Immunoblastic Large cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoma Plasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) Large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, and or a combination of the methods described herein with a subject having a hematological malignancy selected from lymphomatoid granulomatosis. Disclosed herein are methods for administering modified effector cells that are a subject having a hematological malignancy selected from AML or CML, Methods of administering cells are disclosed.

[0287] In other cases, the methods described herein include administering to a subject having an infection due to an infectious disease. The infectious disease is a disease resulting from a bacterial infection, a viral infection, or a fungal infection. In other cases, exemplary viral pathogens include Adenoviridae. idae), Epstein-Barr virus (EBV), cytomegalovirus (CMV), R SV (respiratory syncytial virus), JC virus, B K virus, HSV, HHV family virus, Picornaviridae, He Herpesviridae, Hepadnaviridae, Flaviviridae Flaviviridae, Retroviridae, Orthomyxovirus Orthomyxoviridae, Paramyxoviridae, Papovair Family Papovaviridae, genus Polyomavirus, family Rhabdoviridae Exemplary pathogens include pathogens of the families Habdoviridae, and Togaviridae. Infectious viruses include smallpox, influenza, mumps, measles, chickenpox, Ebola, and Exemplary pathogenic fungi include Candida, Aspergillus, Aspergillus, Cryptococcus, Histoplasma asma, Pneumocystis, and Stachybotrys Exemplary pathogenic bacteria include Streptococcus, Pseudomonas, seudomonas, Shigella, Campylobacter, grapes Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella lmonella).

[0288] Viral-based delivery systems The present disclosure also provides delivery systems, such as virus-based systems, that incorporate the nucleic acids described herein. Exemplary viral expression vectors include adeno-associated viral vectors, adeno-associated viral vectors, and adeno-associated viral vectors. Viral-based vectors (e.g., Crucell, Inc. (Leiden, Netw.) The adenovirus-based Per.C6 system is commercially available from Herlands. Antiviral-based vectors (e.g., Life Technologies (Cardi B.) lentivirus-based pLPI, manufactured by Islington, Calif., and retrovirus vector (e.g., pFB-ERV with pCFB-EGSH), and herpes simplex virus In one embodiment, the vectors include, but are not limited to, virus-based vectors. Vectors are lentiviral vectors derived from retroviruses such as lentiviruses. The vectors used allow for long-term, stable integration and replication of the transgene in daughter cells. This allows for the development and propagation of the cells, making it a suitable tool for achieving long-term gene transfer. Lentiviral vectors have the advantage that they can transduce non-proliferating cells such as hepatocytes. have additional advantages over vectors derived from oncoretroviruses, such as murine leukemia viruses. Lentiviral vectors also have the added advantage of low immunogenicity. In further embodiments, the viral vector is an adeno-associated viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, a suitable vector comprises a replication origin functional in at least one organism. a promoter sequence, convenient restriction endonuclease sites, and one or more select Selection markers (e.g., WO 01 / 96584, WO 01 / 29058, and U.S. Pat. No. 6,326,193) .

[0289] Further suitable vectors may be capable of randomly integrating into the DNA of a host cell. and contains recombination sites that allow specific recombination between the expression vector and the host cell chromosome. Such integrative expression vectors may be used to express the vector in a host cell. The endogenous expression control sequences of the cell's chromosomes can be used to effect expression of the desired protein. Examples of site-specific integrating vectors include those available from, for example, Invitrogen (Carlsbad, flp-in system from Biosciences, Calif. (e.g., pcDNA™5 / FRT), and pExchange-6 from Stratagene (LaJolla, Calif.) cre-lox systems such as those found in Core Vectors Examples of vectors that randomly integrate into host cell chromosomes include For example, pcDNA3.1 from Invitrogen (Carlsbad, Calif.) (when introduced in the absence of T antigen), and Promega (Madison, Wisconsin). pCI or pFN10A(ACT)FLEXI™ manufactured by s. Promoter elements, such as enhancers, regulate the frequency of transcription initiation. , the promoter elements are located within the region 30–110 bp upstream of the start site, Recently, it has been shown that many promoters also contain functional elements downstream of the initiation site. The elements may also be inverted or displaced relative to each other. , the spacing between promoter elements is In the case of the thymidine kinase (tk) promoter, activity begins to decline Before the transcriptional transition, the spacing between promoter elements increases to a distance of 50 bp. Individual elements may be required to activate transcription, depending on the promoter. It is thought that they may function synergistically or independently.

[0290] One example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter. This promoter sequence is a promoter sequence that can be used to express any polynucleotide sequence operably linked to it. A strong constitutive promoter capable of driving high level expression of the nucleic acid sequence - It is an array.

[0291] Another example of a suitable promoter is human elongation growth factor 1 alpha 1 (hEF1a1). In embodiments, the vector constructs comprising the CAR and / or TCR of the present disclosure are , containing hEF1a1 functional mutants.

[0292] However, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus ( MMTV), human immunodeficiency virus (HIV) LTR (long terminal repeat epeat promoter, MoMuLV promoter, avian leukosis virus promoter -, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter In addition, actin promoter, myosin promoter, hemoglobin promoter, and and creatine kinase promoter. Other constitutive promoter sequences may also be used, including, but not limited to, the .alpha. promoter. Furthermore, the present disclosure is not intended to be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of this disclosure. Use may be made of a polynucleotide to which it is operably linked when such expression is desired. Turning on expression of the gene sequence or turning off expression when expression is not desired An example of an inducible promoter is metallothionein (meth) allothionine promoter, glucocorticoid promoter, progesterone promoter promoter, and the tetracycline promoter. Other examples of inducible promoters include tissue-specific promoters, as described herein. In some embodiments, the promoter contains, but is not limited to, an NF-κB binding site ( SEQ ID NOs: 44 to 46), nuclear factor of activated T cells (NFAT) response element (SEQ ID NO: 5 1), a six-site GAL4-inducible proximal factor binding element (PFB) (SEQ ID NO: 62), and comprises a synthetic 5'UTR based on RPL6 (SEQ ID NO: 64). The promoters are the IL-2 core promoter, the IL-2 minimal promoter, and the IL-2 endonucleaser. promoter mutants, (NF-κB)1-IL2 promoter mutants, (NF (NF-κB)3-IL2 promoter mutant, (NF-κB)6-IL2 promoter mutant , 1×NFAT response element-IL2 promoter mutant, 3×NFAT response element To-IL2 promoter mutant, 6×NFAT response element-IL2 promoter mutant Human EEF1A1 promoter variants, human EEF1A1 promoter / enhancer any one of the following promoters: human UBC promoter, and synthetic minimal promoter 1; In certain embodiments, the promoter nucleotides are those disclosed in Table 2. The promoter nucleotides are included.

