WPRE variant construct, composition, and method thereof

Abstract

To provide a safe and effective mutated woodchuck post-transcriptional regulatory element (WPRE) for use in a retroviral vector.SOLUTION: Provided is a vector comprising a mutated woodchuck post-transcriptional regulatory element (WPRE), wherein the vector does not include an X protein promoter, and the mutated WPRE does not include an X protein open reading frame (ORF).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This is an international application under the Patent Cooperation Treaty, claiming priority to U.S. Provisional Patent Application No. 62 / 988,202, filed on 11 March 2020, the entirety of which is incorporated herein by reference.

[0002] References to submitted sequence listings The official copy of the sequence listing is a file named "3000011-017977_Seq_Listing_ST25.txt", created on March 8, 2020, with a size of 247,691 bytes, and submitted electronically via EFS-Web as an ASCII format sequence listing, submitted together with this specification. The sequence listing contained in this ASCII format document is part of the specification, and the entire document is incorporated herein by reference. [Background technology]

[0003] 1. Field This disclosure relates to vectors comprising mutant post-transcriptional regulatory elements. In particular, the present invention relates to mutant WPRE sequences capable of efficiently expressing a target nucleotide in a retroviral vector system. The present invention also relates to a method for delivering and expressing a target nucleotide in target cells.

[0004] 2.Background Retroviral vectors, such as lentiviral vectors, have been proposed, in particular, as delivery systems for transporting target nucleotides to one or more target sites.

[0005] One drawback of retroviral vectors, whether retrovirus-based or lentivirus-based, is that they often fail to generate high levels of gene expression, especially in vivo. Many transcriptional and post-transcriptional steps are involved in the regulation of gene expression. Therefore, it is possible to enhance the expression of transgenes delivered by retroviral vectors by adding elements known to increase gene expression post-transcriptionally. One example is the inclusion of introns within the expression cassette (Choi, T. et al, (1991) Mol. Cell. Biol. 9:3070-3074). Numerous gene transfer experiments, both in vitro and in vivo, have demonstrated that the presence of introns can promote gene expression.

[0006] Other types of elements can also be used to post-transcriptional stimulate the expression of heterologous genes. These elements have the advantage of not requiring splicing events, unlike introns. For example, previous studies have suggested that post-transcriptional regulatory elements (PREs) and introns of hepatitis B virus (HBV) are functionally equivalent (Huang, Z.Mand Yen, TS (1995) Mol.Cell.Biol.15:3864-3869). Woodchuck hepatitis virus (WHV), a close relative of HBV, also possesses PREs (hereinafter referred to as WPREs; see U.S. Patent Nos. 6,136,597 and 6,287,814). WPREs have been shown to be more active than their HBV counterparts and this has been correlated with the presence of additional cis-acting sequences not found in HBV PREs. Insertion of WPRE into lentiviral vectors resulted in significant stimulation of reporter gene expression, including luciferase and green fluorescent protein (GFP), in various cells across different species (Zufferey, R. et al. (1999) J. Virol 73:2886-2892). Stimulation occurred regardless of the cyclic state of the transduced cells.

[0007] WPRE contains three cis-acting sequences that are important for increasing expression levels. However, it also contains a fragment of approximately 180 base pairs (bp) that includes the 5' end of the WHV X protein open reading frame, as well as its associated promoter. The full-length X protein is known to be involved in tumorigenesis (Flajolet, M. et al. (1998) J. Virol. 72:6175-6180). The cis-activation of myc family oncogenes by insertion of viral DNA into the host genome is known to be an important mechanism of WHV-mediated carcinogenesis (Buendia, MA (1994) In C. Brechot (ed.), Primary liver cancer: etiological and progression factors, pp. 211-224: CRC Press, Boca Raton, Fla.; Fourel, G. (1994) In F. Tronche and M. Yaniv (ed.), Liver gene expression, pp. 297-343; RGLandes Company, Austin, Tex.). The tumorigenic potential of the WHV X protein has raised concerns about incorporating WPRE into retroviral vectors, particularly regarding in vivo applications.

[0008] Previous studies have suggested that mutations in the X protein open reading frame (ORF) within WPRE reduce the oncogenic activity of the X protein, thereby improving its safety profile for incorporation into retroviral vectors. (See, for example, U.S. Patent No. 7,419,829; Donello, J.E et al. (1998) J.Virol.72(6):5085-5092; Schambach, A. et al. (2006) Gene Ther.13:641-645; Zanta-Boussif, MA et al. (2009) Gene Ther.16:605-619; Ou L. et al. (2016) Mol.Gen.Metab.Rep.8:87-93). However, inconsistent effects of heterologous gene expression on post-transcriptional stimulation have been observed in various mutant WPREs. Generally, the greater the degree of mutation introduced into WPRE, the less effective the mutant WPRE is when post-transcriptional heterologous gene expression is stimulated. [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, the need for safe and effective WPREs for use with retroviral vectors still exists. [Means for solving the problem]

[0010] In one embodiment, the present application relates to a mutant WPRE sequence for use in retroviral vectors in which, for example, WHV X protein expression is attenuated or absent. In some embodiments, the start codon of any open reading frame (ORF) within the WPRE is mutated, the WHV X protein promoter is deleted, and the WHV X protein OFR is deleted. In some embodiments, the WHV X protein promoter and the WHV X protein start codon are mutated.

[0011] In some embodiments, the mutant WPRE sequence contains mutations in one or more start codons corresponding to nucleotide positions 106-108, 152-154, 245-247, 272-274, 283-285, 362-364, and 603-605 in the WT WPRE nucleotide sequence described in SEQ ID NO: 1. In some embodiments, the mutant WPRE sequence contains mutations in one or more start codons corresponding to nucleotide positions 70-72, 108-110, 121-123, 138-140, 187-189, and 428-430 in the WT WPRE nucleotide sequence described in SEQ ID NO: 2.

[0012] A start codon may be mutated at one, two, or all three positions within one or more start codons. If two or more start codons are mutated, each start codon mutation may be independent of the other start codon mutations. In other words, each start codon mutated within a WPRE does not need to be mutated in the same manner. In some embodiments, each of one or more start codons is mutated at one position within the start codon. For example, the first nucleotide of the start codon may be mutated from "A" to "C", "G", or "T"; or the second nucleotide of the start codon may be mutated from "T" to "A", "C", or "G"; or the third nucleotide of the start codon may be mutated from "G" to "A", "C", or "T". In some embodiments, one or more start codons are mutated from "ATG" to "TTG". In some embodiments, each of one or more start codons is mutated from "ATG" to "TTG".

[0013] In some embodiments, the variant WPRE is selected from SEQ ID NOs: 3 and 4.

[0014] This application also provides vectors, such as retroviral or lentiviral vectors, comprising variant WPREs of the present invention. Such vectors may be used in therapeutic procedures such as gene delivery systems for genome function analysis, drug discovery, target validation, protein production (e.g., therapeutic proteins, vaccines, monoclonal antibodies), gene therapy, and adaptive cell therapy.

[0015] In some embodiments, lentiviral transduction vectors and constructs for their manufacture are provided, which can be used to introduce an expressible nucleotide sequence of interest (NOI) into a host cell. Lentiviral transduction vectors are enveloped virion particles containing an expressible nucleotide sequence, capable of entering a target host cell and thereby delivering the expressible sequence into the cell. The enveloped particles are preferably pseudotyped with an engineered or native viral envelope protein derived from another viral species, including a non-lentivirus, thereby altering the host range and infectivity of the native lentivirus. As detailed below, transduction vectors can be used in a wide range of applications, including, for example, protein production (including vaccine production), gene therapy, delivery of therapeutic polypeptides, and delivery of siRNA, ribozymes, antisense, and other functional polynucleotides. Such transduction vectors have the ability to carry one or more genes and to contain inhibitory sequences (e.g., RNAi or antisense).

[0016] In some embodiments, the vector comprises two or more NOIs. Such a vector may be used, for example, to produce a multimeric protein in a host cell. In some embodiments, the vector comprises a first nucleotide sequence S1 encoding protein Z1 and a second nucleotide sequence S2 encoding protein Z2, wherein the Z1 and Z2 types are dimers. The vector may further comprise a third nucleotide sequence S3 encoding protein Y1 and a fourth nucleotide sequence S4 encoding protein Y2, wherein the Y1 and Y2 types are second dimers.

[0017] In another embodiment, the vector may further include a fifth nucleotide sequence S5 encoding the 2A peptide and a sixth nucleotide sequence S6 encoding the linker peptide, where S5 and S6 are positioned between S1 and S2, S1 and S3, S1 and S4, S2 and S3, S2 and S4, and / or S3 and S4.

[0018] In some embodiments, the 2A peptide may be selected from P2A (SEQ ID NO: 6), T2A (SEQ ID NO: 7), E2A (SEQ ID NO: 8), or F2A (SEQ ID NO: 9).

[0019] In some embodiments, the linker peptide is any peptide having a length of 3 to 10 amino acids. In some embodiments, the linker peptide may be GSG or SGSG (SEQ ID NO: 5).

[0020] In another embodiment, the vector may further include a seventh nucleotide sequence S7 that encodes a frin peptide (sequence number 10), located between S1 and S2, S1 and S3, S1 and S4, S2 and S3, S2 and S4, and / or S3 and S4.

[0021] In another embodiment, the vector may further include promoter sequences that control the transcription of S1, S2, S3, S4, S5, S6 and / or S7, the promoter sequences being selected from the cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein (MBP) promoter, glial fibrillary acidic protein (GFAP) promoter, modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer (MNDU3), ubiquitin C promoter, EF-1α promoter, or mouse stem cell virus (MSCV) promoter.

[0022] In some embodiments, the first dimer Z1Z2 is represented by sequence numbers 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 25 and 92, 91 and 92, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 You may choose from 48 and 49, 50 and 51, 52 and 53, 54 and 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, or 89 and 90.

[0023] In some embodiments, the second dimer Y1Y2 is described in Sequence IDs 11 and 12.

[0024] In another embodiment, the viral vector is selected from adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, or picornaviruses.

[0025] In another embodiment, the vector is pseudotyped with the envelope protein of a virus selected from natural feline endogenous virus (RD114), a chimeric version of RD114 (RD114TR), gibbon leukemia virus (GALV), a chimeric version of GALV (GALV-TR), bitrophozoic mouse leukemia virus (MLV4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), avian plague virus (FPV), Ebola virus (EboV), baboon retrovirus envelope glycoprotein (BaEV), or lymphocytic choriomeningitis virus (LCMV).

[0026] In one embodiment, the present disclosure relates to a method for preparing T cells for immunotherapy, comprising the steps of: isolating T cells from a blood sample of a human subject; activating the isolated T cells in the presence of an aminobisphosphonate; transducing the activated T cells with a vector described herein; and growing the transduced T cells.

[0027] In another embodiment, T cells may be isolated from a human leukocyte apheresis sample.

[0028] In another embodiment, the aminobisphosphonate may be selected from pamidronic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, their salts and / or hydrates.

[0029] In another embodiment, T cells can be activated with OKT3 and anti-CD28.

[0030] In another embodiment, activation may be further carried out in the presence of human recombinant interleukin 2 (IL-2), human recombinant interleukin 15 (IL-15), and human recombinant interleukin 7 (IL-7).

[0031] In another embodiment, proliferation may occur in the presence of IL-2 and IL-15 or IL-15 and IL-7.

[0032] In another embodiment, the T cells may be γδT cells or αβT cells.

[0033] In another embodiment, the first dimer Z1Z2 and the second dimer Y1Y2 are co-expressed on the surface of proliferated T cells.

[0034] In another aspect, the disclosure relates to a population of proliferated T cells prepared by the method of the above aspect.

[0035] In some embodiments, the composition further comprises an adjuvant.

[0036] In some embodiments, the adjuvant is selected from one or more of the following: anti-CD40 antibody, imiquimod, reciquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-α, interferon-β, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulate formulations with poly(lactidocoglycol) (PLG), virosoms, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

[0037] In one embodiment, the present disclosure relates to a method for treating a patient having cancer, comprising administering to the patient a composition comprising a population of proliferated T cells as described herein, wherein the T cells kill cancer cells that present a peptide on their surface in complex with an MHC molecule, the peptide being selected from any of SEQ ID NOs: 99-256, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

[0038] In one embodiment, the present disclosure relates to a composition comprising T cells as described herein or a population of proliferated T cells as described herein for use in the treatment of cancer, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

[0039] In further embodiments, this disclosure refers to the use of the T cells described herein, or compositions comprising the T cells described herein, for the manufacture of a drug.

[0040] In further embodiments, the Disclosure refers to the use of T cells described herein or compositions comprising T cells described herein for the manufacture of agents for treating cancers such as those described herein.

[0041] In one embodiment, the present disclosure relates to a method for inducing an immune response in a patient with cancer, comprising administering to the patient a composition comprising a population of proliferated T cells as described herein, wherein the T cells kill cancer cells that present a peptide on their surface in complex with an MHC molecule, the peptide being selected from any of SEQ ID NOs: 99-256, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

[0042] In another embodiment, the immune response comprises a cytotoxic T cell response.

[0043] Finally, the present invention also provides a kit comprising at least one vector of the present invention. In one embodiment, the kit comprises at least one vector of the present invention, optionally packaging material, and optionally a label or packaging insert contained within the packaging material.

[0044] In one embodiment, the present disclosure relates to a method for preparing T cells for immunotherapy, comprising the steps of isolating T cells from a blood sample of a human subject, activating the isolated T cells in the presence of a statin, transducing the activated T cells with the vector of the present disclosure, and growing the transduced T cells, wherein the vector may be pseudotyped with vesicular stomatitis virus (VSV-G) and any envelope protein described herein, including RD114TR.

[0045] In another embodiment, T cells may include CD4+ T cells, CD8+ T cells, γδ T cells, and / or natural killer T cells.