[0293] To assess the expression of a CAR or TCR polypeptide or portion thereof, The expression vector to be introduced may also be transfected or infected via a viral vector. and a selection method for facilitating the identification and selection of expressing cells from a cell population for which expression is desired. The gene may also contain a marker gene or a reporter gene, or both. In this case, the selectable marker is carried on a separate piece of DNA and is then co-transfected. Both selectable markers and reporter genes may be used to express the It can be flanked by appropriate regulatory sequences to allow expression. Useful selectable markers include , for example, antibiotic resistance genes such as the neomycin resistance gene (neo) and the ampicillin resistance gene In some embodiments, the truncated epidermal growth factor receptor (HER1t) gene is a HER1-dependent agonist. The gene can be used as a selectable marker gene.

[0294] The reporter gene is used to identify potentially transfected cells and to identify the regulatory sequences. Reporter genes can be used to assess the functionality of a gene. not present in or expressed by the organism or tissue of the spirient, A polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. The DNA is then transferred to the recipient cell, where it is then inserted into the recipient cell. At each time point, the reporter gene expression is assayed. The enzymes used in the assay include luciferase, beta-galactosidase, and chloramphenicol acetyltransferase. a gene encoding secreted alkaline phosphatase, or green fluorescent protein Containing protein genes (e.g., Ui-Tei et al., FEBS Letters 479: 79-82 (2000)) Suitable expression systems are well known and can be prepared using known techniques, or commercially available. Generally, the smallest number of reporter genes that show the highest level of reporter gene expression is used. The construct with the 5' flanking region of such a gene is identified as a promoter. The promoter region can be linked to a reporter gene, and a drug can be administered to the promoter. It can be used to assess the ability to modulate driven transcription.

[0295] In some embodiments, the vector drives expression of the transgene hEF1a1 Promoter, bovine growth hormone poly(A) sequence that enhances transcription, woodchuck hepatitis virus In addition to the post-transcriptional regulatory element (WPRE), the LTR sequence derived from the pFUGW plasmid Contains columns.

[0296] Methods for introducing and expressing genes into cells are known in the art. In this context, the vector can be introduced into a host cell, e.g., a mammalian cell, by any method in the art. Can be easily introduced into mammalian, bacterial, yeast, or insect cells For example, an expression vector can be introduced into a host cell by physical means or chemical means. The gene may be introduced by environmental or biological means.

[0297] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation. Transplantation, lipofection, gene gun, microinjection, electroporation, etc. The art provides methods for generating cells containing vectors and / or exogenous nucleic acids. For example, see Sambrook et al. (Molecular Cloning: A Laboratory Manual). ual, Cold Spring Harbor Laboratory, New York (2001). Multiple Implementations In some embodiments, the method for introducing polynucleotides into host cells is by calcium phosphate transfection. transfection or polyethyleneimine (PEI) transfection.

[0298] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA vectors. This includes the use of viral vectors and, in particular, retroviral vectors. A vector is the most common method for inserting genes into mammalian cells, e.g., human cells. It has become a widely used method. Other viral vectors include lentivirus, poxvirus, viruses, herpes simplex virus I, adenovirus, and adeno-associated virus, etc. For example, U.S. Patent Nos. 5,350,674 and 5,585,3 See specification No. 62.

[0299] Chemical means for introducing polynucleotides into host cells include polymer complexes, nanoparticles, and Capsules, microspheres, beads, as well as oil-in-water emulsions, micelles, and mixed emulsions cells, and colloidal dispersion systems such as lipid-based systems including liposomes. Exemplary colloids for use as delivery vehicles in vitro and in vivo The liquid-like system is a liposome (eg, an artificial membrane vesicle).

[0300] When a viral delivery system is used, an exemplary delivery vehicle is a liposome. Introduction into cells (in vitro, ex vivo, or in vivo) For this purpose, the use of lipid formulations is contemplated. In another embodiment, the nucleic acid can be associated with a lipid. Lipid-associated nucleic acids are dispersed in aqueous liposomes interspersed within the lipid bilayer of the liposome. It can also be encapsulated inside a liposome and associate with both liposomes and oligonucleotides. It can also be attached to liposomes via a linking molecule, and can be incorporated into liposomes. It can be complexed with liposomes, dispersed in a solution containing lipids, or It can be mixed with lipids, it can be combined with lipids, it can be suspended in lipids. It may be contained as a suspension, or may be contained with or complexed with micelles. or otherwise associated with lipids. The compositions related to the expression vectors are not limited to any particular structure in solution. For example, the composition may exist in a bilayer structure as a micelle, or in a "collapsing" structure. The composition may also be simply dispersed in a solution. Lipids can also form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be natural or synthetic. In nature, lipid droplets occur within the cytoplasm, as well as long-chain aliphatic hydrocarbons and fatty acids. , alcohols, amines, amino alcohols, and aldehydes, as well as their derivatives. This includes a class of compounds having the following structure:

[0301] Lipids suitable for use can be purchased from commercial sources. Dimethylpropional phospholipid ("DMPC") was purchased from Sigma, St. Louis, Mo. Dicetyl phosphate ("DCP") is available from K & K Laboratories Cholesterol ("Ch") can be purchased from Pharmacia (Plainview, NY). oi") is available commercially from Calbiochem-Behring, and Jimilis Dimethylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Pharmaceuticals. It can be purchased from Color Lipids, Inc. (Birmingham, Ala.). Stock lipid solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform evaporates more readily than methanol, so it is the only "Liposome" means an encapsulated lipid bilayer or lipid aggregate. In general, the present invention encompasses a variety of single lipid vehicles and multi-layer lipid vehicles formed by the creation of Liposomes are vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes can be characterized as having: Multilamellar liposomes have multiple lipid layers formed by suspending phospholipids in an excess amount of aqueous solution. The lipid components undergo a self-transition before forming the closed structure. , which traps water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, there are also compositions in solution that have structures different from the normal vesicle structure. For example, lipids may take on a micelle structure, and the lipid molecules may form heterogeneous structures. Lipofectamine-nucleic acid complexes are also envisioned. do.

[0302] Non-viral-based delivery systems Optionally, for expressing a CAR and / or TCR as described herein The polynucleotide encoding the gene switch polypeptide also can be used to encode a DNA sequence in a vertebrate. "Sleepi" refers to a synthetic DNA transposon system for introduction into animal chromosomes. Use non-viral-based delivery systems, such as the Sympathomimetic Beauty (SB) transposon system. Some exemplary implementations of the SB transposon system are: The form is described in, for example, U.S. Patent Nos. 6,489,458 and 8,227,4 32, 9,228,180, and WO 2016 / 1451 Sleeping Beauty Trans The transposon system is composed of Sleeping Beauty (SB) transposase and SB transposase. In some embodiments, the Sleeping Beauty transposon The transposon system is the SB11 transposon system, the SB100X transposon system, or the S B110 transposon system, and any other variants of these.

[0303] DNA transposons are simple cuts from one DNA site to another. Translocation occurs by a chain-and-paste method. Transposition involves the transfer of a defined DNA segment to a single DNA fragment. molecules and can be separated into the same DNA molecule or genome, or into different DNA molecules. This is a precise process that often moves the Tc1 / marine to another site in the genome. Like r-type transposases, SB transposases also transfer transposons to recipients. The insertion site is located at a TA dinucleotide base pair within the target DNA sequence. It may be at another site within the offspring, or it may be in another DNA molecule (or chromosome). There are approximately 200 million TA sites in the mammalian genome, including the genome. During the transposon integration process, the TA sequence is duplicated. This duplication is the most prominent site of transposition. It is a characteristic feature and is used in some experiments to confirm the mechanism. Alternatively, the transposase may be encoded within a transposon, and the transposase may be derived from another source, e.g. It may be supplied by a DNA or mRNA source, in which case the transposon Non-autonomous transposons are non-autonomous elements that cannot be independently excised after insertion. It is most useful as a genetic tool because it cannot be extracted and reinserted. Posons are non-viral vectors for the introduction of genes into the genome of vertebrates and for gene therapy. In short, it is intended to be used as a target for genetically modifying T cells. , Sleeping Beauty (SB) series (Hackett et al., Mol Ther 18:674-83, (2010)) was adapted from (Cooper et al., Blood 105:1622-31, (2005)). Steps: (i) SB transposon for redirecting T cell specificity [i.e. DNA plus expressing the chimeric antigen receptor (CAR) and SB transposase Electrotransfer of mide (Jin et al., Gene Ther 18:849-56, (2011), Kebriae i et al., Hum Gene Ther 23:444-50, (2012)), and (ii) derived from the K562 cell line. Designer artificial antigen-presenting cells (AaPCs) In contact with a microbial cell (also known as a microbial and propagating cell), This involved propagation and expansion of T cells stably expressing the integrated sequence. The SB transposon system expresses tdIL-15, IL-21, and / or chimeric antigen receptors. Such systems include coding sequences that encode the polypeptides. Singh et al., Cancer Res (8):68 (2008). Ap Ril 15, 2008 and Maiti et al., J Immunother. 36(2): 112-123 (2013) It has been done.

[0304] In some embodiments, the CAR or TCR, one or more gene switch polypeptides The polynucleotide encoding mbIL-15 is selected from one or more Sleep The SB transposase is encoded within the ing Beauty transposon and is a distinct In some embodiments, the CD19-specific CAR is encoded within a vector. The mb-IL15 gene is encoded in a transposon DNA plasmid vector, and the mb-IL15 gene is inserted into a second transposon. The SB transposase is encoded within a third DNA plasmid vector. In some embodiments, the mbIL-15 is encoded within a plasmid vector. Examples of cell tags include truncated epidermal growth factor receptor tag (HER1t), HER1t1, HER1t2, HER1t3, HER1t4, HER1t5, HER1t 6, HER1t7, HER1t8, HER1t9, HER1t10, HER1t11, C D20 and CD20t1, CD20 tag, or depletion or kill switch or any other suitable cell tag for use as an enrichment marker Non-limiting exemplary gene switch vector systems including cell tags are shown in Figures 2A-2 D, Fig. 3, Figs. 19A-19B, Fig. 20A, Fig. 22, Figs. 24A-24D, and Figs. 25A- An example is given in 25B.

[0305] In some embodiments, HER1t is cetuximab, which is approved by the FDA; depletion of infused CAR-T cells via administration of any antibody that recognizes HER1t In some embodiments, CD20 also acts as a CAR-T cells infused via administration of FDA-approved rituximab therapy Allowing for cell depletion allows for a safety mechanism.

[0306] Exogenous nucleic acids can be introduced into host cells or cells can be otherwise transfected with the inhibitors of the present disclosure. Regardless of the method used to expose the recombinant DNA sequence to the host cell, Various assays can be performed to identify the specificity of the protein. , Southern and Northern blotting, RT-PCR and PCR, etc., well-known molecular assays; e.g., immunological means (ELISA and Western blot) or the assays described herein for identifying agents within the scope of this disclosure. Assays that fit within the confines of a lab can be used to detect "biochemical" assays, such as those that detect the presence or absence of specific peptides. " assay.

[0307] In some embodiments, the SB11 transposon system, the SB100X transposon system, the S B110 transposon system, piggyBac transposon system (see, e.g., Wilson et al., "PiggyBac Transposon-mediated See, “Ed Gene Transfer in Human Cells,” Molecular Therapy 15:...

Claims

1. Encoding a gene switch polypeptide for ligand-induced control of heterologous gene expression a composition comprising a polynucleotide that encodes a gene switch polypeptide, a) a DNA-binding domain fused to a first nuclear receptor ligand-binding domain a first gene switch polypeptide, and b) a transactivation domain fused to a second nuclear receptor ligand binding domain a second gene switch polypeptide comprising wherein the first gene switch polypeptide and the second gene switch polypeptide A composition in which the tide is linked by a linker.

2. One or more nucleotides encoding a gene switch system for ligand-inducible control of heterologous gene expression. A composition comprising a plurality of polynucleotides, wherein the gene switch system comprises: a) a first gene switch polypeptide comprising a transactivation domain; b) a second gene comprising a DNA-binding domain fused to a ligand-binding domain; a child switch polypeptide; and c) at least one heterologous polypeptide wherein the first gene switch polypeptide, the second gene switch polypeptide and one of the heterologous polypeptides is connected by a polypeptide linker the first gene switch polypeptide, the second gene switch polypeptide, and and another one of said heterologous polypeptides, said polypeptides A composition wherein the linker comprises a cleavable linker or a ribosome-skipping linker sequence. 。

3. The DNA binding domain may be GAL4 (GAL4 DBD), LexA DBD, or transcription factor. Transcription factor DBD, steroid / thyroid hormone nuclear receptor superfamily member DB D, a bacterial LacZ DBD, and a yeast DBD. Or the composition described in 2.