[0046] In another embodiment, the statins atorvastatin, cerivastatin, dalvastatin, fluindostatin, fluvastatin, mevastatin, pravastatin, simvastatin, berostatin, and rosuvastatin may be selected. [Brief explanation of the drawing]

[0047] [Figure 1] Alignment of wild-type (WT) WPRE derived from the woodchuck hepatitis virus genome, provided under GenBank registration number J02442.1 (SEQ ID NO: 1); WT WPRE derived from woodchuck hepatitis B virus (WHV8 strain), provided under GenBank registration number J04514.1 (SEQ ID NO: 2); a mutant WPRE (SEQ ID NO: 4) in which the X protein promoter and start codon are mutated; and a mutant WPRE (SEQ ID NO: 3) in which multiple start codons within the WPRE are mutated and both the X protein promoter and ORF are deleted. [Figure 2-1] Alignment of wild-type (WT) WPRE derived from the woodchuck hepatitis virus genome, provided under GenBank registration number J02442.1 (SEQ ID NO: 1), and a mutant WPRE (SEQ ID NO: 3) in which multiple start codons within the WPRE are mutated and both the X protein promoter and ORF are deleted. The X protein promoter is underlined, and the X protein start codon is italicized. [Figure 2-2] Same as above [Figure 3] Schematic diagrams of vector constructs according to several embodiments of this disclosure are shown. [Figure 4] The following are exemplary lentiviral constructs according to several embodiments of the present disclosure. [Figure 5]The HEK-293T titers obtained after transduction using lentiviral constructs according to several embodiments of this disclosure are shown. Mutant A contains wild-type (WT) WPRE (positive control); Mutant B does not contain WPRE (negative control); Mutant C contains mutant WPRE in which the X protein promoter and start codon are mutated (SEQ ID NO: 4); Mutant D contains mutant WPRE in which the start codon is mutated and both the X protein promoter and ORF are deleted (SEQ ID NO: 3). [Figure 6] The expression of the TCR on the surface of CD8+ cells 6 days after transduction with the R4-B4 lentiviral construct is shown in several embodiments of this disclosure. Expression was detected by tetramers using lentiviral titer in two separate donors. Panel A is donor #1, and Panel B is donor #2. Logarithmic viral dilution factors are presented along the X axis. Mutant A contains wild-type (WT) WPRE (positive control); Mutant B does not contain WPRE (negative control); Mutant C contains mutant WPRE with mutated X protein promoter and start codon (SEQ ID NO: 4); Mutant D contains mutant WPRE with mutated start codon and deletion of both X protein promoter and ORF (SEQ ID NO: 3). [Figure 7] The expression of the TCR on the surface of CD8+ cells 4 days after transduction with the R4-A1B4 lentiviral construct according to several embodiments of this disclosure is shown. Expression was detected by tetramers using lentiviral titer in two separate donors. Panel A is donor #1, and Panel B is donor #2. Logarithmic viral dilution factors are presented along the X axis. Mutant A contains wild-type (WT) WPRE (positive control); Mutant B does not contain WPRE (negative control); Mutant C contains mutant WPRE with mutated X protein promoter and start codon (SEQ ID NO: 4); Mutant D contains mutant WPRE with mutated start codon and deletion of both X protein promoter and ORF (SEQ ID NO: 3). [Figure 8]The expression of TCR on the surface of CD8+ cells (A) or CD4+ cells (B) four days after transduction with the R4-B4 lentiviral construct according to several embodiments of this disclosure. Expression was detected by tetramers using lentiviral titration. Logarithmic viral dilution factors are presented along the X axis. Mutant A contains wild-type (WT) WPRE (positive control); Mutant B does not contain WPRE (negative control); Mutant C contains mutant WPRE with mutated X protein promoter and start codon (SEQ ID NO: 4); Mutant D contains mutant WPRE with mutated start codon and deletion of both X protein promoter and ORF (SEQ ID NO: 3). [Figure 9] The expression of TCR on the surface of CD4+ cells (A) or CD4+ cells (B) four days after transduction with the R4-A1B4 lentiviral construct according to several embodiments of this disclosure. Expression was detected by tetramers using lentiviral titration. Logarithmic viral dilution factors are presented along the X axis. Mutant A contains wild-type (WT) WPRE (positive control); Mutant B does not contain WPRE (negative control); Mutant C contains mutant WPRE with mutated X protein promoter and start codon (SEQ ID NO: 4); Mutant D contains mutant WPRE with mutated start codon and deletion of both X protein promoter and ORF (SEQ ID NO: 3). [Figure 10]This demonstrates that replication ratios are unaffected by WPRE mutations. Cell viability exceeded 90% for all lentiviral constructs tested at the optimal MOI (data not shown). Explanations of the lentiviral abbreviations shown along the X axis can be found in Figure 4. Briefly, the last letter of the abbreviation for each construct corresponds to the WPRE used. Variant A contains wild-type (WT) WPRE (positive control); Variant B does not contain WPRE (negative control); Variant C contains a mutant WPRE in which the X protein promoter and start codon are mutated (SEQ ID NO: 4); Variant D contains a mutant WPRE in which the start codon is mutated and both the X protein promoter and ORF are deleted (SEQ ID NO: 3). [Figure 11] This study demonstrates that WPRE variants do not alter TCR tetramer expression normalized to vector copy number. The data presented are mean / - standard deviation (SD) for all donors. Panel A shows results for CD8+ tetramers only. Panel B shows results for total CD3+ tetramers. A = wild-type (WT) WPRE (positive control); B = no WPRE (negative control); C = mutant WPRE with mutations in the X protein promoter and start codon (SEQ ID NO: 4); and D = mutant WPRE with mutations in the start codon and deletions of both the X protein promoter and ORF (SEQ ID NO: 3). [Figure 12] This study demonstrates that WPRE variants exhibit equivalent TCR tetramer expression normalized to viral titer. The presented data are mean / - standard deviation (SD) for all donors. A = wild-type (WT) WPRE (positive control); B = no WPRE (negative control); C = mutant WPRE with mutations in the X protein promoter and start codon (SEQ ID NO: 4); and D = mutant WPRE with mutations in the start codon and deletions of both the X protein promoter and ORF (SEQ ID NO: 3). [Figure 13]This shows that WPRE mutants exhibit equivalent TCR tetramer surface expression as determined by flow cytometry. Panel A shows CD4-CD8+ / tetramer+ data. Panel B shows CD4+CD8- / tetramer+ data. A = wild-type (WT) WPRE (positive control); B = no WPRE (negative control); C = mutant WPRE with mutations in the X protein promoter and start codon (SEQ ID NO: 4); and D = mutant WPRE with mutations in the start codon and deletion of both the X protein promoter and ORF (SEQ ID NO: 3). [Figure 14-1] This panel shows cytokine production by CD4+ or CD8+ T cells in the presence of target-positive tumor cells. Panel A shows interferon-γ (IFN-γ) production in CD8+ T cells. Panel B shows IFN-γ production in CD4+ T cells. Panel C shows tumor necrosis factor-α (TNF-α) production in CD8+ T cells. Panel D shows TNF-α production in CD4+ T cells. MCF7 = negative; SW982 = 460 CpC. Explanations of lentiviral abbreviations shown along the X-axis can be found in Figure 4. Briefly, the last letter of the abbreviation for each construct corresponds to the WPRE used. Mutant A contains wild-type (WT) WPRE (positive control); mutant B does not contain WPRE (negative control); mutant C contains mutant WPRE with mutated X protein promoter and start codon (SEQ ID NO: 4); mutant D contains mutant WPRE with mutated start codon and deletion of both X protein promoter and ORF (SEQ ID NO: 3). [Figure 14-2] Same as above [Figure 15] The present disclosure describes a process for producing γδT cells according to one embodiment. The γδT cell production may include, for example, the steps of collecting or obtaining leukocytes or PBMCs such as leukocyte apheresis products, depleting αβT cells from the PBMCs or leukocyte apheresis products, and subsequently activating, transducing, and proliferating γδT cells. [Figure 16]The following describes a T cell production process according to another embodiment of the present disclosure. The T cell production may include steps of collecting or obtaining leukocytes or PBMCs, such as leukocyte apheresis products, and subsequently activating, transducing, and proliferating T cells. [Figure 17] This disclosure shows a γδT cell manufacturing process according to one embodiment. [Figure 18A] This disclosure demonstrates the effect of WPRE on transgene expression in γδT cells according to one embodiment of this disclosure. [Figure 18B] The effect of WPRE on transgene expression in γδT cells is shown according to another embodiment of this disclosure. [Figure 19A] The effect of WPRE on transgene expression in γδT cells is shown according to another embodiment of this disclosure. [Figure 19B] The effect of WPRE on transgene expression in γδT cells is shown according to another embodiment of this disclosure. [Figure 20] This invention demonstrates the effect of WPRE on the copy number of an introduced gene incorporated into γδT cells, according to one embodiment of this disclosure. [Figure 21] This invention demonstrates the effect of WPRE on the transgene expression / copy number ratio of the incorporated transgene in γδT cells according to one embodiment of this disclosure. [Figure 22] Another embodiment of this disclosure demonstrates the effect of WPRE on the transgene expression / integrated transgene copy number ratio in γδT cells. [Figure 23] The effect of WPRE on transgene expression in γδT cells is shown according to another embodiment of this disclosure. [Figure 24] Another embodiment of this disclosure demonstrates the effect of WPRE on the transgene expression / integrated transgene copy number ratio in γδT cells. [Figure 25] Another embodiment of this disclosure demonstrates the effect of WPRE on the transgene expression / integrated transgene copy number ratio in γδT cells. [Modes for carrying out the invention]

[0048] Before further explanation of the subject matter, it should be understood that this disclosure is not limited to the specific embodiments of the disclosure described below, as variations of certain embodiments still fall within the scope of the attached claims. It should also be understood that the terminology used is for the purpose of describing specific embodiments and is not intended to be limiting. Rather, the scope of this disclosure is established by the attached claims.

[0049] As used herein, the term “operably connected” means that the components described are in a relationship that enables them to function in the manner they are intended.

[0050] In the use of this specification, the term “autocleaved 2A peptide” refers to a relatively short peptide (approximately 20 amino acids long, depending on the virus of origin) that acts cotranslatically by preventing the formation of a normal peptide bond between glycine and the final proline, resulting in ribosome skipping to the next codon and nascent peptide cleavage between Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the “upstream” protein, while the proline is added to the N-terminus of the “downstream” protein. The autocleaved 2A peptide may be selected from porcine rhinitis virus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot-and-mouth disease virus (F2A), or any combination thereof (see, for example, Kim et al., PLOS One 6:e18556, 2011; its contents, including the 2A nucleic acid and amino acid sequence, are incorporated herein by reference in their entirety). By adding a linker sequence (GSG or SGSG (SEQ ID NO: 5)) before the self-cleaving 2A sequence, this may enable the efficient synthesis of biologically active proteins such as TCRs.

[0051] As used herein, the term “promoter” refers to a regulatory region of DNA, generally located upstream (towards the 5' region of the sense strand), that enables the transcription of a gene. Promoters contain specific DNA sequences and response elements recognized by proteins known as transcription factors. These factors bind to the promoter sequence and recruit RNA polymerase, an enzyme that synthesizes RNA from the coding region of a gene. For example, promoter sequences used herein may be selected from the cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein (MBP) promoter, glial fibrillary acidic protein (GFAP) promoter, modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer (MNDU3), ubiquitin C promoter, EF-1α promoter, or mouse stem cell virus (MSCV) promoter.

[0052] As used herein, the term “cistron” refers to a portion of a DNA molecule that specifies the formation of one polypeptide chain, i.e., encodes one polypeptide chain. For example, “bi-cistron” refers to two sections of a DNA molecule that specify the formation of two polypeptide chains, i.e., encodes two polypeptide chains; “tri-cistron” refers to three sections of a DNA molecule that specify the formation of three polypeptide chains, i.e., encodes three polypeptide chains.

[0053] In the context of this specification, the terms “multicistronic RNA” or “multicistronic mRNA” refer to RNA containing genetic information for translation into several proteins. In contrast, monocistronic RNA contains genetic information for conversion into only one protein. In the context of this disclosure, multicistronic RNA transcribed from a lentivirus may be translated into two proteins, for example, the TCRα and TCRβ chains.

[0054] As used herein, the term “tandem-located” refers to a situation where genes are adjacent to each other within a single file on a nucleic acid sequence, with one following or behind the other. Genes are ligated adjacently on the nucleic acid sequence, and the coding strands (sense strands) of each gene are ligated together on the nucleic acid sequence.

[0055] As used herein, the term “sense strand” refers to the DNA strand of a gene that is translated or is translatable into a protein. When a gene is oriented in the “sense direction” relative to the promoter of its nucleic acid sequence, the “sense strand” is located at the 5' end of the promoter, where the first codon of the nucleic acid encoding the protein is proximal to the promoter and the last codon is distal to the promoter.

[0056] As used herein, the term “viral vector” refers to a nucleic acid vector construct that comprises at least one element of viral origin, has the ability to be packaged into a viral vector particle, and encodes at least an exogenous nucleic acid. Vectors and / or particles may be used for the purpose of transferring any nucleic acid into a cell, either in vitro or intra vivo. Numerous forms of viral vectors are known in the art. The term “virion” is used to refer to a single infectious viral particle. “Viral vector,” “viral vector particle,” and “viral particle” also refer to a complete viral particle having its DNA or RNA core and protein coat as present outside a cell. For example, a viral vector may be selected from adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, or picornaviruses.

[0057] The terms “T cell” or “T lymphocyte” are recognized in the art and are intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. Exemplary T cell populations suitable for use in a particular embodiment include, but are not limited to, helper T cells (HTLs; CD4+ T cells), cytotoxic T cells (CTLs; CD8+ T cells), CD4+CD8+ T cells, CD4-CD8-T cells, natural killer T cells, γδ T cells, or any other subset of T cells. Other exemplary T cell populations suitable for use in a particular embodiment include, but are not limited to, T cells expressing one or more markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR, which may be further isolated by positive or negative selection techniques if desired.

[0058] The terms “statin,” “vastatin,” or “3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor,” as used herein synonymously, refer to pharmaceuticals that inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. This enzyme is involved in the conversion of HMG-CoA to mevalonic acid, which is one of the steps in cholesterol biosynthesis. Such inhibition can be readily determined by standard assays well known to those skilled in the art.

[0059] Preferred statins that may be used in accordance with this disclosure include atorvastatin as disclosed in U.S. Patent No. 4,681,893; atorvastatin calcium as disclosed in U.S. Patent No. 5,273,995; cerivastatin as disclosed in U.S. Patent No. 5,502,199; dalvastatin as disclosed in U.S. Patent No. 5,316,765; fluindostatin as disclosed in U.S. Patent No. 4,915,954; fluvastatin as disclosed in U.S. Patent No. 4,739,073; U.S. Patent Examples include lovastatin disclosed in U.S. Patent No. 4,231,938; mevastatin disclosed in U.S. Patent No. 3,983,140; pravastatin disclosed in U.S. Patent No. 4,346,227; simvastatin disclosed in U.S. Patent No. 4,444,784; berostatin disclosed in U.S. Patents No. 4,448,784 and 4,450,171; and rosuvastatin disclosed in U.S. Patents No. 6,858,618 and 7,511,140. The contents of each of these patents are incorporated herein by reference in their entirety. Preferred 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors may include atorvastatin, atorvastatin calcium (also known as Liptor®), lovastatin (also known as Mevacor®), pravastatin (also known as Pravachol®), symvastatin (also known as Zocor®), and rosuvastatin.

[0060] Post-transcriptional regulatory elements The woodchuck hepatitis virus (WHV) post-transcriptional regulator (WPRE) can enhance expression from several different vector types, including lentiviral vectors (U.S. Patent No. 6,136,597; 6,287,814; Zufferey, R., et al. (1999). J. Virol. 73:2886-92). Although theoretical constraints are undesirable, this enhancement is thought to be due to improved RNA processing at the post-transcriptional level, resulting in an increase in nuclear transcript levels. A doubling of mRNA stability also contributes to this enhancement (Zufferey, R., et al. ibid.). The level of protein expression enhancement from transcripts containing WPRE has been reported to be approximately 2-5 times compared to those without WPRE, and this correlates well with the increase in transcription levels. This has been demonstrated with many different transgenes (Zufferey, R., et al. ibid.).

[0061] WPRE contains three cis-acting sequences that are crucial for increasing expression levels. Furthermore, it contains a fragment of approximately 180 bp (the full-length ORF is 425 bp) comprising the 5' end of the WHV X protein ORF, along with its associated promoter. Translation from the transcript initiated by the X promoter results in the formation of a protein representing the 60 amino acids at the NH2 terminus of the X protein. This truncated X protein can promote tumorigenesis, especially when the truncated X protein sequence is integrated into the host cell genome at a specific locus (Balsano, C. et al., (1991) Biochem. Biophys Res. Commun. 176:985-92; Flajolet, M. et al. (1998) J. Virol. 72:6175-80; Zheng, YW, et al. (1994) J. Biol. Chem. 269:22593-8; Runkel, L., et al. (1993) Virology 197:529-36). Therefore, expression of the truncated X protein, when delivered to target cells, may promote tumorigenesis and interfere with the safe use of the wild-type WPRE sequence.

[0062] In the use of this specification, the “X region” of a WPRE is defined as comprising at least the first 60 amino acids of an X protein ORF containing the translation start codon and its associated promoter. “X protein” is defined herein as a truncated X protein encoded by an X protein ORF as described herein.

[0063] The inventors have prevented the expression of the X protein by introducing mutations into the WPRE sequence. In some embodiments, these mutations are introduced into one or more start codons that occur with the WPRE sequence. In some embodiments, the X protein promoter and ORF are deleted from the WPRE sequence to obtain a shortened WPRE sequence. In other embodiments, the X protein promoter and X protein start codon are mutated.