4. 1 to 3, wherein the DNA binding domain has the sequence set forth in SEQ ID NO:

184.

3. The composition according to any one of claims 1 to 3.

5. The transactivation domain comprises a VP16 transactivation domain and a B42 acidic activation domain.

5. The method of claim 1, further comprising at least one of the activator transactivation domains. The composition according to any one of claims 1 to 4.

6. 10. The method of claim 1, wherein the transactivation domain has the sequence set forth in SEQ ID NO:

181.

6. The composition according to any one of claims 1 to 5.

7. the first nuclear receptor ligand-binding domain, the second nuclear receptor ligand-binding domain domain, and at least one of the ligand-binding domains is an ecdysone receptor. receptor (EcR), ubiquitous receptor, orphan receptor 1, NER-1, steroid hormone receptor monocyte nuclear receptor 1, retinoid X receptor-interacting protein 15, liver X receptor β, Steroid hormone receptor-like protein, liver X receptor, liver X receptor α, farnesoid X receptor, receptor-interacting protein 14, and farnesol receptor 7. The composition of claim 1, comprising at least one of:

8. the first nuclear receptor ligand-binding domain, the second nuclear receptor ligand-binding domain domain, and at least one of the ligand-binding domains is SEQ ID NO: 18 5 to 186. The composition according to item .

9. the first gene switch polypeptide comprises an EcR nuclear receptor ligand binding domain; wherein the second gene switch polypeptide comprises a GAL4 DBD fused to a GAL4 gene. VP16 fused to the thyroid receptor X (RXR) nuclear receptor ligand binding domain The composition of claim 1 , comprising a transactivation domain.

10. the Gal4 DBD fused to the EcR nuclear receptor ligand binding domain , a sequence shown in any one of SEQ ID NOs: 185 to 186 or 187 to 188 fused to the retinoid receptor X (RXR) nuclear receptor ligand binding domain. The VP16 transactivation domain has the sequence set forth in SEQ ID NO:

183. The composition of claim 9.

11. The linker may be a cleavable linker, a ribosome skipping linker sequence, or an IR The composition of claim 1, which is an ES linker.

12. The linker is an IRES linker, and the IRES linker is selected from the group consisting of SEQ ID NOs: 18 to 19.

12. The composition of claim 11, having a sequence shown in any one of Tables 19.

13. the linker is a cleavable linker or a ribosome-skipping linker sequence; The composition of claim 11.

14. The cleavable linker or the ribosome skipping linker sequence is a 2A linker , p2A linker, T2A linker, F2A linker, E2A linker, GSG-2A linker linker, GSG linker, SGSG linker, furin linker mutant and 14. The composition of claim 2 or 13, comprising one or more of these derivatives.

15. The cleavable linker or the ribosome skipping linker sequence is SEQ ID NO: 14 6 to 162 of the sequence of claim 2 or 13 composition.

16. The polynucleotide or the one or more polynucleotides is an antigen-binding polynucleotide.

16. The composition of any one of claims 1 to 15, further encoding a peptide.

17. The antigen-binding polypeptide is a chimeric antigen receptor (CAR) and a T cell receptor cytoplasmic domain. The composition of claim 16, comprising at least one of:

18. The antigen-binding polypeptide comprises a CAR, and the CAR binds to CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP -2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, I L-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1 , MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvII I, CD123, and VEGF-R2. The composition of claim 17.

19. the antigen-binding polypeptide comprises a CAR, the CAR being selected from the group consisting of SEQ ID NOs: 210-24 18. The composition of claim 17, having a sequence as set forth in any one of Table 4.

20. The polynucleotide or the one or more polynucleotides further comprise a cell tag.

20. The composition of any one of claims 16 to 19, encoding

21. The cell tag is at least one of a HER1 truncated mutant and a CD20 truncated mutant.

21. The composition of claim 20, further comprising one or more of:

22. The cell tag has a sequence set forth in any one of SEQ ID NOs: 189 to 202. The composition of claim 20.

23. the first gene switch polypeptide, the second gene switch polypeptide, The expression of the antigen-binding polypeptide and at least one of the cell tags is The promoter is modulated by a promoter, and the promoter is a tissue-specific promoter or is an EF1A promoter or a functional variant thereof. Finished product.

24. The promoter has a sequence shown in any one of SEQ ID NOs: 58 to 60.

24. The composition of claim 23, wherein the EF1A promoter is a promoter of the EF1A gene, or a functional variant thereof. 。

25. The promoter is a tissue-specific promoter, , a T cell specific response element.

26. the tissue-specific promoter comprises one or more NFAT response elements. Item 26. The composition according to item 25.

27. The NFAT response element is represented by any one of SEQ ID NOs: 51 to 57.

27. The composition of claim 26, having the sequence:

28. The composition further comprises a second polynucleotide encoding a heterologous gene polypeptide. The composition of claim 1 comprising:

29. The heterologous polypeptides include cytokines, cell tags, and chimeric antigen receptors (e.g., 29. The composition of claim 2 or 28, comprising at least one of:

30. The cytokines include one cytokine, and the cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-19, IL-19 L-2, IL-15, IL-12, IL-21, IL-15, IL-15Rα, or I 30. The composition of claim 29, comprising at least one of a fusion of an L-15 mutant.

31. 31. The composition of claim 30, wherein the cytokine is in a secreted form.

32. 31. The composition of claim 30, wherein the cytokine is in a membrane-bound form.

33. The cytokine has a sequence set forth in any one of SEQ ID NOs: 203 to 209.

33. The composition of any one of claims 30 to 32, comprising:

34. The expression of said at least one heterologous gene polypeptide is controlled by an inducible promoter.

34. The composition of any one of claims 2 and 28 to 33, wherein the composition is hydrated.

35. The inducible promoter is a sequence shown in any of SEQ ID NOs: 40 to 64.

34. The composition of claim 33, having an array.

36. the inducible promoter is coupled to the first gene switch polypeptide and the second gene switch polypeptide; and wherein the gene switch polypeptide of claim 1 is modulated by at least one of the gene switch polypeptides of claim 1. Item 36. The composition according to item 34 or 35.

37. The polynucleotide of claim 1 or one or more of the polynucleotides of claim 2. A vector comprising at least one of the nucleotides.