[0064] As used herein, “mutation” may consist of one or more nucleotide deletions, additions, or substitutions.

[0065] In some embodiments, the mutant WPRE sequence contains mutations in one or more start codons corresponding to nucleotide positions 106-108, 152-154, 245-247, 272-274, 283-285, 362-364, and 603-605 in the WT WPRE nucleotide sequence described in SEQ ID NO: 1. In some embodiments, the mutant WPRE sequence contains mutations in all 1, 2, 3, 4, 5, 6, or 7 start codons corresponding to nucleotide positions 106-108, 152-154, 245-247, 272-274, 283-285, 362-364, and 603-605 in the WT WPRE nucleotide sequence described in SEQ ID NO: 1. In some embodiments, the mutated WPRE sequence contains mutations in each start codon corresponding to nucleotide positions 106-108, 152-154, 245-247, 272-274, 283-285, 362-364, and 603-605 in the WT WPRE nucleotide sequence described in SEQ ID NO: 1.

[0066] In another embodiment, the mutant WPRE sequence contains mutations in one or more start codons corresponding to nucleotide positions 70-72, 108-110, 121-123, 138-140, 187-189, and 428-430 in the WT WPRE nucleotide sequence described in SEQ ID NO: 2. In some embodiments, the mutant WPRE sequence contains mutations in all 1, 2, 3, 4, 5, or 6 start codons corresponding to nucleotide positions 70-72, 108-110, 121-123, 138-140, 187-189, and 428-430 in the WT WPRE nucleotide sequence described in SEQ ID NO: 2. In some embodiments, the mutated WPRE sequence contains mutations in each start codon corresponding to nucleotide positions 70-72, 108-110, 121-123, 138-140, 187-189, and 428-430 in the WT WPRE nucleotide sequence described in SEQ ID NO: 2.

[0067] One or more start codons may be mutated at one, two, or all three positions within the start codon. If two or more start codons are mutated, each start codon mutation may be independent of the other start codon mutations. In other words, each start codon mutated within a WPRE does not need to be mutated in the same manner. In some embodiments, each of one or more start codons is mutated at one position within the start codon. For example, the first nucleotide of the start codon may be mutated from "A" to "C", "G", or "T"; or the second nucleotide of the start codon may be mutated from "T" to "A", "C", or "G"; or the third nucleotide of the start codon may be mutated from "G" to "A", "C", or "T".

[0068] In some embodiments, each of one or more start codons is mutated at two or all three positions within the start codon. For example, the first nucleotide of the start codon may be mutated from "A" to "C", "G", or "T"; and / or the second nucleotide of the start codon may be mutated from "T" to "A", "C", or "G"; and / or the third nucleotide of the start codon may be mutated from "G" to "A", "C", or "T".

[0069] In some embodiments, one or more start codons are mutated from "ATG" to "TTG". In some embodiments, each of one or more start codons is mutated from "ATG" to "TTG".

[0070] In one embodiment, the mutant WPRE sequence is selected from SEQ ID NO: 3 and SEQ ID NO: 4. In another embodiment, the mutant WPRE sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 3 or 4. In some embodiments, the mutant WPRE sequence is 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99% or more identical to SEQ ID NO: 3, where the mutant WPRE sequence does not contain "ATG". In some embodiments, the mutant WPRE sequence is 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99% or more identical to SEQ ID NO: 3, where the mutant WPRE sequence does not contain "ATG" except at nucleotide positions 65-67.

[0071] In some embodiments, the WPRE sequence is identical to sequence number 4 by 95%, 96%, 97%, 98%, 98%, 99%, or 100%, where nucleotide positions 413–417 are "ATCAT" and nucleotide positions 428–430 are not "ATG".

[0072] Retrovirus The concept of using viral vectors in gene or cell therapy is recognized, for example, in Verma and Somia (1997) Nature 389:239-242, the entire content of which is cited.

[0073] In one embodiment, "virus" refers to both naturally occurring viruses and artificially created viruses. The viruses in some embodiments of this disclosure may be either enveloped viruses or non-enveloped viruses. Parvoviruses (such as AAV) are an example of non-enveloped viruses. In preferred embodiments, the virus may be an enveloped virus. In preferred embodiments, the virus may be a retrovirus, particularly a lentivirus. Examples of viral envelope proteins that can facilitate viral infection of eukaryotic cells include HIV-1-derived lentiviral vectors (LV) pseudotyped with envelope glycoproteins (GP) from vesicular stomatitis virus (VSV-G), modified feline endogenous retrovirus (RD114TR) (SEQ ID NO: 95), and modified gibbon leukemia virus (GALVTR). These envelope proteins can efficiently facilitate the entry of other viruses, such as parvoviruses and adeno-associated viruses (AAV), thereby demonstrating their broad-spectrum efficiency. For example, Moloney mouse leukemia virus (MLV) 4070 env (as described in Merten et al., J. Virol. 79:834-840, 2005, the contents of which are incorporated herein by reference), RD114 env, chimeric envelope protein RD114pro or RDpro (RD114-HIV chimera constructed by substituting the R peptide cleavage sequence of RD114 with the HIV-1 matrix / capsid (MA / CA) cleavage sequence, as described in Bell et al., Experimental Biology and Medicine 2010;235:1269-1276, the contents of which are incorporated herein by reference), or baculovirus GP64 env (as described in Wang et al., J. Virol. 81:10869-10878, 2007, the contents of which are incorporated herein by reference), or GALV Other viral envelope proteins may be used, including env (as described in Merten et al., J.Virol.79:834-840, 2005, the details of which are incorporated herein by reference) or derivatives thereof.

[0074] The term "retrovirus" includes, but is not limited to, all other retroviridae, including mouse leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney's mouse leukemia virus (Mo-MLV), FBR mouse osteosarcoma virus (FBRMSV), Moloney's mouse sarcoma virus (Mo-MSV), Abelson's mouse leukemia virus (A-MLV), avian myelocytosis virus-29 (MC29), and avian erythroblastosis virus (AEV) and lentivirus.

[0075] A detailed list of retroviruses can be found in Coffin et al. ("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).

[0076] Lentiviruses also belong to the retroviridae family, but they can infect both dividing and non-dividing cells.

[0077] Lentiviruses are divided into "primate" and "non-primate" groups. An example of a primate lentivirus is the human immunodeficiency virus (HIV). Non-primate lentiviruses include the prototype "slow virus" Visna / Maedivirus (VMV), as well as related Caprine arthritis encephalitis virus (CAEV), Equine infectious anemia virus (EIAV), and the recently reported feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0078] Detailed genome structures of several lentiviruses can be found in the NCBI Genbank database, among others (i.e., GenBank registration numbers AF033819 and AF033820, respectively). Details of HIV variants are also available in the HIV database managed by Los Alamos National Laboratory.

[0079] During infection, retroviruses first attach to specific cell surface receptors. Once inside a susceptible host cell, the retrovirus's RNA genome is copied into DNA by a reverse transcriptase encoded by the virus, which is carried into the parent virus. This DNA is then transported to the host cell nucleus, where it is integrated into the host genome. At this stage, it is typically called a provirus. Proviruses remain stable within the host chromosome during cell division and are transcribed like other cellular genes. The provirus encodes other elements necessary to produce more viruses and can then leave the cell through a process called "budding."

[0080] The genome of each retrovirus contains genes called gag, pol, and env that encode virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs). LTRs are responsible for proviral integration and transcription. They also function as enhancer-promoter sequences. In other words, LTRs can regulate viral gene expression. Capsid formation of retroviral RNA is carried out by a psi sequence located at the 5' end of the viral genome.

[0081] LTR itself is an identical sequence that can be divided into three elements called U3, R, and U5. U3 originates from the sequence specific to the 3' end of the RNA. R originates from the repeat sequences at both ends of the RNA, and U5 originates from the sequence specific to the 5' end of the RNA. The sizes of the three elements can vary considerably depending on the retrovirus.

[0082] In the viral genome, the transcription start site is located at the U3-R boundary of the left LTR, and the poly(A) addition (termination) site is located at the R-U5 boundary of the right LTR. U3 contains most of the proviral transcription regulators, including the promoter and several enhancer sequences that respond to cellular, and sometimes viral, transcription activator proteins. Some retroviruses have one or more of the following genes encoding proteins involved in gene expression regulation: tat, rev, tax, and rex.

[0083] Regarding the structural genes gag, pol, and env themselves: gag encodes the internal structural proteins of the virus. The Gag protein is degraded by proteolysis into mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene encodes reverse transcriptase (RT), which, along with DNA polymerase, associated RNase H, and integrase (IN), mediates genome self-replication. The env gene encodes the virion's surface (SU) glycoprotein and transmembrane (TM) protein, which form a complex that specifically interacts with cell receptor proteins. This interaction ultimately leads to infection through the fusion of the viral membrane with the cell membrane.

[0084] Retroviruses may also contain “additional” genes encoding proteins other than gag, pol, and env. Examples of additional genes in HIV include one or more of vif, vpr, vpx, vpu, tat, rev, and nef. EIAV, for example, contains additional genes S2 and dUTPase.

[0085] Proteins encoded by additional genes perform a variety of functions, some of which may overlap with functions provided by cellular proteins. For example, in EIAV, tat functions as a transcriptional activator of the viral LTR. It binds to a stable stem-loop RNA secondary structure called the TAR. Rev regulates and coordinates the expression of viral genes via the rev response element (RRE). The mechanism of action of these two proteins is thought to be broadly similar to a similar mechanism in primate viruses. The function of S2 is unknown, but it does not appear to be essential. Furthermore, an EIAV protein, Ttm, has been identified, encoded by the first exon of tat, which is spliced ​​into the env coding sequence at the transmembrane protein origin.

[0086] delivery system Retroviral vector systems have been proposed, among other things, as delivery systems for transferring target nucleotides (NOIs) to one or more target sites. Phase transfer can occur in vitro, in vitro, in vivo, or a combination thereof. Retroviral vector systems are even used to study various aspects of the retroviral life cycle, including receptor use, reverse transcription, and RNA packaging (a review of which is incorporated herein by reference by Miller, 1992 Curr Top Microbiol Immunol 158:1-24).

[0087] Recombinant retroviral vector particles can transduce NOI into receptor cells. Once inside the cell, the RNA genome from the vector particle is reverse transcribed into DNA and integrated into the receptor cell's DNA.

[0088] As used herein, the term “vector genome” refers to the RNA construct and / or incorporated DNA construct present in a retroviral vector particle. The term also encompasses a separate or isolated DNA construct capable of encoding such an RNA genome. A retroviral or lentiviral genome should comprise at least one component that is inducible from a retroviral or lentiviral. The term “inducible” is used in its usual sense, meaning a nucleotide sequence or a portion thereof, which may be derived from, but not necessarily from, a virus such as a lentivirus. For example, sequences may be prepared synthetically or by the use of recombinant DNA technology. Preferably, the genome comprises a psi region (or a similar component capable of causing capsid formation).

[0089] The viral vector genome is preferably "replication-deficient," meaning that the genome alone does not contain enough genetic information to enable independent self-replication for the production of infectious viral particles within the receptor cell. In a preferred embodiment, the genome lacks a functional env, gag, or pol gene.

[0090] The viral vector genome may contain some or all of a long terminal repeat (LTR). Preferably, the genome contains at least some of an LTR or similar sequence that can mediate proviral integration and transcription. This sequence may also contain, or function as, an enhancer-promoter sequence.

[0091] Viral vector genomes according to some aspects of the present invention may be provided as a kit of components. For example, the kit may include (i) one or more plasmids containing an NOI and an internal regulatory sequence, such as a promoter or IRES sequence; and (ii) a retroviral genome construct having appropriate restriction enzyme recognition sites for cloning the NOI and the internal regulatory sequence into a viral genome.

[0092] It is known that when the components necessary for the production of retroviral vector particles are co-introduced into separate DNA sequences within the same cell and expressed separately, retroviral particles containing a deficient retroviral genome with a therapeutic gene are produced. These cells are called production cells.

[0093] There are two common procedures for generating producible cells. In one, sequences encoding the retroviral Gag, Pol, and Env proteins are introduced into cells and stably integrated into the cell genome; this generates a stable cell line called a packaging cell line. Packaging cell lines produce the proteins necessary for packaging retroviral RNA, but because they lack the psi region, they cannot produce capsids. However, when a vector genome containing the psi region is introduced into a packaging cell line, the helper proteins can package psi-positive recombinant vector RNA to produce a recombinant viral stock. This can be used to transduce NOIs into receptor cells. A recombinant virus whose genome lacks all the genes necessary to produce viral proteins can infect only once and cannot replicate. Thus, NOIs are introduced into the host cell genome without the generation of potentially harmful retroviruses. An overview of available packaging strains can be found in "Retroviruses" (the entire content of which is referenced by reference, 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus, pp 449).

[0094] The present invention also provides a packaging cell line comprising the viral vector genome of the present invention. For example, the packaging cell line may be transduced with a viral vector system comprising a genome, or transfused with a plasmid having a DNA construct capable of encoding an RNA genome. The present invention also provides retroviral (or lentiviral) vector particles produced by such cells.

[0095] The second approach involves the simultaneous introduction of a deficient retroviral genome containing three distinct DNA sequences necessary for generating retroviral vector particles—namely, the env coding sequence, the gag-pol coding sequence, and one or more NOIs—into cells via transient transfusion. This procedure is called transient triple transfusion (Landau & Littman 1992; Pear et al. 1993). The triple transfusion procedure has been optimized (Soneoka et al. 1995; Finer et al. 1994). International publication pamphlet No. 94 / 29438 describes the generation of producible cells in vitro using this multiple DNA transient transfusion method.

[0096] The viral components necessary to complement the vector genome may reside on one or more "production plasmids" for translocation into cells.

[0097] The present invention also provides a vector system for producing retroviral particles comprising (i) a retroviral genome according to several aspects of the present invention; (ii) nucleotide sequences encoding retroviral gag and pol proteins; and (iii) nucleotide sequences encoding other essential viral packaging components not encoded by the nucleotide sequences of (ii).

[0098] In one embodiment, a nucleic acid sequence from which a particle encodes at least one of Vpr, Vif, Tat, Nef, or similar accessory genes from a retrovirus derived therefrom is disrupted or removed from the system so that the nucleic acid sequence can no longer encode a functional Vpr, Vif, Tat, Nef, or similar accessory protein.

[0099] The present invention also provides cells transfused with such vector systems and retroviral vector particles produced by such cells. Preferably, the gag-pol sequence is codon-optimized for use with specific producing cells (see below).

[0100] The env protein encoded by the nucleotide sequence in (iii) may be a homologous retroviral or lentiviral env protein. Alternatively, it may be a heterologous env, or an env from a non-retroviral or non-lentiviral (see "Pseudotyping" below).

[0101] The term "viral vector system" is generally used to refer to a kit of components that can be used in combination with other components necessary for viral particle production to produce viral particles within a host cell. For example, a retroviral vector genome may lack one or more genes necessary for viral replication. This may be combined in the kit with, for example, a further complementary nucleotide sequence or sequence set on one or more production plasmids. Co-introducing the genome together with the production plasmid should provide the components necessary for the production of infectious viral particles.

[0102] Alternatively, the complementary nucleotide sequence may be stably present within the packaging cell line included in the kit.

[0103] The present invention also relates to a retroviral vector system capable of delivering an RNA genome to a recipient cell, wherein the genome is longer than the wild-type genome of a lentivirus.

[0104] In some embodiments, the RNA genome of the vector system has up to 5%, preferably up to 10%, or even up to 30% more bases than the wild-type genome. Preferably, the RNA genome is about 10% longer than the wild-type genome. For example, wild-type EIAV comprises an RNA genome of about 8 kb. The EIAV vector system of the present invention may have an RNA genome of up to (preferably about) 8.8 kb.

[0105] In some embodiments, the retroviral vector systems of the present invention are self-inactivating (SIN) vector systems. For example, a self-inactivating retroviral vector system is constructed by deleting the transcriptional enhancer in the U3 region of the 3'LTR, or by deleting both the enhancer and the promoter. After a round of reverse transcription and integration of the vector, these modifications are copied to both the 5'LTR and 3'LTR, producing a transcriptionally inactive provirus. However, any promoters within the LTRs in such a vector still retain transcriptional activity. This strategy is employed to eliminate the influence of the viral LTR's enhancer and promoter on transcription from genes located within it. Such effects include increased or suppressed transcription. This strategy can also be used to eliminate downstream transcription from the 3'LTR to genomic DNA. This is of particular concern in human gene therapy, where preventing accidental activation of endogenous oncogenes may be important.

[0106] In some embodiments, a recombinase-assisted mechanism is used to promote the production of high-titer controlled lentiviral vectors from the cells producing the present invention.

[0107] In some embodiments, the present disclosure includes obtaining T cells from at least one donor, patient, or individual; activating the T cells with an anti-CD3 antibody and / or an anti-CD28 antibody; transducing the activated T cells with a viral vector; optionally, expanding the transduced T cells; optionally, measuring the amount of amplified T cells that express the transgene and / or the copy number of the integrated transgene in each of the T cells at a plurality of volume concentrations; optionally, identifying a volume concentration that results in a maximum average value of the amount of expanded T cells that express the transgene and / or a maximum average value of the copy number of the integrated transgene, without exceeding five copies of the integrated transgene, in each of the expanded T cells from a plurality of healthy donors; and transducing the T cells obtained from a patient with a viral vector at the volume concentration identified for immunotherapy, as described in U.S. Patent Application Publication No. 20190216852, the entire contents of which are incorporated herein by reference.

[0108] In some embodiments, the plurality of volume concentrations are from about 10

[0109] , , 7 , about 0.01 μl per cell to about 10 6 about 1 ml per cell; about 2×10 6 about 0.01 μl per cell to about 2×10 6 about 1 ml per cell; about 5×10 6 about 0.01 μl per cell to about 5×10 6 about 1 ml per cell; about 10 7 about 0.01 μl per cell to about 10 7 about 1 ml per cell; about 10 7 about 1 μl per cell to about 10 7 about 500 ml per cell; about 10 7 about 5 μl per cell to about 10 7 about 150 ml per cell; about 10 7 about 8 μl per cell to about 10 7 about 12 ml per cell.

[0109] As used herein, the term “recombinase-assisted system” includes, but is not limited to, systems that catalyze recombination events between 34 bp FLP recognition targets (FRTs) using the Cre recombinase / loxP recognition site of bacteriophage P1 or site-specific FLP recombinase of S. cerevisiae.

[0110] A site-specific FLP recombinase from S. cerevisiae, which catalyzes recombination events between 34 bp FLP recognition targets (FRTs), has been constructed in a DNA construct, and high-level lentivirus-producing cell lines have been created using recombinase-assisted recombination (Karreman et al. (1996) NAR 24:1616-1624). A similar system has been developed using the Cre recombinase / loxP recognition site of bacteriophage P1 (Vanin et al. (1997) J. Virol 71:7820-7826). This was incorporated into a lentiviral genome, resulting in the creation of high-titer lentivirus-producing cell lines.

[0111] By using production / packaging cell lines, it is possible to grow and isolate quantities of retroviral vector particles (e.g., prepare retroviral vector particles of the appropriate titer) for subsequent transduction in, for example, a target site (such as a specific organ or tissue) or target cells (such as T cells). Production cell lines are typically suitable for large-scale production of vector particles.

[0112] Transient translocation offers certain advantages over the cell packaging method. In this regard, transient translocation avoids the longer time required to create stable vector-producing cell lines and is used when the vector genome or retroviral packaging components are toxic to cells. If the vector genome encodes toxic genes or genes that interfere with the host cell's self-renewal, such as cell cycle inhibitors or apoptosis-inducing genes, creating a stable vector-producing cell line may be difficult; however, transient translocation can be used to produce the vector before the cells die. Furthermore, cell lines have been developed using transient translocation to produce vector titer levels comparable to those obtained from stable vector-producing cell lines (Pear et al. 1993, PNAS 90:8392-8396).

[0113] The producing / packaging cells can be any suitable cell type. While the producing cells are generally mammalian cells, they could also be insect cells, for example.

[0114] In the context of this specification, the terms “producing cell” or “vector-producing cell” refer to a cell that contains all the elements necessary for the production of retroviral vector particles.

[0115] In some embodiments, the producing cells can be obtained from a stable producing cell line, an induced stable producing cell line, or an induced producing cell line.

[0116] As used herein, the term “induced-producing cell line” refers to a transdependence-producing cell line that has been screened and selected for high expression of a marker gene. Such cell lines support high levels of expression from retroviral genomes. The term “induced-producing cell line” is used synonymously with the terms “induced-stable-producing cell line” and “stable-producing cell line.”

[0117] In some embodiments, the induced producing cell lines include, but are not limited to, retrovirus and / or lentivirus-producing cells.

[0118] In some embodiments, the envelope protein sequence and the nucleocapsid sequence are all stably integrated into the production and / or packaging cell. However, one or more of these sequences may also exist in episomal form, and gene expression may originate from the episome.

[0119] As used herein, the term “packaging cell” refers to a cell containing the missing elements in the RNA genome necessary for the production of infectious recombinant virus. Typically, such packaging cells contain one or more production plasmids capable of expressing viral structural proteins (such as codon-optimized gag-pol and env), but they do not contain packaging signals.

[0120] The term "packaging signal," often used synonymously with "packaging sequence" or "psi," is used to refer to a non-coding sequence necessary for capsid formation of the retroviral RNA chain during viral particle formation. In HIV-1, this sequence maps to a locus extending from upstream of the major splice donor site (SD) to at least the gag start codon.

[0121] Suitable packaging cell lines for use with the above vector constructs are readily prepared (see also International Publication No. 92 / 05266, whose contents are referenced by reference) and may be used to create production cell lines for producing retroviral vector particles. As mentioned above, an overview of available packaging lines can be found in "Retroviruses".

[0122] Furthermore, as mentioned above, simple packaging cell lines containing proviruses lacking the packaging signal have been found to rapidly produce undesirable self-replicating viruses through recombination. To improve safety, second-generation cell lines lacking the 3'LTR of the provirus have been created. In such cells, two recombinations are required to produce wild-type viruses. Further improvements involve introducing the gag-pol and env genes into separate constructs, so-called third-generation packaging cell lines. These constructs are introduced sequentially to prevent recombination during translocation.

[0123] In some embodiments, the packaging cell line is a second-generation packaging cell line or a third-generation packaging cell line.

[0124] In these split constructs, in third-generation cell lines, further reduction of recombination may be achieved by modifying codons. This technique, based on genetic coding redundancy, aims to reduce homology between separate constructs, for example, between overlapping regions of the gag-pol and env open reading frames.

[0125] Packaging cell lines are useful for providing gene products necessary to encapsulate and deliver membrane proteins for the production of high-titer vector particles. Packaging cells may be cells cultured in vitro, such as tissue culture cell lines. Suitable cell lines include, but are not limited to, mammalian cells such as mouse fibroblast-derived cell lines or human cell lines. In some embodiments, packaging cell lines are primate or human cell lines such as HEK293, 293-T, TE671, and HT1080.

[0126] For both experimental and practical applications, it is desirable to use high-titer viral preparations. Techniques for increasing viral titer include the use of psi and packaging signals as described above, and the concentration of viral stocks.

[0127] As used herein, the term “high titer” means an effective amount of retroviral vector or particle that can be transduced into a target site such as a cell.

[0128] As used herein, the term “effective dose” means a sufficient amount of retroviral or lentiviral vector or vector particle to induce NOI expression at a target site.

[0129] High titer viral preparations from producing / packaging cells typically contain approximately 10 per mL. 5 ~10 7 This is the degree of retrovirus particles. In another embodiment, the preparation is at least 10 8 TU / mL, preferably 10 8 ~10 9 TU / mL, more preferably at least 10 9 It has a titer of TU / mL (the titer is expressed as the number of units of introduction per 1 mL (TU / mL) measured on a standard D17 cell line). Other concentration methods, such as ultrafiltration or binding to and elution from the matrix, may be used.

[0130] The expression product encoded by NOI may be a protein secreted from the cell. Alternatively, the NOI expression product is not secreted and is active intracellularly. In some applications, it is preferable that the NOI expression product exhibits a bystander effect or a distant bystander effect; that is, the production of the expression product in one cell resulting in the regulation of additional related cells, adjacent or distant (e.g., transmissible), that share a common phenotype (each of which is invoked by reference, Zennou et al., (2000) Cell 101:173; Folleuzi et al., (2000) Nat. Genetics 25:217; Zennou et al., (2001) Nat. Biotechnol. 19:446).

[0131] The presence of a sequence called a central polyprint lacte (cPPT) may improve the efficiency of gene delivery to non-dividing cells. This cis-acting element is located, for example, in the viral polymerase coding region element. In some embodiments, the viral genome of the present invention comprises a cPPT sequence.

[0132] Furthermore, the viral genome may include translation enhancers.

[0133] NOIs may be operably linked to one or more promoter / enhancer elements. The transcription of one or more NOIs may be under the control of a viral LTR or / or a promoter-enhancer element. In some embodiments, the promoter may be a potent viral promoter such as CMV, or a cytoconstitutive promoter such as PGK, β-actin, or EF1α. The promoter may be regulated or tissue-specific. Control of expression may also be achieved by using a system such as a tetracycline system that switches gene expression on or off in response to an exogenous agent (e.g., tetracycline or an analogue thereof).

[0134] Pseudotyping In designing retroviral vector systems, it is desirable to manipulate particles with different target cell specificities compared to the native virus to enable the delivery of genetic material to a range of replicated or modified cell types. One way to achieve this is to manipulate the viral envelope protein to alter its specificity. Another approach is to introduce heterologous envelope proteins into the vector particles, thereby replacing or adding the native viral envelope protein.

[0135] The term pseudotyping refers to the incorporation, partial replacement, or complete replacement of the env genes in a viral genome with a heterologous env gene, such as an env gene from another virus. Pseudotyping is not a new phenomenon, and examples can be found in International Publication No. 99 / 61639, International Publication No. A-98 / 05759, International Publication No. A-98 / 05754, International Publication No. A-97 / 17457, International Publication No. A-96 / 09400, International Publication No. A-91 / 00047 and Mebatsion et al. (1997) Cell 90:841-847, the contents of which are incorporated herein by reference in their entirety.

[0136] In some embodiments, the vector system is pseudotyped with a gene encoding at least a portion of the rabies G protein. An example of a rabies G pseudotyped retrovirus vector is found in International Publication No. 99 / 61639. In further embodiments, the vector system is pseudotyped with a gene encoding at least a portion of the VSV-G protein. An example of a VSV-G pseudotyped retrovirus vector is found in U.S. Patent No. 5,817,491, the entirety of which is incorporated herein by reference. In another embodiment, the vector is pseudotyped with the envelope protein of a virus selected from natural feline endogenous virus (RD114), a chimeric version of RD95 (RD114TR; SEQ ID NO: 95), gibbon leukemia virus (GALV), a chimeric version of GALV (GALV-TR), bitrophozoic mouse leukemia virus (MLV4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), avian plague virus (FPV), Ebola virus (EboV), baboon retrovirus envelope glycoprotein (BaEV), or lymphocytic choriomeningitis virus (LCMV).

[0137] Minimal retroviral or lentiviral systems have been demonstrated to be constructed from HIV, SIV, FIV, and EIAV viruses. Such systems do not require any additional genes, such as vif, vpr, vpx, Vpu, tat, rev, and nef, for vector production or transduction of dividing and non-dividing cells. It has also been demonstrated that a minimal EIAV vector system can be constructed that does not require S2 for either vector production or transduction of dividing or non-dividing cells. Deleting additional genes is advantageous. Firstly, it allows for the production of vectors without genes associated with disease in lentiviral (e.g., HIV) infection. Tat, in particular, is associated with disease. Secondly, the deletion of additional genes allows the vector to package more heterologous DNA. Thirdly, genes with unknown functions, such as S2, may be omitted, thus reducing the risk of undesirable effects. Examples of minimal lentiviral vectors are disclosed in International Publication No. A-99732646 and in International Publication No. A-98 / 17815, the entire contents of which are incorporated herein by reference.

[0138] The absence of functional co-genes from a retroviral vector production system means that those functional genes are also not present in the retroviral vector particles produced by the system. Furthermore, the co-proteins encoded by those genes and incorporated into the vector particles are not present in the vector particles. In known retroviral vector production systems, co-genes may be present as part of the vector genome encoding DNA or together with the packaging components. The location of co-genes in the vector production system also depends to some extent on their relationship to other retroviral components. For example, vif is often part of the gag-pol packaging cassette within the packaging cell. Therefore, removing functional co-genes for the purposes of this invention may involve their removal from the packaging components, or from the vector genome, or possibly both.

[0139] To remove a functional accessory gene, it may not be necessary to remove the entire gene. Usually, it is sufficient to remove a portion of the gene or to disrupt it in other ways. The absence of a functional accessory gene is understood herein to mean that the gene does not exist in a form capable of encoding a functional accessory protein.

[0140] In some embodiments, the vector particles lack both the functional vpr and tat genes or similar genes that are typically present in the lentivirus based on them. These two accessory genes are associated with lentiviral characteristics that are particularly undesirable for gene or cell therapy vectors. However, except as otherwise provided above, the present invention is not limited to combinations of accessory genes that are absent in the system according to the present invention for producing HIV-1-based vector particles, and any combination of three or more, more preferably four genes, may not be present in their functional form. Most preferably, all five accessory genes, vpr, vif, tat, nef, and vpu, are absent in their functional form. Similarly, in other lentivirus-related systems, it is most preferable that all accessory genes are absent in their functional form (except rev, which is desirable to have present unless replaced by a system similar to the rev / RRE system).

[0141] Therefore, in some embodiments, the delivery system according to the present invention lacks at least tat and S2 (in the case of an EIAV vector system), and possibly also lacks vif, vpr, vpx, vpu, and nef. Preferably, the system of the present invention also lacks rev. Rev has previously been considered essential for efficient virus production in some retroviral genomes. For example, in the case of HIV, it was thought that the sequences of rev and RRE should be included. However, it has been found that the requirements for rev and RRE can be reduced or eliminated by codon optimization (see below) or by substitution with other functional equivalents such as the MPMV system. Since codon-optimized gag-pol expression is rev-independent, RRE can be removed from the gag-pol expression cassette, thus eliminating the possibility of recombination with RRE contained in the vector genome.

[0142] In some embodiments, the viral genome of the present invention lacks a Rev response element (RRE). In other embodiments, the nucleic acid sequence encoding Rev or its functional equivalent is disrupted so that the nucleic acid sequence is unable to encode a functional Rev or is removed from the vector genome.

[0143] In some embodiments, the systems used in the present invention are based on so-called "minimal systems" from which some or all of the additional genes have been removed. Preferably, the viral vector of the present invention has a minimal viral genome.

[0144] As used herein, the term “minimal viral genome” means that a viral vector has been engineered to remove non-essential elements and retain essential elements, thereby providing the necessary functions to infect, transduce, and deliver NOI to target host cells. Preferably, a viral vector having a minimal viral genome is a minimal lentiviral vector.

[0145] Codon optimization Codon optimization is previously described in International Publication No. 99 / 41397, which is incorporated herein by reference in its entirety. Different cells utilize different codons. This codon bias corresponds to a bias in the relative abundance of specific tRNAs in different cell types. By modifying codons in the sequence to match the relative abundance of the corresponding tRNAs, it is possible to increase expression levels. Similarly, it is possible to decrease expression by intentionally selecting codons that are known to be rare in certain cell types for the corresponding tRNAs. Thus, more sophisticated translational control is possible.