38. The vector may be a lentiviral vector, a retroviral vector, or a non-viral vector.

38. The vector of claim 37, which is a vector.

39. The non-viral vector is a Sleeping Beauty transposon.

39. The vector of claim 38.

40. 1. A method for modulating expression of a heterologous gene in an effector cell, comprising: (i) binding of a DNA fused to a ligand-binding domain to said effector cell; (ii) a first gene switch polypeptide comprising a transactivation domain; (iii) a second gene switch polypeptide encoded by the heterologous gene, and (iv) a cleavable linker sequence or ribosome. One or more polypeptides encoding a polypeptide linker containing a skipping linker sequence. introducing a polynucleotide, wherein the polypeptide linker is a gene switch polypeptide, said second gene switch polypeptide, and said heterologous and combining one of the gene switch polypeptides with the first gene switch polypeptide, the second gene switch polypeptide, and the and another one of said heterologous gene polypeptides. and connecting with; The effector cells are incubated with a ligand in an amount sufficient to induce expression of the heterologous gene. contacting the A method comprising:

41. At least one of the one or more polynucleotides is an antigen-binding polypeptide. The method of claim 40, further encoding a tide.

42. The antigen-binding polypeptide selectively binds to a predetermined cell surface protein of a target cell.

42. The method of claim 41 , wherein

43. 43. The method of claim 42, wherein the target cell is a mammalian cell.

44. 44. The method of claim 42 or 43, wherein the target cell is a tumor cell.

45. 45. The method of claim 44, wherein the selected cell surface protein is a tumor antigen.

46. The effector cells are contacted with the ligand prior to the step of contacting the effector cells with the antigen-binding The synthetic polypeptide selectively binds to the predetermined cell surface protein of the target cell.

46. ​​The method of any one of claims 42 to 45,

47. at least one of the following steps: The effector cells were incubated with the target cells for 7 days.

47. The method of claim 46, wherein the subject is exposed to a substance.

48. Binding of the antigen-binding polypeptide to the predetermined cell surface protein is 48. The method of any one of claims 42 to 47, wherein effector cells are activated.

49. The antigen-binding polypeptide is a chimeric antigen receptor (CAR) and a T cell receptor cytoplasmic domain.

49. The method of any one of claims 38 to 48, comprising at least one of:

50. The antigen-binding polypeptide comprises a CAR, and the CAR binds to CD19, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP -2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, I L-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1 , MAGE-A1, MUC-16, h5T4, PSMA, TAG-72, EGFRvII I, CD123, and VEGF-R2.

50. The method of claim 49.

51. the antigen-binding polypeptide comprises a CAR, the CAR being selected from the group consisting of SEQ ID NOs: 210-24 50. The method of claim 49, having a sequence as set forth in any one of Table 4.

52. 39. The method of claim 38, wherein the heterologous polypeptide comprises the antigen-binding polypeptide.

51. The method of any one of claims 51 to 51.

53. At least one of the one or more polynucleotides further comprises a cell tag.

53. The method of any one of claims 37 to 52, wherein the

54. The cell tag is at least one of a HER1 truncated mutant and a CD20 truncated mutant.

54. The method of claim 53, further comprising:

55. The cell tag has a sequence set forth in any one of SEQ ID NOs: 189 to 202.

54. The method of claim 53,

56. 56. Any of claims 53 to 55, wherein the heterologous gene polypeptide comprises the cell tag.

10. The method according to claim 1.

57. At least one of the one or more polynucleotides further comprises a cytokine.

57. The method of any one of claims 37 to 56, wherein the method encodes:

58. The cytokines include IL-1, IL-2, IL-15, IL-12, 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-34, IL-35, IL-36, IL-37 at least one of IL-15, IL-15Rα, or a fusion of an IL-15 mutant; 58. The method of claim 57, comprising:

59. 59. The method of claim 58, wherein the cytokine is in a secreted form.

60. 59. The method of claim 58, wherein the cytokine is in a membrane-bound form.

61. The cytokine has a sequence set forth in any one of SEQ ID NOs: 203 to 209.

61. The method of any one of claims 57 to 60, comprising:

62. 62. Any of claims 57 to 61, wherein the heterologous polypeptide comprises the cytokine. The method according to any one of claims 1 to 5.

63. The expression of the heterologous gene polypeptide is modulated by an inducible promoter.

63. The method of any one of claims 37 to 62.

64. The inducible promoter has a sequence shown in any one of SEQ ID NOs: 40 to 64.

64. The method of claim 63, comprising:

65. The DNA binding domain may be GAL4 (GAL4 DBD), LexA DBD, or transcription factor. Transcription factor DBD, steroid / thyroid hormone nuclear receptor superfamily member DB D, a bacterial LacZ DBD, and a yeast DBD.

65. The method of any one of claims 7 to 64.

66. 65. The method of claim 64, wherein the DNA binding domain has the sequence set forth in SEQ ID NO:

184. The method described.

67. The transactivation domain comprises a VP16 transactivation domain and a B42 acidic activation domain.

67. The method of claim 37, further comprising at least one of the activator transactivation domains.

10. The method according to any one of claims 1 to 9.

68. 67. The transactivation domain has the sequence set forth in SEQ ID NO:

181. The method described below.

69. The first gene switch polypeptide and the second gene switch polypeptide At least one of them is a response element capable of binding to the DNA binding domain.

69. The method of any one of claims 37 to 68, further comprising:

70. The ligand-binding domain may be selected from the group consisting of ecdysone receptor (EcR), ubiquitous receptors, and oncogenes. -phan receptor 1, NER-1, steroid hormone nuclear receptor 1, retinoid X receptor interacting protein 15, liver X receptor beta, steroid hormone receptor-like protein, Liver X receptor, liver X receptor α, farnesoid X receptor, receptor-interacting protein 14, and at least one of the farnesol receptors.

10. The method according to any one of the preceding claims.

71. The ligand-binding domain is set forth in any one of SEQ ID NOs: 185-186.

71. The method of claim 70, wherein the sequence is

72. the first gene switch polypeptide comprises an EcR nuclear receptor ligand binding domain; wherein the second gene switch polypeptide comprises a GAL4 DBD fused to a GAL4 gene. VP16 fused to the thyroid receptor X (RXR) nuclear receptor ligand binding domain 72. The method of any one of claims 37 to 71, comprising a transactivation domain.

73. The Gal4 DBD fused to EcR is any one of SEQ ID NOs: 185-186. a retinoid receptor X (RXR) nuclear receptor ligand having a sequence shown in any one of The VP16 transactivation domain fused to the binding domain is shown in SEQ ID NO:

183.

73. The method of claim 72, wherein the sequence is

74. The ligand is (2S, 3R, 5R, 9R, 10R, 13R, 14S, 17R) -17-[(2S, 3R )-3,6-dihydroxy-6-methylheptan-2-yl]-2,3,14-trihydro Roxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17- Decahydro-1H-cyclopenta[a]phenanthren-6-one; N'-(3,5-dimethylbenzoyl)-N'-[(3R)-2,2-dimethyl-3- xanyl]-2-ethyl-3-methoxybenzohydrazide; 5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-( 3,5-dimethyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl) -hydrazides; 5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-( 3,5-dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl (propyl)-hydrazide; 5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-( 1-tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl)-hydrazine Do; 5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-( 1-tert-butyl-butyl)-N'-(3,5-dimethoxy-4-methyl-benzoyl (I)-hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-( 3,5-dimethyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl-propyl) -hydrazides; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-( 3,5-dimethoxy-4-methyl-benzoyl)-N'-(1-ethyl-2,2-dimethyl (propyl)-hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-( 1-tert-butyl-butyl)-N'-(3,5-dimethyl-benzoyl)-hydrazine Do; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid N'-( 1-tert-butyl-butyl)-N'-(3,5-dimethoxy-4-methyl-benzoyl (I)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'- (3-Methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl) (pyr)-N'-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(3-methylbenzoic acid (2-methyl-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-butyl)-N '-(3-Methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'- (2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl) (2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl) (3-methoxy-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-butyl)-N '-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 2-Methoxy-nicotinic acid N-(1-tert-butyl-pentyl)-N'-(4-ethyl) (I-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(2,2-dimethyl-1-phenyl-propyl)-N' -(4-ethyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-pentyl)-N'-(3-methylbenzoic acid (2-methyl-benzoyl)-hydrazide; and 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-pentyl)- N'-(3-methoxy-2-methyl-benzoyl)-hydrazide 74. The method of any one of claims 37 to 73, comprising at least one of:

75. The expression of the heterologous gene is expressed in the presence of the ligand in the absence of the ligand.

75. The method of claim 37, wherein the expression of the gene is reduced or eliminated compared to that of the gene in 1. The method according to claim 1.

76. the expression of the heterologous gene is restored by administering an additional amount of the ligand.

76. The method of claim 75.

77. The first gene switch polypeptide and the second gene switch polypeptide The expression of at least one of the genes is modulated by a promoter, However, the present invention is not limited to the use of tissue-specific promoters or EF1A promoters, or functional variants thereof.

77. The method of any one of claims 37 to 76, wherein the compound is a variant.

78. The promoter has a sequence shown in any one of SEQ ID NOs: 58 to 60.

78. The method of claim 77, wherein the promoter is an EF1A promoter or a functional variant thereof.

79. The promoter is a tissue-specific promoter, , a T cell specific response element.

80. the tissue-specific promoter comprises one or more NFAT response elements.

80. The method according to paragraph 79.

81. The NFAT response element is represented by any one of SEQ ID NOs: 50 to 57.

81. The method of claim 80, having the sequence:

82. 81. The one or more polynucleotides are contained within a vector.

10. The method according to any one of the preceding claims.

83. The vector may be a lentiviral vector, a retroviral vector, or a non-viral vector.

83. The method of claim 82, wherein the vector is a vector.

84. The non-viral vector is a Sleeping Beauty transposon.

84. The method of claim 83.

85. A gene for ligand-inducible control of heterologous gene expression comprising one or more expression cassettes. A gene switch system, wherein the one or more expression cassettes comprise: a) a DNA-binding domain fused to a first nuclear receptor ligand-binding domain a sequence encoding a first gene switch polypeptide, comprising: b) a transactivation domain fused to a second nuclear receptor ligand binding domain a sequence encoding a second gene switch polypeptide comprising: Including, the first gene switch polypeptide and the second gene switch polypeptide; are linked by a polypeptide linker; Gene switch system.

86. A gene for ligand-inducible control of heterologous gene expression comprising one or more expression cassettes. A gene switch system, wherein the one or more expression cassettes comprise: a) encoding a first gene switch polypeptide comprising a transactivation domain array; b) a second gene comprising a DNA-binding domain fused to a ligand-binding domain; a sequence encoding a child switch polypeptide; and c) a sequence encoding a heterologous polypeptide Including, the transactivation domain fused to the ligand binding domain, DNA binding a polypeptide linker, wherein one of the heterologous polypeptides is a polypeptide linker; The transactivation domain, DN, fused to the ligand binding domain by A binding domain, and linked to another one of the heterologous polypeptides. wherein the polypeptide linker comprises a cleavable linker sequence or a ribosomal skipping including a linker sequence, Gene switch system.

87. The one or more expression cassettes further comprise a sequence encoding a heterologous gene polypeptide.

86. The gene switch system of claim 85, comprising:

88. The one or more expression cassettes include: a) one or more recombinase junction sites; and b) a sequence encoding a serine recombinase 88. The gene of any one of claims 85 to 87, further comprising one or more of: Switch system.

89. The one or more expression cassettes include: a) a non-inducible promoter; and b) Inducible promoters 89. The gene of any one of claims 85 to 88, further comprising one or more of: Switch system.

90. 85 or 86, wherein the DNA binding domain has the sequence set forth in SEQ ID NO:

184.

89. A gene switch system according to any one of claims 89 to 90.

91. 85. The method of claim 85, wherein the transactivation domain has the sequence set forth in SEQ ID NO:

181.

91. The gene switch system of any one of claims 1 to 90.

92. The first nuclear receptor ligand binding domain and the second nuclear receptor ligand binding domain and at least one of the ligand-binding domains is selected from the group consisting of SEQ ID NO: 18 92. Any of claims 85 to 91, having a sequence as set forth in any one of claims 5 to 186. A gene switch system according to any one of claims 1 to 4.

93. The non-inducible promoter is selected from the group consisting of a sequence shown in any one of SEQ ID NOs: 40 to 64.

90. The gene switch system of claim 89, having an array.

94. The inducible promoter has a sequence shown in any one of SEQ ID NOs: 40 to 64.

90. The gene switch system of claim 89, comprising:

95. The polypeptide linker is set forth in any one of SEQ ID NOs: 146 to 162.

86. The gene switch system of claim 85, having an array comprising:

96. The polypeptide linker is any one of SEQ ID NOs: 18-19 and 146-162. a cleavable linker, a ribosomal skipping linker, or is an IRES linker.