[0146] Many viruses, including HIV and other lentiviruses, use a large number of rare codons, and by modifying these to correspond to commonly used mammalian codons, increased expression of packaging components in mammalian production cells can be achieved. Codon frequency tables are publicly known in the art not only for mammalian cells but also for various other organisms.

[0147] Codon optimization also offers many other advantages. By modifying these sequences, RNA instability sequences (INS) are removed from the nucleotide sequences encoding the packaging components of the viral particle necessary for assembly in the producing / packaging cells. Simultaneously, the amino acid sequence coding sequences of the packaging components are preserved, so the viral components encoded by the sequences remain the same, or at least sufficiently similar to not impair the function of the packaging components. Codon optimization also overcomes the requirements for Rev / RRE export, making the optimized sequences Rev-independent. Codon optimization also reduces homologous recombination between different constructs within the vector system (e.g., between the gag-pol and env open reading frame overlapping regions). Therefore, the overall effect of codon optimization is a significant increase in viral titer and improved safety.

[0148] In one embodiment, only codons related to INS are codon-optimized. However, in a more preferred and practical embodiment, the sequences are codon-optimized in their entirety, except for sequences containing frameshift regions.

[0149] The gag-pol gene consists of two overlapping reading frames, one encoding the gag protein and the other the pol protein. The expression of both proteins depends on a frameshift during translation. This frameshift occurs when ribosomes "slip" during translation. This slip is thought to be caused, at least partially, by RNA secondary structures that cause ribosome dysfunction. Such secondary structures are located downstream of the frameshift region of the gag-pol gene. In the case of HIV, the overlapping region extends from nucleotide 1222 downstream of the gag start (where nucleotide 1 is A in gag ATG) to the end of gag (nt 1503). Therefore, the 281 bp fragment spanning the frameshift region and the overlapping region of the two reading frames is preferably not codon-optimized. Retaining this fragment allows for more efficient expression of the gag-pol protein.

[0150] Derivatives from optimal codon usage frequencies may be created, for example, to correspond to convenient restriction sites, and conservative amino acid changes may be introduced into the gag-pol protein.

[0151] In some embodiments, codon optimization is based on highly expressed mammalian genes. The third base, and possibly the second and third bases, may be modified.

[0152] Due to the degenerate nature of the genetic code, it will be understood that a skilled person of the art can achieve a large number of gag-pol sequences. Furthermore, there are many described retroviral variants that can be used as starting points for constructing codon-optimized gag-pol sequences. Lentiviral genomes can vary considerably; for example, there are many pseudo-species of HIV-1 that still function. This is also true for EIAV. These variants may be used to enhance specific parts of the transduction process. Details of HIV variants can be found in the HIV database maintained by Los Alamos National Laboratory. Details of EIAV clones can be found in the NCBI database maintained by the National Institutes of Health.

[0153] The codon-optimized gag-pol sequence strategy can be used in relation to any retrovirus. This applies to all lentiviruses, including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1, and HIV-2. Furthermore, this method can be used to increase gene expression from HTLV-1, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERV), MLV, and other retroviruses.

[0154] Codon optimization can make gag-pol expression Rev-independent. However, to enable the use of anti-rev or RRE factors in retroviral vectors, the viral vector production system must be completely Rev / RRE-independent. Therefore, the genome must also be modified. This can be achieved by optimizing the vector genome components. Advantageously, these modifications can lead to the production of a safer system that does not contain any additional proteins in both production cells and transduced cells.

[0155] As described above, the packaging components of retroviral vectors include the expression products of the gag, pol, and env genes. Furthermore, efficient packaging depends on the short sequences of the four stem-loops followed by the gag and env subsequences ("packaging signals"). Therefore, including deleted gag sequences in the retroviral vector genome (in addition to the complete gag sequence on the packaging construct) optimizes the vector titer. To date, it has been reported that efficient packaging requires 255-360 nucleotides of gag in the vector that still retains the env sequence, or about 40 nucleotides of gag in specific combinations of gag and env deletions that are splice donor mutations. It has been found that all deletions in the gag except for the approximately 360 nucleotides at the N-terminus result in an increase in vector titer. Therefore, preferably, the retroviral vector genome includes a gag sequence comprising one or more deletions, and more preferably, the gag sequence comprises approximately 360 nucleotides that can be derived from the N-terminus.

[0156] NOI In the present invention, the term NOI (Nucleotide of Interest) includes any suitable nucleotide sequence, which does not necessarily have to be a complete natural DNA or RNA sequence. Therefore, an NOI may be, for example, a synthetic RNA / DNA sequence, a codon-optimized RNA / DNA sequence, a recombinant RNA / DNA sequence (i.e., prepared using recombinant DNA technology), a cDNA sequence, or a partial genomic DNA sequence, including combinations thereof. The sequence does not have to be a coding region. If it is a coding region, it does not have to be the entire coding region. Furthermore, the RNA / DNA sequence may be sense-oriented or antisense-oriented. Preferably, it is sense-oriented. Preferably, the sequence is cDNA, comprises cDNA, or is transcribed from cDNA.

[0157] NOIs, also called heterologous sequences, heterologous genes, or transgenes, may be any heterologous sequence of interest delivered to a host cell via a lentiviral transvestment vector, including, but not limited to, sequences encoding therapeutic proteins, enzymes, antibodies, etc.; siRNA; antisense; microRNA, aptamers; ribozymes, any gene repression or silencing sequences; and one or more of the following: selection genes, marker genes, and therapeutic genes.

[0158] NOIs may be candidate genes that are potentially important in disease processes. Therefore, the vector system of the present invention may be used, for example, for target validation purposes.

[0159] NOIs may have therapeutic or diagnostic applications. Suitable NOIs include, but are not limited to, sequences encoding enzymes, cytokines, chemokines, hormones, antibodies, antioxidant molecules, engineered immunoglobulin-like molecules, single-chain antibodies, fusion proteins, immunocostimulatory molecules, immunomodulatory molecules, antisense RNA, small interfering RNA (siRNA), transdominant-negative variants of target proteins, toxins, conditional toxins, antigens, antigen receptors, chimeric antigen receptors, T cell receptors, tumor suppressor proteins, and growth factors, membrane proteins, pro-angiogenic and anti-angiogenic proteins and peptides, vasoactive proteins and peptides, antiviral proteins and ribozymes, and their derivatives (such as associated reporter groups). NOIs may also encode prodrug-activating enzymes. When used in a research context, NOIs may also encode, but are not limited to, green fluorescent protein (GFP); luciferase; β-galactosidase; or reporter genes such as resistance genes to antibiotics such as ampicillin, neomycin, bleomycin, zeosin, chloramphenicol, hygromycin, and kanamycin.

[0160] The NOI may encode all or part of the target protein ("POI"), or a variant, homolog, or mutant thereof. For example, the NOI may encode a fragment of POI that can function in vivo in a manner similar to that of the wild-type protein.

[0161] The term "mutant" includes point-of-instance (POIs) that contain one or more amino acid mutations from the wild-type sequence. For example, a mutant may include the addition, deletion, or substitution of one or more amino acids.

[0162] Here, the term "homologous entity" refers to an entity that encodes a protein that has a specific homology to NOI or a certain degree of homology to POI. Here, the term "homologous entity" can be synonymous with "identity."

[0163] In one embodiment, the vectors, constructs, or sequences described herein may contain at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the reference sequence. A sequence "having at least 85% identity with the reference sequence" is a sequence that, in its entire length, has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the entire length of the reference sequence. In one embodiment, the vectors, constructs, or sequences described herein may contain at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the sequence numbers 1 to 95.

[0164] In the context of this application, “percentage of identity” or “% identity” is calculated using global pairwise alignment (i.e., two sequences are compared over their entire lengths). Methods for comparing the identity of two or more sequences are well known in the art. For example, the “needle” program may be used to find the optimal alignment (including gaps) of two sequences, considering their entire lengths, using the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J.Mol.Biol.48:443-453). The Needle program is available, for example, on the ebi.ac.uk World Wide Web site and is further described in the following publication (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp.276-277). The percentage of identity between two polypeptides according to the present invention is calculated using the EMBOSS:needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5, and a Blosum62 matrix.

[0165] A protein consisting of an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" to a reference sequence may contain mutations such as deletions, insertions, and / or substitutions compared to the reference sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence may correspond to a homologous sequence originating from a different species than the reference sequence.

[0166] "Amino acid substitutions" may be conservative or non-conservative. Preferably, the substitution is a conservative substitution in which one amino acid is replaced by another amino acid having similar structural and / or chemical properties.

[0167] In one embodiment, a conservative substitution may be one described by Dayhoff in "The Atlas of Protein Sequence and Structure, Vol. 5," Natl. Biomedical Research, the entire content of which is referenced by means of this invention. For example, in one embodiment, amino acids belonging to one of the following groups can be exchanged with each other, thus constituting a conservative exchange: Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine (S), threonine (T); Group 2: cysteine ​​(C), serine (S), tyrosine (Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamic acid (E). In one embodiment, the conservative amino acid substitution may be selected from T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and / or T→S.

[0168] In further embodiments, conservative amino acid substitutions may include substitutions of amino acids with other amino acids of the same class, such as (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) non-charged: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative amino acid substitutions may also be made as follows: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Patent No. 10,106,805, whose entire contents are incorporated by reference).

[0169] In another embodiment, conservative substitutions may be performed according to Table A. A method for predicting resistance to protein modification is found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the entire contents of which are referenced by means of this document.

[0170] [Table A]

[0171] In one embodiment, the sequences described herein may include mutations, substitutions, or deletions of 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids or nucleotides. In one embodiment, any 266 of sequence numbers 1 to 95 may include mutations, substitutions, or deletions of 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30. In yet another embodiment, the mutation or substitution is a conservative amino acid substitution.

[0172] In another embodiment, the conservative substitutions may be those shown in Table B under the heading “Conservative Substitutions.” If such substitutions result in changes to biological activity, more substantial changes, labeled “Exemplary Substitutions” in Table B, may be introduced, and the products may be screened as necessary.

[0173] [Table B]

[0174] Internal ribosome entry site (IRES) The viral genome of the present invention comprises at least one NOI, but may optionally comprise two or more NOIs. To express two or more NOIs, two or more transcription units may be present in the vector genome, one for each NOI. However, as is evident from the literature, retroviral vectors achieve the highest titer and most potent gene expression characteristics if they are genetically simple. (PCT / GB96 / 01230; Bowtell et al.,1988 J.Virol.62,2464;Correll et al.,1994 Blood 84,1812;Emerman and Temin 1984 Cell 39,459;Ghattas et al.,1991 Mol.Cell.Biol.11,5848;Hantzopoulos et al. al.,1989 PNAS 86,3519;Hatzoglou et al.,1991 J.Biol.Chem 266,8416;Hatzoglou et al.,1988.J.Biol.Chem 263,17798;Li et al.,1992 Hum.Gen.Ther.3,381;McLachlin et al.,1993 Virol.195,1;Overell (Adam et al., 1988 Mol.Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1, 307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197). Therefore, it is preferable to use the internal ribosome entry site (IRES) to initiate translation of the second (and subsequent) coding sequence with a polycistronic (or, as used herein, "multicistronic") message (Adam et al. 1991 J.Virol. 65, 4985).

[0175] Insertion of IRES factors into retroviral vectors allows for adaptation to the retroviral self-renewal cycle and enables the expression of multiple coding regions from a single promoter (Adam et al. (as above); Koo et al. (1992) Virology 186:669-675; Chen et al. 1993 J. Virol 67:2142-2148). IRES factors were first discovered at the untranslated 5' end of picornaviruses and promote cap-independent translation of viral proteins (Jang et al. (1990) Enzyme 44;292-309). When located between open reading frames of RNA, IRES factors enable efficient translation of downstream open reading frames by promoting ribosome entry at the IRES element and subsequently initiating translation downstream.

[0176] The term "cistron" refers to a portion of a DNA molecule that specifies the formation of one polypeptide chain, i.e., encodes one polypeptide chain. For example, "bicistron" refers to two sections of a DNA molecule that specify the formation of two polypeptide chains, i.e., encode two polypeptide chains; "tricistrone" refers to three sections of a DNA molecule that specify the formation of three polypeptide chains, i.e., encode three polypeptide chains. The term "multicistronic RNA" refers to RNA that contains genetic information to be translated into several proteins. In contrast, monocistronic RNA contains genetic information to be converted into only one protein. In the context of this disclosure, multicistronic RNA transcribed from a lentivirus may be translated into two proteins, for example, the TCRα and TCRβ chains.

[0177] A review of IRESs is presented by Mountford and Smith (TIG May 1995 vol 11, No 5:179-184). Several different IRES sequences are known, including the following: See also those derived from encephalomyocarditis virus (EMCV) (Ghattas, IR, et al., Mol. Cell. Biol., 11:5848-5859 (1991)); BiP protein (Macejak and Sarnow, Nature 353:91 (1991)); the Antennapedia gene (exons d and e) of Drosophila (Oh, et al., Genes & Development, 6:1643-1653 (1992)); and those in poliovirus (PV) (Pelletier and Sonenberg, Nature 334:320-325 (1988)); and Mountford and Smith, TIG 11, 179-184 (1985).

[0178] According to International Publication No. A-97 / 14809, IRES sequences are typically found in the 5' non-coding region of a gene. In addition to those in the literature, they can be found empirically by searching for gene sequences that affect expression and then determining whether those sequences affect DNA (i.e., function as promoters or enhancers) or only RNA (function as IRES sequences).

[0179] IRES factors from PV, EMCV, and porcine vesiculosis virus have been previously used in retroviral vectors (Coffin et al., as above).

[0180] The term "IRES" includes any sequence or combination of sequences that function as an IRES or enhance the function of an IRES. IRESs may be of viral origin (e.g., EMCV IRES, PV IRES, or FMDV 2A-like sequences) or of cellular origin (e.g., FGF2 IRES, NRF IRES, Notch 2 IRES, or EIF4 IRES).

[0181] For the IRES to initiate translation of each NOI, it must be located between or before the NOIs in the vector genome. For example, in the case of a multicistronic sequence containing NOIs, the genome may look like this: [NOI1-IRES1]...NOI n n = any integer

[0182] For bicistronic and tricistronic sequences, the order may be as follows: NOI1-IRES1-NOI2 NOI1-IRES1-NOI2-IRES2-NOI3

[0183] Alternative configurations of RES and NOI may also be used. For example, transcripts containing IRES and NOI do not need to be driven by the same promoter.

[0184] One example of this arrangement is as follows: IRES1-NOI1-promoter-NOI2-IRES2-NOI3.

[0185] In some embodiments, in any construct utilizing an internal cassette having two or more IRESs and NOIs, the IRESs may be of different origins, i.e., heterogeneous. For example, one IRES may be derived from EMCV and the other IRES may be derived from poliovirus.

[0186] Other methods for expressing multiple genes from a single vector While IRES is an efficient method for simultaneously expressing multiple genes from a single vector, other methods are also useful and may be used alone or in combination with IRES. These include the use of multiple internal promoters within the vector (Overell et al., Mol Cell Biol. 8:1803-8 (1988)) or the use of alternating splicing patterns that result in multiple RNA species derived from a single viral genome expressing different genes. This strategy has previously been used alone for two genes (Cepko et al. Cell 37:1053 (1984)).

[0187] For example, a multi-cloning site (MCS) that facilitates the insertion of NOIs can be further incorporated into the vector. This MCS facilitates the introduction of any promoter, a single gene, two genes, and optionally, gene-inhibiting sequences such as antisense, ribozymes, shRNA, RNAi, microRNA, aptamers, and transdominant mutant proteins. A preferred embodiment is the expression of a target gene whose nucleotide sequence has been modified to be codon-degenerate with respect to the endogenous gene in the cell, and further, the same vector expresses a gene-inhibiting or silencing sequence that can inhibit or silence the native gene of interest. This approach is very useful for understanding the function of various protein domains by expressing a gene-inhibiting or silencing sequence that suppresses or silences the expression of a native, unmodified target gene while simultaneously expressing a target protein with a modified domain. This application can also be used in gene therapy approaches for the treatment of diseases. For example, a lentiviral vector expressing an RNAi that targets β-hemoglobin can suppress or silence sickle hemoglobin in patients with sickle cell anemia. The same lentiviral vector can also express normal hemoglobin molecules, codon-degenerate at the RNAi-targeted site. Thus, erythrocytes expressing sickle globin can suppress sickle globin expression while expressing native hemoglobin, potentially correcting genetic abnormalities. Lentiviral vectors can be delivered to stem cell populations that produce erythrocytes expressing hemoglobin, which ultimately becomes red blood cells. This approach could be used to treat a wide variety of diseases, including cancer, genetic disorders, and infectious diseases.

[0188] Transduced cells The present invention also relates to cells transduced with a vector system comprising a viral genome according to the present invention.

[0189] Cells may be transduced in vivo, in vitro, or in vitro by any suitable means. For example, if the cells are from a mammalian subject, the cells may be isolated from the subject and transduced in preparation for re-implantation into the subject (extravitational transduction). Alternatively, cells may be transduced in vivo by direct gene transfer using the vector system of the present invention according to standard techniques (e.g., via injection of a vector stock expressing NOI). If the cells are part of a cell line that is stable in culture (i.e., can survive multiple passages and multiple times in vitro), they may be transduced in vivo by standard techniques, for example, by exposing the cells to a viral supernatant containing a vector expressing NOI.

[0190] The cells may be any cells that are readily transducible. The cells may also be non-dividing cells if the vector system can transduce non-dividing cells (for example, if it is a lentiviral system).

[0191] In one embodiment, the disclosure relates to the activation, transduction, and / or proliferation of immune cells such as lymphocytes, neutrophils, and / or monocytes. In some embodiments, the immune cells are lymphocytes such as T cells (e.g., tumor-infiltrating lymphocytes, CD8+ T cells, CD4+ T cells, and γδT cells), B cells, and / or NK cells, which may be used for transgene expression. In another embodiment, the disclosure relates to the activation, transduction, and proliferation of γδT cells while depleting α- and / or β-TCR-positive cells.

[0192] In one embodiment, the entire PBMC population may be activated and proliferated without prior depletion of specific cell populations such as monocytes, αβ T cells, B cells, and NK cells. In another embodiment, γδ T cells may be isolated from a composite sample cultured in vitro. In another embodiment, an enriched population of γδ T cells may be prepared prior to their specific activation and proliferation. In another embodiment, T cell activation and proliferation may be carried out without the presence of native or engineered APCs. In another embodiment, T cell isolation and proliferation from tumor specimens may be carried out using immobilized T cell mitogens, including antibodies specific to the TCR, and other TCR activators, including lectins. In another embodiment, T cell isolation and proliferation from tumor specimens may be carried out in the absence of immobilized T cell mitogens, including antibodies specific to the TCR, and other TCR activators, including lectins.

[0193] In one embodiment, T cells are isolated from leukocyte apheresis of a target, such as a human subject. In another embodiment, T cells are not isolated from PBMCs (Primary Bacterial Cells).

[0194] The preparation of T cells may be carried out using the method disclosed in U.S. Patent No. 20190247433, which is incorporated herein by reference in its entirety.

[0195] In one embodiment, the present disclosure provides a method for transducing T cells, comprising the steps of: thawing frozen PBMCs; resting the thawed PBMCs; activating T cells in cultured PBMCs using an anti-CD3 antibody and an anti-CD28 antibody; transducing the activated T cells with a viral vector; growing the transduced T cells; and obtaining the grown T cells.

[0196] In another aspect, the present disclosure relates to a method for preparing a T cell population, comprising the steps of: obtaining fresh PBMCs (i.e., PBMCs are not obtained by thawing cryopreserved PBMCs); activating T cells in the fresh PBMCs with an anti-CD3 antibody and an anti-CD28 antibody; transducing the activated T cells with a viral vector; growing the transduced T cells; and collecting the grown T cells.

[0197] In another embodiment of the present disclosure, resting may not be necessary for fresh PBMCs, i.e., unfrozen PBMCs. Thus, fresh PBMCs without resting may be activated with anti-CD3 and anti-CD28 antibodies, followed by viral vector transduction to obtain transducible T cells.

[0198] In another embodiment, the thawing step, the resting step, the activation step, the transduction step, the propagation step, and / or the acquisition step may be carried out in a closed system.

[0199] In another embodiment, the activation step, the transduction step, the propagation step, and the harvesting step may be carried out in a closed or semi-closed system.

[0200] In another embodiment, the closed system may be a CliniMACS Prodigy®, a WAVE(XURI®) bioreactor, a WAVE(XURI®) bioreactor combined with BioSafe Sepax® II, a G-Rex / GatheRex® closed system, or a G-Rex / GatheRex® closed system combined with BioSafe Sepax® II.

[0201] To produce T cells with improved adoptive immunotherapy efficacy, the T cells may be prepared using the method disclosed in U.S. Patent No. 20190292520, the entirety of which is incorporated herein by reference.

[0202] In another embodiment, a method for producing T cells with improved efficacy for adoptive immunotherapy may include the steps of obtaining T cells from at least one healthy donor, patient, or individual; activating the T cells; transducing the activated T cells with a viral vector; proliferating the transduced T cells for approximately 3 to 5 days after activation; and collecting the transduced T cells for injection into at least one healthy donor, patient, or individual, wherein the efficacy for adoptive immunotherapy of T cells proliferated for approximately 3 to 5 days is improved compared to activated transduced T cells proliferated for approximately 7 days or more after activation.

[0203] In another embodiment, the proliferated T cells are naive T cells (TN) and / or stem memory T cells (T). scm ) / T central memory (T cm ) Present the phenotype.

[0204] In another embodiment, a method for producing T cells with improved efficacy for adoptive immunotherapy may include the steps of: obtaining a population of CD8+ T cells from a patient or donor; determining the percentage of CD28+CD8+ T cells in the obtained population; activating the determined population, which comprises at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, with an anti-CD3 antibody or anti-CD28 antibody; transducing the activated T cell population with a viral vector; and growing the transduced T cell population.

[0205] In another aspect, the present disclosure relates to an in vitro method for producing T cells with improved efficacy in immunotherapy, comprising the steps of: determining the percentage of CD28+CD8+ T cells in an isolated CD8+ T cell population; activating the determined population with an anti-CD3 antibody or anti-CD28 antibody, provided that the determined population contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% CD28+CD8+ T cells; transducing the activated T cell population with a viral vector; and growing the transduced T cell population.

[0206] In another aspect, the disclosure relates to a method for producing T cells with improved efficacy in immunotherapy, comprising the steps of: obtaining a population of CD8+ T cells from a patient or donor; determining the percentage of CD28+CD8+ T cells in the obtained population; activating the determined TCR population with an anti-CD3 antibody in the absence of an anti-CD28 antibody, provided that the determined population contains less than approximately 50%, less than approximately 45%, less than approximately 40%, less than approximately 35%, less than approximately 30%, less than approximately 25%, less than approximately 20%, less than approximately 15%, less than approximately 10%, less than approximately 9%, less than approximately 8%, less than approximately 7%, less than approximately 6%, less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 2%, or less than 1% CD28+CD8+ T cells; transducing the activated T cell population with a viral vector; and growing the transduced T cell population.

[0207] In another aspect, the disclosure relates to an in vitro method for producing T cells with improved efficacy in immunotherapy, comprising the steps of: determining the percentage of CD28+CD8+ T cells in an isolated CD8+ T cell population; activating the determined TCR population with an anti-CD3 antibody in the absence of an anti-CD28 antibody, provided that the determined population contains less than approximately 50%, less than approximately 45%, less than approximately 40%, less than approximately 35%, less than approximately 30%, less than approximately 25%, less than approximately 20%, less than approximately 15%, less than approximately 10%, less than approximately 9%, less than approximately 8%, less than approximately 7%, less than approximately 6%, less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 2%, or less than 1% CD28+CD8+ T cells; transducing the activated T cell population with a viral vector; and growing the transduced T cell population.

[0208] In another embodiment, transduction and proliferation may be carried out in the presence of at least one cytokine.

[0209] In one embodiment, isolated γδT cells may proliferate rapidly in response to contact with one or more antigens. Some γδT cells, such as Vγ9Vδ2+ T cells, may proliferate rapidly in vitro in response to contact with certain antigens, such as prenyl pyrophosphate, alkylamines, and metabolites or microbial extracts, during tissue culture. Stimulated γδT cells may exhibit numerous antigen-presenting, stinging, and adhesion molecules, which may facilitate the isolation of γδT cells from a composite sample. γδT cells in a composite sample may be stimulated in vitro with at least one antigen for 1, 2, 3, 4, 5, 6, 7 days, or another suitable period. Stimulation of γδT cells with appropriate antigens may cause a γδT cell population to proliferate in vitro.

[0210] Non-limiting examples of antigens that may be used to stimulate the proliferation of γδ T cells from complex samples in vitro include prenyl pyrophosphates such as isopentenyl pyrophosphate (IPP), alkylamines, metabolites of human microbial pathogens, metabolites of commensal bacteria, methyl-3-butenyl-1-pyrophosphate (2M3B1PP), (E)-4-hydroxy-3-methyl-buto-2-enyl pyrophosphate (HMB-PP), ethyl pyrophosphate (EPP), farnesyl pyrophosphate (FPP), dimethylallyl phosphate (DMAP), dimethylallyl pyrophosphate (DMAPP), ethyl adenosine triphosphate (EPPPA), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), Examples may include isopentenyl adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), 3-formyl-1-butyl pyrophosphate (TUBAg1), X-pyrophosphate (TUBAg2), 3-formyl-1-butyl-uridine triphosphate (TUBAg3), 3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg4), monoethylalkylamines, allyl pyrophosphates, clotyl pyrophosphates, dimethylallyl-γ-uridine triphosphates, clotyl-γ-uridine triphosphates, allyl-γ-uridine triphosphates, ethylamines, isobutylamines, sec-butylamines, isoamylamines, and nitrogen-containing bisphosphonates.

[0211] Activation and proliferation of γδT cells can be carried out using the activators and co-stimulants described herein to initiate specific γδT cell proliferation and sustained populations. In one embodiment, activation and proliferation of γδT cells from different cultures may achieve different clonal population subsets or mixed polyclonal population subsets. In another embodiment, different agonists may be used to identify activators that provide specific γδ activation signals. In another embodiment, the activators that provide specific γδ activation signals may be different monoclonal antibodies (MAb) against the γδTCR. In another embodiment, companion co-stimulants may be used that help initiate specific γδT cell proliferation without induction of cellular energy and apoptosis. These co-stimulants may include ligands that bind to receptors expressed on γδ cells, such as NKG2D, CD161, CD70, JAML, DNAX submolecule-1 (DNAM-1), ICOS, CD27, CD137, CD30, HVEM, SLAM, CD122, DAP, and CD28. In another embodiment, the co-stimulator may be an antibody specific to unique epitopes on the CD2 and CD3 molecules. CD2 and CD3 may have different three-dimensional structures when expressed on αβ or γδ T cells. In another embodiment, specific antibodies against CD3 and CD2 may result in clear activation of γδ T cells.

[0212] Prior to manipulating the γδT cells, a population of γδT cells may be proliferated in vitro. Non-limiting examples of reagents that may be used to promote the proliferation of γδ T cell populations in vitro include anti-CD3 or anti-CD2, anti-CD27, anti-CD30, anti-CD70, anti-OX40 antibodies; IL-2, IL-15, IL-12, IL-9, IL-33, IL-18, or IL-21, CD70 (CD27 ligand), phytohemagglutinin (PHA), concavalin A (ConA), pokeweed (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), lentil (Lens culinaris) agglutinin (LCA), pea (Pisum sativum) agglutinin (PSA), apple snail (Helix pomatia) agglutinin (HPA), Vicia graminea lectin (VGA), or other suitable mitogens that can stimulate T cell proliferation.

[0213] In one embodiment, engineered (or transduced) γδ T cells can be proliferated in vitro without stimulation by antigen-presenting cells or aminobisphosphonates. The antigen-reactive engineered T cells of the Disclosure may be proliferated in vitro and in vivo. In another embodiment, the active population of engineered γδ T cells of the Disclosure may be proliferated in vitro without antigen stimulation by aminobisphosphonates such as antigen-presenting cells, antigenic peptides, non-peptide molecules, or small molecule compounds, using specific antibodies, cytokines, mitogens, or fusion proteins such as IL-17Fc fusion protein, MICAFC fusion protein, and CD70Fc fusion protein. Examples of antibodies that may be used to proliferate γδ T cell populations include anti-CD3, anti-CD27, anti-CD30, anti-CD70, anti-OX40, anti-NKG2D, or anti-CD2 antibodies; examples of cytokines may include IL-2, IL-15, IL-12, IL-21, IL-18, IL-9, IL-7, and / or IL-33; examples of mitogens include CD70, a ligand for human CD27; phytohemagglutinin (PHA); concavalin A (ConA); pokeweed mitogen (PWM); protein peanut agglutinin (PNA); soybean agglutinin (SBA); lentil (Lens culinaris) agglutinin (LCA); pea (Pisum sativum) agglutinin (PSA); apple snail (Helix pomatia) agglutinin (HPA); and Vicia graminea. Examples include graminea lectin (VGA) or another suitable mitogen capable of stimulating T cell proliferation. In another embodiment, the engineered γδ T cell population may proliferate for less than 60 days, less than 48 days, less than 36 days, less than 24 days, less than 12 days, or less than 6 days. In another embodiment, the engineered γδ T cell population may proliferate for about 7 to about 49 days, about 7 to about 42 days, about 7 to about 35 days, about 7 to about 28 days, about 7 to about 21 days, or about 7 to about 14 days.

[0214] In another aspect, the Disclosure provides a method for growing an engineered T cell population in vitro for adoptive immunization therapy. The engineered T cells of the Disclosure may be grown in vitro. The engineered T cells of the Disclosure may be grown in vitro without activation by APC or without co-culture with APC and aminophosphates.

[0215] The ability of T cells to recognize broad-spectrum antigens can be enhanced by genetic engineering of T cells. In one embodiment, T cells can be engineered to provide a universal allogeneic therapy that recognizes selected antigens in vivo. Genetic engineering of T cells may include stably incorporating constructs expressing tumor-recognition portions such as αβTCR, γδTCR, or chimeric antigen receptors (CARs), their antigen-binding fragments, or lymphocyte-activating domains into the genome of isolated T cells or cytokines (e.g., IL-15, IL-12, IL-2, IL-7, IL-21, IL-18, IL-19, IL-33, IL-4, IL-9, IL-23, or IL-1β) that combine both antigen-binding and T-cell activation functions into a single receptor, thereby enhancing T cell proliferation, survival, and function in vitro and in vivo. Genetic engineering of isolated T cells may also include deleting or disrupting gene expression from one or more endogenous genes in the genome of isolated T cells, such as MHC loci.

[0216] Chimeric antigen receptor (CAR) Embodiments of this disclosure may include introducing nucleic acids encoding one or more CARs into T cells. The T cells may be αβT cells, γδT cells, or natural killer T cells. In various embodiments, this disclosure provides T cells genetically engineered with vectors designed to express CARs that redirect cytotoxicity to tumor cells. CARs are molecules that combine antibody-based specificity against a target antigen, such as a tumor antigen, with a T cell receptor activating intracellular domain to produce chimeric proteins that exhibit specific anti-tumor cell immune activity. As used herein, the term “chimeric” describes a substance composed of different protein or DNA portions from different origins.