97. The one or more expression cassettes contain a sequence encoding a chimeric antigen receptor (CAR). wherein expression of the chimeric antigen receptor is driven by a non-inducible promoter.

86. The gene switch system of claim 85, wherein said gene switch system is modulated.

98. The CAR is selected from the group consisting of CD19, CD33, BCMA, CD44, α-folate receptor, and CAIX. , CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folic acid Binding proteins, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelial cell membrane protein Lin, CD22, EGFR, MUC-1, MAGE-A1, MUC-16, h5T4, P Among SMA, TAG-72, EGFRvIII, CD123, and VEGF-R2 98. The gene switch system of claim 97, which is capable of binding to at least one

99. 98. The method of claim 97, wherein the non-inducible promoter comprises EF1A or a mutant thereof. Gene switch system described above.

100. 98. The gene of claim 97, wherein the expression cassette has the sequence shown in SEQ ID NO:

131. Gene switch system.

101. The expression cassette further comprises a sequence encoding a cell tag, the cell tag being a phosphorylated 98. The gene switch system of claim 97, wherein the gene switch system is linked to the CAR by a CAR.

102. The cell tag is at least one of a HER1 truncated mutant and a CD20 truncated mutant.

102. The gene switch system of claim 101, comprising one or more of:

103. 103. The method of claim 102, wherein the expression cassette has the sequence set forth in SEQ ID NO:

132. Gene switch system.

104. the expression cassette comprises the sequence encoding the first gene switch polypeptide and and said sequence encoding said second gene switch polypeptide, one of the first gene switch polypeptide and the second gene switch polypeptide is linked to the CAR by a linker. Chi type.

105. the first gene switch polypeptide and the second gene switch polypeptide, and the polypeptide linker is a truncated form of The gene switch system of claim 104, which is a linker.

106. 106. The method of claim 105, wherein the expression cassette has the sequence set forth in SEQ ID NO:

133. Gene switch system.

107. the first gene switch polypeptide and the second gene switch polypeptide, are linked by the polypeptide linker, and the polypeptide linker is an IRE The gene switch system of claim 106, which is an S linker.

108. 108. The method of claim 107, wherein the expression cassette has the sequence set forth in SEQ ID NO:

134. Gene switch system.

109. The one or more expression cassettes may comprise the sequence encoding the heterologous gene polypeptide. an expression cassette containing a sequence, wherein expression of the heterologous gene polypeptide is controlled by an inducible promoter 88. The gene switch system of claim 86 or 87, wherein the gene switch system is modulated by

110. 110. The gene sequence of claim 109, wherein the heterologous gene polypeptide comprises a cytokine. Itchy type.

111. The cytokines include IL-1, IL-2, IL-15, IL-12, 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-34, IL-35, IL-36, IL-37 at least one of IL-15, IL-15Rα, or a fusion of an IL-15 mutant; 111. The gene switch system of claim 110, comprising:

112. 110. The method of claim 109, wherein the expression cassette has the sequence set forth in SEQ ID NO:

135. Gene switch system.

113. the expression cassette comprises a sequence encoding a second heterologous polypeptide, 110. The gene switch of claim 109, wherein the two heterologous gene polypeptides comprise a cell tag. system.

114. The cell tag is at least one of a HER1 truncated mutant and a CD20 truncated mutant.

114. The gene switch system of claim 113, comprising one or more of:

115. 12. The method of claim 11, wherein the cell tag is linked to the cytokine by a linker.

5. The gene switch system according to claim 4.

116. 116. The method of claim 115, wherein the expression cassette has the sequence set forth in SEQ ID NO:

136. Gene switch system.

117. A gene switch system for integrating a heterologous gene into a host cell, said host cell , the presence of said serine recombinase, and said one or more recombinase junction sites. When contacted with the one or more expression cassettes under 89. The gene switch system of claim 88, which is incorporated into a cell.

118. The system further comprises a ligand, and contacting the host cells with the ligand induces 118. The gene switch system of claim 117, wherein the heterologous gene is expressed in the host cell.

119. The gene of claim 117 or 118, wherein the host cell is a T cell or an NK cell. Gene switch system.

120. 9. The inducible promoter is activated by the transactivation domain.

10. The gene switch system according to claim 9.

121. 121. The method of any one of claims 85 to 120, contained within one or more vectors. Gene switch system.

122. 122. The gene switch system of claim 121, contained within a single vector.

123. 90. One or more of the components of the gene switch system of any one of claims 85 to 89. A polynucleotide encoding one or more of:

124. A vector comprising the polynucleotide of claim 123.

125. The vector may be a lentiviral vector, a retroviral vector, or a non-viral vector.

125. The vector of claim 124, which is any one of the vectors.

126. The vector is a non-viral vector, and the non-viral vector is a Sleep The vector of claim 125, which is a ing Beauty transposon.

127. a polynucleotide encoding a first gene switch polypeptide; a polynucleotide encoding a second gene switch polypeptide, and Polynucleotide encoding a gene of interest (GOI) A polynucleotide construct comprising: The polynucleotide encoding the GOI is the polynucleotide encoding the second gene switch polypeptide and the second gene switch polypeptide. A continuous open reading frame ( and the polynucleotide construct comprises a polynucleotide encoding a linker. wherein the GOI is linked to the first gene switch polypeptide by the linker. and a second gene switch polypeptide, Polynucleotide constructs.

128. (i) a DNA-binding domain fused to a first nuclear receptor ligand-binding domain; and (ii) a first sequence encoding a first gene switch polypeptide comprising: Contains a transactivation domain fused to a second nuclear receptor ligand binding domain , at least one of the second sequences encoding a second gene switch polypeptide Including; The expression of at least one of the first sequence and the second sequence is modulated by the NFAT response element of Polynucleotide.

129. 1. A method of stimulating proliferation and / or survival of engineered cells, comprising: (a) obtaining a cell sample from a subject; (b) detecting in cells of the cell sample one or more transposons containing one or more transposons; transfecting a polynucleotide, said one or more transfectants The spozone Chimeric Antigen Receptor (CAR) or TCR, cytokine, one or more cell tags a gene switch polypeptide for ligand-inducible control of said cytokine, and the one or more polynucleotides are integrated into the genome of the cell, Transposases effective in bringing about populations Steps to code Including, the gene switch polypeptide binds to i) a first nuclear receptor ligand binding domain; a first gene switch polypeptide comprising a DNA-binding domain fused to ii) a transactivation domain fused to a second nuclear receptor ligand binding domain a second gene switch polypeptide comprising the first gene switch polypeptide the second gene switch polypeptide is linked by a linker; method.