[0217] A CAR may contain an extracellular domain (also called a binding domain or antigen-specific binding domain) that binds to a specific target antigen, a transmembrane domain, and an intracellular signaling domain. A key feature of a CAR may be its ability to redirect the specificity of immune effector cells, thereby inducing the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner, and to utilize the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific coreceptors.

[0218] In certain embodiments, the CAR may contain an extracellular binding domain, including but not limited to an antibody or its antigen-binding fragment, anchoring ligand, or coreceptor extracellular domain, which specifically binds to a target antigen that is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In certain embodiments, the TAA or TSA may be expressed on hematological cancer cells. In other embodiments, the TAA or TSA may be expressed on solid tumor cells. In certain embodiments, the solid tumor may be glioblastoma, non-small cell lung cancer, lung cancer other than non-small cell lung cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, colon cancer, gastric cancer, splenic cancer, skin cancer, brain cancer other than glioblastoma, kidney cancer, thyroid cancer, etc.

[0219] In certain embodiments, TAA or TSA may include α-folate receptor, 5T4, ανβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD 2. The group may be selected from the following: GD3, *glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, λ, Lewis-Y, κ, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, and VEGFR2.

[0220] CAR binding domain In certain embodiments, the CARs envisioned herein include an extracellular binding domain that specifically binds to a target polypeptide, such as a target antigen, expressed on tumor cells. In the use herein, the terms “binding domain,” “extracellular domain,” “extracellular binding domain,” “antigen-specific binding domain,” and “extracellular antigen-specific binding domain” are used synonymously and may provide a CAR having the ability to specifically bind to a target antigen of interest. The binding domain may include any protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a biomolecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or its components). The binding domain may include any natural, synthetic, semi-synthetic, or recombinant binding partner to the biomolecule of interest.

[0221] In certain embodiments, the extracellular binding domain of the CAR may include an antibody or its antigen-binding fragment. “Antibody” refers to a binder which is a polypeptide containing at least a light-chain or heavy-chain immunoglobulin variable region, and which specifically recognizes and binds to an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, such as those recognized by immune cells; such antibodies may also include their antigen-binding fragments. The term may also include genetically modified forms such as chimeric antibodies (e.g., humanized mouse antibodies), heterocoupled antibodies such as bispecific antibodies, and their antigen-binding fragments. See also Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0222] In certain embodiments, the target antigens include α-folate receptor, 5T4, ανβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, the EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, and GD3. *Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, λ, Lewis-Y, κ, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, or epitopes of the VEGFR2 polypeptide.

[0223] The variable regions of the light and heavy chains may contain a "framework" region interrupted by three hypervariable regions, also referred to as "complementarity-determining regions" or "CDRs." The CDRs are sequenced according to Kabat et al (Wu, TT and Kabat, EA, J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat EA, PNAS, 84:2440-2443 (1987) (see Kabat et al, Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services, 1991, incorporated herein by reference); or Chothia et al (Choithia, C. and Lesk, AM, J Mol. Biol, 196(4):901-917 (1987), Chothia, C. et al. The structure may be defined or identified by conventional methods, such as those described in al., Nature, 342:877-883 (1989). The contents of the aforementioned references are incorporated herein by reference in their entirety. The sequences of different light chain or heavy chain framework regions may be relatively conserved within species such as humans. The framework region of an antibody, which is a combined framework region of the light and heavy chains of its constituent elements, may help to arrange and align the CDRs in three-dimensional space. The CDRs may be primarily responsible for binding the antigen to the epitope. The CDRs of each chain are typically numbered sequentially starting from the N-terminus, such as CDR1, CDR2, They may also be referred to as CDR3, and may be typically identified by the chain in which a particular CDR is located. Therefore, CDRs located in the variable domain of the antibody's heavy chain may be referred to as CDRH1, CDRH2, and CDRH3, while CDRs located in the variable domain of the antibody's light chain are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different binding sites for different antigens) may have different CDRs. While CDRs differ from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. These positions within a CDR are called specificity-determining residues (SDRs).

[0224] A reference to "VH" or "VH" refers to the variable region of the immunoglobulin heavy chain, including antibodies, Fv, scFv, dsFv, Fab, and other antibody fragments. A reference to "VL" or "VL" refers to the variable region of the immunoglobulin light chain, including antibodies, Fv, scFv, dsFv, Fab, and other antibody fragments.

[0225] A "monoclonal antibody" is an antibody produced by a single clone of a B lymphocyte, or by a cell into which the light and heavy chain genes of a single antibody have been transfused. Monoclonal antibodies may be produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immunosplenic cells. Monoclonal antibodies may include humanized monoclonal antibodies.

[0226] A "chimeric antibody" has a framework residue from one species, such as a human, and a CDR (generally antigen-binding-constituting) from another species, such as a mouse. In certain preferred embodiments, the CAR disclosed herein may contain an antigen-specific binding domain, which is the chimeric antibody or its antigen-binding fragment.

[0227] In certain embodiments, the antibody may be a humanized antibody (such as a humanized monoclonal antibody) that specifically binds to surface proteins on tumor cells. The “humanized” antibody is an immunoglobulin comprising a human framework region and one or more CDRs derived from non-human (e.g., mouse, rat, or synthetic) immunoglobulins. The humanized antibody may be constructed by genetic engineering (see, for example, U.S. Patent No. 5,585,089, the entirety of which is incorporated herein by reference).

[0228] In embodiments, the extracellular binding domain of CAR may contain antibodies or antigen-binding fragments, including but not limited to camel Ig (camelid antibody (VHH)), IgNAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), bis-scFv, (scFv)2, small antibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv protein ("dsFv"), and single-domain antibodies (sdAb, nanobodies).

[0229] In the context of this specification, "Camel Ig" or "Camelid VHH" refers to the smallest known antigen-binding unit of a heavy chain antibody (the entire content of which is incorporated herein by reference: Koch-Nolte, et al, FASEB J., 21:3490-3498 (2007)). "Heavy chain antibody" or "Camelid antibody" refers to an antibody that contains two VH domains and does not contain a light chain (the entire content of which is incorporated herein by reference: Riechmann L. et al, J.Immunol.Methods 231:25-38 (1999); International Publication No. 94 / 04678; International Publication No. 94 / 25591; U.S. Patent No. 6,005,079).

[0230] "IgNAR," in "immunoglobulin novel antigen receptor," refers to a class of antibodies from the shark immune repertoire that consists of a homodimer of one variable novel antigen receptor (VNAR) domain and five constant novel antigen receptor (CNAR) domains.

[0231] Papain digestion of antibodies produces two identical antigen-binding fragments, referred to as "Fab" fragments, each having a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to readily crystallize. Fab fragments contain variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the heretical name for Fab' in which the cysteine ​​residue of the constant domain supports a free thiol group. F(ab')2 antibody fragments were originally produced as a pair of Fab' fragments with a hinge cysteine ​​between them. Other chemical conjugations of antibody fragments are also known.

[0232] "Fv" is the smallest antibody fragment containing a complete antigen-binding site. In single-chain Fv (scFv) species, one heavy chain and one light chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimer" structure similar to that in double-chain Fv species.

[0233] The term "diabody" refers to an antibody having two antigen-binding sites, comprising a heavy-chain variable domain (VH) bound to a light-chain variable domain (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain, creating two antigen-binding sites. A diabody may be bivalent or bispecific. Diabodies are described in more detail, for example, in European Patent No. 404,097; International Publication No. 1993 / 01161 pamphlet; Hudson et al, Nat. Med. 9:129-134 (2003); and Hollinger et al, PNAS USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al, Nat. Med. 9:129-134 (2003). The content of the foregoing references is hereby incorporated by reference in its entirety into this specification.

[0234] "Single-domain antibody" or "sdAb" or "nanobody" refers to an antibody fragment consisting of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL domain) (the entire content of which is hereby incorporated by reference into this specification, Holt, L., et al, Trends in Biotechnology, 21(11):484-490).

[0235] A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, where these domains are present in either orientation in a single polypeptide chain (e.g., VL-VH or VH-VL). Generally, an scFv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315, the entire contents of which are incorporated herein by reference.

[0236] In certain embodiments, the scFv binds to an α-folate receptor, 5T4, ανβ6 integrin, BCMA, B7-H3, B7-H6, CALX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, the EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, *glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, λ, Lewis-Y, κ, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM, or VEGFR2 polypeptide.

[0237] Linker of the CAR In certain embodiments, the CAR may contain linker residues between various domains, such as the VH domain and the VL domain, added for proper spacing and conformation of the molecule. The CAR may contain one, two, three, four, or five or more linkers. In certain embodiments, the length of the linker may be about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening amino acid length. In some embodiments, the linker may have an amino acid length of 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, or more. Examples useful for describing linkers include glycine polymer (G)n; glycine-serine polymer (Gi_sSi_5)n, where n is at least an integer of 1, 2, 3, 4, or 5; glycine-alanine polymer; alanine-serine polymer; and other flexible linkers are known in the art. Glycine and glycine-serine polymers are relatively unstructured and may therefore function as neutral tethers between domains of fusion proteins such as CARs. Glycine may have significantly more phi-psi space than alanine and may be far less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992), the entire content of which is incorporated herein by reference). A typical skilled technician may recognize that, in order to provide a desired CAR structure, the design of the CAR in a particular embodiment may include a flexible linker and a linker that may be fully or partially flexible, such that one or more parts may be included to give the structure less flexibility.

[0238] In certain embodiments, the CAR may further include an scFV containing a variable region linker. The "variable region linker" is an amino acid sequence that links the heavy chain variable region to the light chain variable region and provides a spacer function that fits the interaction of the two subbinding domains so that the resulting polypeptide maintains specific binding affinity to the same target molecule as an antibody which may also contain the same light and heavy chain variable regions. In one embodiment, the variable region linker may have an amino acid length of 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, or more. In certain embodiments, the variable region linker may contain a glycine-serine polymer (Gi_sSi_5)n, where n is at least an integer of 1, 2, 3, 4, or 5. In another embodiment, the variable region linker comprises a (G4S)3 amino acid linker.

[0239] CAR spacer domain In certain embodiments, the CAR binding domain may be followed by one or more “spacer domains,” which refer to regions that move the antigen-binding domain away from the effector cell surface to enable proper intercellular contact, antigen binding, and activation (the entire content of which is incorporated herein by reference, Patel et al, Gene Therapy, 1999;6:412-419). The spacer domains may be natural, synthetic, semi-synthetic, or derived from recombinant sources. In certain embodiments, the spacer domains may include, but are not limited to, one or more heavy chain constant regions such as CH2 and CH3 of an immunoglobulin. The spacer domains may comprise amino acid sequences of a natural immunoglobulin hinge region or a modified immunoglobulin hinge region. In one embodiment, the spacer domains may comprise CH2 and CH3 of IgG1.

[0240] CAR hinge domain The binding domain of a CAR may generally be followed by one or more "hinge domains," which may play a role in positioning the antigen-binding domain away from the effector cell surface, enabling proper intercellular contact, antigen binding, and activation. A CAR may generally contain one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be natural, synthetic, semi-synthetic, or derived from a recombinant source. The hinge domain may contain amino acid sequences of a natural immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in a CAR may include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8a, CD4, CD28, and CD7, which may be wild-type hinge regions from these molecules or modified. In another embodiment, the hinge domain may include a CD8α hinge region.

[0241] CAR membrane permeable domain (TM) The “transmembrane domain” may be a portion of the CAR that can fuse an extracellular binding portion and an intracellular signaling domain to attach the CAR to the plasma membrane of an immunoeffector cell. The TM domain may be natural, synthetic, semi-synthetic, or derived from a recombinant source. Exemplary TM domains (including at least their transmembrane region) may be derived from the α, β, or ζ chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the CAR may contain a TM domain derived from CD8a. In another embodiment, the CAR envisioned herein comprises a TM domain derived from CD8α and a short oligolinker or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, linking the TM domain to the intracellular signaling domain of the CAR. Glycine-serine linkers provide particularly suitable linkers.

[0242] CAR intracellular signaling domain In certain embodiments, the CAR may contain an intracellular signaling domain. The “intracellular signaling domain” refers to a portion of the CAR that transmits the message of the CAR, which is effectively bound to a target antigen, into the interior of immune effector cells and is involved in inducing effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-binding target cells, or other cellular responses induced by antigen binding to the extracellular CAR domain.

[0243] The term "effector function" refers to a specific function of a cell. The effector function of a T cell may include, for example, cytokine secretion, or cytolytic activity or assistance to activity. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that can transmit effector function signals and instruct the cell to perform a specific function. While the entire intracellular signaling domain may be used, it is often not necessary to use the entire domain. As long as a truncated portion of the intracellular signaling domain is usable, such a truncated portion may be used in place of the entire domain, provided it can transmit effector function signals. The term "intracellular signaling domain" may also mean that it contains a sufficient truncated portion of the intracellular signaling domain to transmit effector function signals.

[0244] It is known that signals generated via the TCR alone are insufficient for complete activation of T cells, and that secondary or co-stimulatory signals may be required. Therefore, it can be said that T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains (e.g., the TCR / CD3 complex) that initiate antigen-dependent primary activation via the TCR, and co-stimulatory signaling domains that act in an antigen-independent manner to provide secondary or co-stimulatory signals. In preferred embodiments, the CAR may contain an intracellular signaling domain that comprises one or more "co-stimulatory signaling domains" and "primary signaling domains." The primary signaling domain can modulate primary activation of the TCR complex in either a stimulatory or inhibitory manner. A primary signaling domain acting in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples that are particularly useful in describing the ITAM-containing primary signaling domains in the present invention may include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζCD22, CD79a, CD79b, and CD66d. In certain preferred embodiments, the CAR may comprise the CD3ζ primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling domain and the co-stimulatory signaling domains may be tandem-linked to the carboxyl terminus of the transmembrane domain in any order.

[0245] CAR may contain one or more co-stimulatory signaling domains to enhance the efficacy and proliferation of T cells expressing the CAR receptor. In the use herein, the terms co-stimulatory signaling domain or co-stimulatory domain refer to the intracellular signaling domain of a co-stimulatory molecule. Examples useful in describing such co-stimulatory molecules may include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), CTLA4, LFA-1, CD2, CD7, LIGHT, TRIM, LCK3, SLAM, DAP10, LAG3, HVEM and NKD2C, and CD83. In one embodiment, CAR may contain one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain.

[0246] In one embodiment, CAR is α-folate receptor, 5T4, ανβ6 integrin, BCMA, B7-H3, B7-H6, CALX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, *glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-Al+NY-ESO-1, HLA-A2+NY-ESO- It may also contain: 1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, λ, Lewis-Y, κ, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEM, or VEGFR2 polypeptide; a transmembrane domain derived from a polypeptide selected from the group consisting of CD8α, CD4, CD45, PD1, and CD152; and one or more intracellular costimulatory signaling domains selected from the group consisting of CD28, CD54, CD134, CD137, CD152, CD273, CD274, and CD278; and scFv that binds to the CD3ζ primary signaling domain.

[0247] In another embodiment, CAR includes α-folate receptor, 5T4, ανβ6 integrin, BCMA, B7-H3, B7-H6, CALX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, and ErbB2 (HER2). EGFR family, EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, *glypican-3(G PC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+N The formulation may also contain Y-ESO-1, IL-11Rα, IL-13Rα2, λ, Lewis-Y, κ, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEM, or VEGFR2 polypeptide; a hinge domain selected from the group consisting of IgG1 hinge / CH2 / CH3 and CD8α, and CD8α; a transmembrane domain derived from a polypeptide selected from the group consisting of CD8α, CD4, CD45, PD1, and CD152; and one or more intracellular costimulatory signaling domains selected from the group consisting of CD28, CD134, and CD137; and scFv that binds to the CD3ζ primary signaling domain.