130. A method for enhancing the survival of engineered cells in vivo in a subject in need thereof. So, (a) obtaining a cell sample from a subject; (b) detecting in cells of the cell sample one or more transposons containing one or more transposons; transfecting a polynucleotide, said one or more transfectants The spozone Chimeric Antigen Receptor (CAR) or TCR, cytokine, one or more cell tags a gene switch polypeptide for ligand-inducible control of said cytokine, and The DNA is effective to integrate into the genome of the cells, resulting in a population of engineered cells. transposase Steps to code Including, the gene switch polypeptide binds to i) a first nuclear receptor ligand binding domain; a first gene switch polypeptide comprising a DNA-binding domain fused to ii) a transactivation domain fused to a second nuclear receptor ligand binding domain a second gene switch polypeptide comprising the first gene switch polypeptide the second gene switch polypeptide is linked by a linker; method.

131. wherein the step of transfecting the cells comprises electroporating the cells. The method of claim 129 or 130.

132. At least one polynucleotide of the one or more polynucleotides , encoding said gene switch polypeptide, and said at least one polynucleotide is modulated by a promoter, and the promoter is a tissue-specific promoter. or EF1A promoter, or a functional variant thereof.

131. The method of any one of claims 1 to 131.

133. The promoter has a sequence shown in any one of SEQ ID NOs: 58 to 60.

133. The method of claim 132, wherein the EF1A promoter is a promoter encoding the EF1A promoter, or a functional variant thereof. 。

134. The promoter is a tissue-specific promoter, , the method of claim 132, comprising a T cell specific response element.

135. The promoter is a tissue-specific promoter, , the method of claim 132, comprising one or more NFAT response elements.

136. The NFAT response element is represented by any one of SEQ ID NOs: 50 to 57. The method of claim 135, having the sequence:

137. The cytokines include IL-1, IL-2, IL-15, IL-12, 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-34, IL-35, IL-36, IL-37 at least one of IL-15, IL-15Rα, or a fusion of an IL-15 mutant; 137. The method of any one of claims 129 to 136, comprising:

138. 138. The method of claim 137, wherein the cytokine is in a secreted form.

139. 138. The method of claim 137, wherein the cytokine is in a membrane-bound form.

140. The cytokine has a sequence set forth in any one of SEQ ID NOs: 203 to 209.

140. The method of any one of claims 137 to 139, comprising:

141. 10. The method of claim 1, wherein the cell is a NK cell, a NKT cell, a T cell, or a T cell precursor. 29 to 140. The method of any one of claims 29 to 140.

142. The method comprises administering an effective amount of the engineered cells to a subject in need thereof.

142. The method of any one of claims 129 to 141, further comprising:

143. the step of administering includes rapidly infusing the engineered cells into the subject.

143. The method of claim 142.

144. The method comprises administering an effective amount of the ligand to induce expression of the cytokine.

144. The method of claim 142 or 143, further comprising the step of:

145. 145. The method of claim 144, wherein the ligand is berezimex.

146. The CAR is selected from the group consisting of CD19, CD33, BCMA, CD44, α-folate receptor, and CAIX. , CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, folic acid Binding proteins, GD2, GD3, IL-13R-α2, KDR, EDB-F, mesothelial cell membrane protein Lin, CD22, EGFR, MUC-1, MAGE-A1, MUC-16, h5T4, P Among SMA, TAG-72, EGFRvIII, CD123, and VEGF-R2 146. The method of claim 129, wherein the at least one The method described.

147. 147. The method of claim 146, wherein said CAR is capable of binding to said CD19. 。

148. 147. The method of claim 146, wherein said CAR is capable of binding to said CD33. 。

149. 129 to 130, wherein the transposase is a salmonid-type Tc1-like transposase.

148. The method of any one of claims 148 to 148.

150. The transposase is SB11 or SB100x transposase.

150. The method of any one of paragraphs 129 to 149.

151. The one or more cell tags are selected from the group consisting of a HER1 truncation mutant and a CD20 truncation mutant.

151. The method of any one of claims 129 to 150, comprising at least one of:

152. The one or more cell tags are set forth in any one of SEQ ID NOs: 189-202.

152. The method of any one of claims 129 to 151, having a sequence

153. 1. A method of treating a subject with a solid tumor, comprising: (a) obtaining a cell sample from a subject; (b) detecting in cells of the cell sample one or more transposons containing one or more transposons; transfecting a polynucleotide, said one or more transfectants The spozone Chimeric Antigen Receptor (CAR) or TCR, cytokine, one or more cell tags a gene switch polypeptide for ligand-inducible control of said cytokine, and The DNA is effective to integrate into the genome of the cells, resulting in a population of engineered cells. transposase Code the gene switch polypeptide comprising: i) a first nuclear receptor ligand binding domain; a first gene switch polypeptide comprising a DNA-binding domain fused to and ii) a transactivation domain fused to a second nuclear receptor ligand binding domain. a second gene switch polypeptide comprising a first gene switch polypeptide; and the second gene switch polypeptide are linked by a linker. 、 Steps and (c) administering the population of engineered cells to the subject; A method comprising:

154. The cytokines include IL-1, IL-2, IL-15, IL-12, 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-34, IL-35, IL-36, IL-37 at least one of IL-15, IL-15Rα, or a fusion of an IL-15 mutant; 154. The method of claim 153, comprising:

155. 155. The method of claim 154, wherein the cytokine comprises IL-12.

156. At least one polynucleotide of the one or more polynucleotides , encoding said gene switch polypeptide, and said at least one polynucleotide is modulated by a promoter, and the promoter is a tissue-specific promoter. or EF1A promoter, or a functional variant thereof. The method described below.

157. The promoter has a sequence shown in any one of SEQ ID NOs: 58 to 60.

157. The method of claim 156, wherein the EF1A promoter is a promoter encoding the EF1A promoter, or a functional variant thereof. 。

158. The promoter is a tissue-specific promoter, , the method of claim 156, comprising a T cell specific response element.

159. The promoter is a tissue-specific promoter, , the method of claim 156, comprising one or more NFAT response elements.

160. The NFAT response element is represented by any one of SEQ ID NOs: 50 to 57.

160. The method of claim 159, having the sequence:

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