[0248] In another embodiment, CAR is α-folate receptor, 5T4, ανβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, the EGFR family including ErbB2 (HER2), EGFRvIII, EGP2 , EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, *glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+M AGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, λ, Lewis-Y, κ, mesothelin, Muc1, Muc16 The scFv may further comprise a linker that binds to the CD3ζ primary signaling domain, and includes: NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEM, or VEGFR2 polypeptide; a hinge domain selected from the group consisting of IgG1 hinge / CH2 / CH3 and CD8α; a transmembrane domain comprising a TM domain derived from a polypeptide selected from the group consisting of CD8a, CD4, CD45, PD1, and CD152; and a short oligo or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the TM domain to the intracellular signaling domain of the CAR; and one or more intracellular costimulatory signaling domains selected from the group consisting of CD28, CD134, and CD137.

[0249] In certain embodiments, CARs include α-folate receptor, 5T4, ανβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, and the EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, *glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO- 1. It may contain a hinge domain containing a CD8α polypeptide; a CD8α transmembrane domain containing a polypeptide linker of about 3 amino acids; one or more intracellular costimulatory signaling domains selected from the group consisting of CD28, CD134, and CD137; and an scFv that binds to the CD3ζ primary signaling domain.

[0250] Manipulated T cells may be produced by a variety of methods. For example, polynucleotides encoding an expression cassette comprising a tumor recognition region or another type of recognition region can be stably introduced into T cells by transposon / transposase systems; or virus-based gene transfer systems such as lentivirus or retrovirus systems; or other suitable methods such as transtransfer, electroporation, transduction, lipofection, nanoengineered materials such as calcium phosphate (CaPO4) or ormosyl; or by viral delivery methods including adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, or other suitable methods. Human gene therapy employs several viral methods, such as those described in International Publication No. 1993020221, the entire content of which is incorporated herein by reference. Non-limiting examples of viral methods that may be used to manipulate T cells include gamma retrovirus methods, adenovirus methods, lentivirus methods, herpes simplex virus methods, vaccinia virus methods, poxvirus methods, or adenovirus-associated virus methods.

[0251] In one embodiment, the constructs and vectors described herein are used in the manner described in U.S. Patent No. 16 / 200,308, filed November 26, 2018, which is incorporated by reference in its entirety.

[0252] cassette In the present invention, a cassette comprising one or more NOIs may be used, and in the case of two or more NOIs, they may be operably linked by an IRES. These cassettes may be used in a method for producing a vector genome in producing cells.

[0253] The present invention also provides an expression vector comprising such a cassette. Transfusion of appropriate cells with such an expression vector should result in cells expressing each POI encoded by the NOI in the cassette. The present invention also provides such transfected cells.

[0254] Cloning of the cassette into an expression vector and transfection of cells with the vector (to confer expression of the cassette) can be carried out by techniques well known in the art (such as those described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)) and other laboratory textbooks).

[0255] In some embodiments, the cassette comprises a promoter.

[0256] In some embodiments, the cassette comprises a NOI. The NOI can be any NOI described in detail above. For example, the NOI may have a therapeutic or diagnostic use. Suitable NOIs include, but are not limited to, enzymes, cytokines, chemokines, hormones, antibodies, antioxidant molecules, engineered immunoglobulin-like molecules, single-chain antibodies, fusion proteins, costimulatory molecules, immunomodulatory molecules, antisense RNAs, small interfering RNAs (siRNAs), dominant negative mutants of target proteins, toxins, conditional toxins, antigens, antigen receptors, chimeric antigen receptors, T cell receptors, tumor suppressor proteins, and growth factors, membrane proteins, angiogenesis-promoting and anti-angiogenesis proteins and peptides, vasoactive proteins and peptides, antiviral proteins and ribozymes, and sequences encoding derivatives thereof (such as related reporter groups).

[0257] In some embodiments, the cassette comprises two or more NOIs. A cassette comprising two or more NOIs can be bicistronic or tricistronic and can comprise the following elements: Promoter - (NOI1) - (IRES1) - (NOI2) Promoter - (NOI1) - (IRES1) - (NOI2) - (IRES2) - (NOI3)

[0258] In some embodiments, a single lentiviral vector expressing one or more proteins can be constructed using a single lentiviral cassette. In particular, a single lentiviral vector expressing at least four individual monomer proteins of two distinct dimers from a single multicistronic mRNA can be constructed using a single lentiviral cassette to co-express dimers on the cell surface. For example, the incorporation of a single copy of a lentiviral vector has been shown to be sufficient to transform γδT cells to co-express TCRαβ and CD8αβ.

[0259] In one embodiment, the disclosure relates to a vector containing a multicistronic cassette in a single vector capable of expressing more than one, more than two, more than three, more than four, more than five, or more than six genes, wherein polypeptides encoded by these genes may interact with each other or form dimers. The dimers may be homodimers, i.e., two identical proteins forming a dimer, or heterodimers, i.e., two structurally different proteins forming a dimer.

[0260] In one embodiment, the lentiviral vector may contain a first nucleotide sequence S1 encoding protein Z1, a second nucleotide sequence S2 encoding protein Z2, a third nucleotide sequence S3 encoding protein Y1, and a fourth nucleotide sequence S4 encoding protein Y2, where Z1 and Z2 form a first dimer and Y1 and Y2 form a second dimer, where the first dimer Z1Z2 is different from the second dimer Y1Y2.

[0261] In one embodiment, the first lentiviral vector may contain a bisistronic cassette (2-in-1) encoding the dimer Z1Z2, and the second lentiviral vector may contain a bisistronic cassette (2-in-1) encoding the dimer Y1Y2. In the 2-in-1 vector, S1 and S2 may be arranged in tandem in the 5' to 3' direction of S1-S2 or S2-S1. Similarly, in the 2-in-1 vector, S3 and S4 may be arranged in tandem in the 5' to 3' direction of S3-S4 or S4-S3. Z1 and Z2 or Y1 and Y2 may be separated by one or more self-cleaved 2A peptides.

[0262] In another embodiment, a single lentiviral vector (4-in-1) may encode both distinct dimers Z1Z2 and Y1Y2, where Z1, Z2, Y1, and Y2 may be separated by a 1- or 1- or 2A self-cleaving peptide. For example, S1-S2-S3-S4, S1-S2-S4-S3, S1-S3-S2-S4, S1-S3-S4-S2, S1-S4-S3-S2, S1-S4-S2-S3, S2-S1-S3-S4, S2-S1-S4-S3, S2-S3-S1-S4, S2-S3-S4-S1, S2-S4-S3-S1, S2-S4-S1-S3, S3-S1-S2-S4, S3-S1-S4-S2 S1, S2, S3, and S4 may be selected from S3-S2-S1-S4, S3-S2-S4-S1, S3-S4-S1-S2, S3-S4-S2-S1, S4-S1-S2-S3, S4-S1-S3-S2, S4-S2-S1-S3, S4-S2-S3-S1, S4-S3-S1-S2, or S4-S3-S2-S1 and arranged tandem in the 5' to 3' direction.

[0263] In one embodiment, the dimer Z1Z2 and / or dimer Y1Y2 may be a TCR having a TCRα chain and a TCRβ chain, or a TCR having a TCRγ chain and a TCRδ chain.

[0264] In one embodiment, TCRs and antigen-binding proteins that can be used with the constructs, methods, and embodiments described herein include, for example, those listed in Table 3 (SEQ ID NOs: 13-92), and U.S. Patent Publication Nos. 20170267738, 20170312350, 20180051080, 20180164315, and 20180161396. Examples of TCRs and antigen-binding proteins described in U.S. Patent Publication No. 20180162922, U.S. Patent Publication No. 20180273602, U.S. Patent Publication No. 20190016801, U.S. Patent Publication No. 20190002556, and U.S. Patent Publication No. 20190135914 include the contents of each of these publications and the sequence lists contained herein, which are incorporated herein by reference in their entirety.

[0265] In one embodiment, examples of TCRs and antigen-binding proteins that can be used in conjunction with the constructs, methods, and embodiments described herein include TCRs and antigen-binding proteins that bind to "target antigenic (TA) peptides".

[0266] As used in the context of this invention, “target antigenic (TA) peptides” refer to peptides isolated and identified from infected or oncological materials, such as materials isolated from individuals with tuberculosis, from Epstein-Barr virus infection, or from cancer. TA peptides and the proteins from which they are derived undergo antigen processing in infected or tumor cells, and are then presented on the cell surface by MHC molecules and cells, particularly TA peptide / MHC complexes, and subsequently recognized by host immune effector cells such as T cells or NKT cells. In the context of this invention, TA peptides consist of or comprise 10, 12, or 14 amino acids, e.g., 8-14, 8-12, e.g., 9-11 amino acids. In the context of this invention, when a specific TA peptide is referred to, it refers to TA-C. Examples of TA antigenic peptides, such as TA-C peptides, include viral antigenic peptides, bacterial antigenic peptides, or tumor-associated antigen (TAA) antigenic peptides, preferably TAA antigenic peptides. Therefore, in one embodiment, the TA antigenic peptide, particularly TA-C, is a viral peptide, a bacterial peptide, or a tumor-associated antigen (TAA) antigenic peptide, preferably a TAA antigenic peptide.

[0267] In the context of the present invention, “viral antigenic peptide” refers to an antigenic peptide of viral origin presented by MHC molecules on the surface of infected cells, i.e., cells typically infected with the virus. Such viral antigenic peptides have been found in connection with infections from, for example, human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), cytomegalovirus (CMV), human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus infection (HPV), Epstein-Barr virus (EBV), and influenza virus. Therefore, in the context of the present invention, the viral antigenic peptide may be an antigenic peptide selected from the group consisting of HIV antigenic peptide, HCMV antigenic peptide, CMV antigenic peptide, HPV antigenic peptide, HBV antigenic peptide; HCV antigenic peptide; EBV antigenic peptide, influenza antigenic peptide, preferably HIV, HBV, influenza, and HCMV antigenic peptides.

[0268] Examples of viral antigenic peptides that can be used in the methods and embodiments described herein include the viral antigenic peptides listed in the table below. Examples of viral antigenic peptides that can be used in the methods and embodiments described herein include at least one viral antigenic peptide shown in Table 1 below, which comprises or consists of an amino acid sequence selected from the amino acid sequences of SEQ ID NOs. 96 to 98.

[0269] [Table 1]

[0270] In the context of the present invention, "bacterial antigenic peptide" refers to an antigenic peptide of bacterial origin presented by MHC molecules on the surface of infected cells, that is, cells typically infected with the bacterium. Such bacterial antigenic peptides have been found, for example, in the context of infection from Mycobacterium tuberculosis. Therefore, the bacterial antigenic peptide in the context of the present invention may be a Mycobacterium tuberculosis antigenic peptide.

[0271] In this specification, "tumor-associated antigen (TAA) peptides," also referred to as "TAA peptides," mean peptides that have been isolated and identified from tumor material, undergo antigen processing in tumor cells, and are therefore recognizable by host immune effector cells. TAA peptides consist of or comprise 10, 12, or 14 amino acids, e.g., 8-14, 8-12, e.g., 9-11 amino acids. In the context of the present invention, TAA peptides may be, for example, cancer / testis (CT) antigenic peptides. Examples of cancer / testis (CT) antigenic peptides are the MAGE-A antigenic peptide with the amino acid sequence of SEQ ID NO: 216 and the PRAME antigenic peptide with the amino acid sequence of SEQ ID NO: 148. In the context of the present invention, TAA peptides comprise T cell epitopes and may also be referred to as TAA peptides in a general context, and when referring to a particular TAA peptide, they may be referred to as TAA peptide C in the context of the present invention.

[0272] In one embodiment, tumor-associated antigens, TAA peptides that can be used in the methods and embodiments described herein include, for example, U.S. Patent Publication No. 20160187351, U.S. Patent Publication No. 20170165335, U.S. Patent Publication No. 20170035807, U.S. Patent Publication No. 20160280759, U.S. Patent Publication No. 20160287687, U.S. Patent Publication No. 20160346371, U.S. Patent Publication No. 20160368965, U.S. Patent Publication No. 20170022251, U.S. Patent Publication No. 20170002055, U.S. Patent Publication No. 20170029486, U.S. Patent Publication No. 20170037089, and U.S. Patent Publication No. 2017 Examples of tumor-associated antigen (TAA) peptides described in U.S. Patent Application Publication No. 0136108, U.S. Patent Application Publication No. 20170101473, U.S. Patent Application Publication No. 20170096461, U.S. Patent Application Publication No. 20170165337, U.S. Patent Application Publication No. 20170189505, U.S. Patent Application Publication No. 20170173132, U.S. Patent Application Publication No. 20170296640, U.S. Patent Application Publication No. 20170253633, U.S. Patent Application Publication No. 20170260249, U.S. Patent Application Publication No. 20180051080, and U.S. Patent Application Publication No. 20180164315 include tumor-associated antigen (TAA) peptides described in each of these publications and the sequence lists contained therein, which are incorporated herein by reference in their entirety.

[0273] In one embodiment, the bispecific antigen-binding protein described herein, particularly antigen-binding site B in the context of the present invention, selectively recognizes cells presenting the TAA peptide described in one or more of the above-mentioned patents and publications. In another embodiment, the TAA that can be used in the methods and embodiments described herein comprises at least one TAA consisting of an amino acid sequence selected from sequences 99 to 256, preferably from the amino acid sequences of sequences 148 and 216. In one embodiment, the bispecific antigen-binding protein, particularly antigen-binding site B of the bispecific antigen-binding protein, selectively recognizes cells presenting the TAA peptide / MHC complex, wherein the TAA peptide comprises or consists of the amino acid sequences of SEQ ID NOs. 99 to 256, or any of the amino acid sequences described in the patents or applications described herein, preferably the amino acid sequences of SEQ ID NOs. 148 and 216.

[0274] Furthermore, in the context of the present invention, the TAA antigenic peptide is a specific ligand for an MHC class I molecule or an MHC class II molecule, preferably an MHC class I molecule.

[0275] In the context of the present invention, TAA antigenic peptide C is selected from the group of TAA antigenic peptides preferably comprising the amino acid sequences of SEQ ID NOs. 99 to 256, preferably PRAME antigenic peptides comprising or comprising the amino acid sequence "SLLQHLIGL" of SEQ ID NO. 148, or MAGE-A antigenic peptides comprising or comprising the amino acid sequence "KVLEHVVRV" of SEQ ID NO. 216, more preferably SEQ ID NO. 216, where the MHC is preferably HLA-A*02.

[0276] In another embodiment, dimer Z1Z2 and / or dimer Y1Y2 are R11KEA (sequences 13 and 14), R20P1H7 (sequences 15 and 16), R7P1D5 (sequences 17 and 18), R10P2G12 (sequences 19 and 20), R10P1A7 (sequences 21 and 22), R4P1D10 (sequences 23 and 24), R4P3F9 (sequences 25 and 26), R4P3F9-B4 (sequences 25 and 92), R4P3F9-A1B4 (sequences 91 and 92), R4P3H3 (sequences 13 and 14 Numbers 27 and 28), R36P3F9 (sequence numbers 29 and 30), R52P2G11 (sequence numbers 31 and 32), R53P2A9 (sequence numbers 33 and 34), R26P1A9 (sequence numbers 35 and 36), R26P2A6 (sequence numbers 37 and 38), R26P3H1 (sequence numbers 39 and 40), R35P3A4 (sequence numbers 41 and 42), R37P1C9 (sequence numbers 43 and 44), R37P1H1 (sequence numbers 45 and 46), R42P3A9 (sequence numbers 47 and 48), R43P3F2 (sequence numbers Numbers 49 and 50), R43P3G5 (sequences 51 and 52), R59P2E7 (sequences 53 and 54), R11P3D3 (sequences 55 and 56), R16P1C10 (sequences 57 and 58), R16P1E8 (sequences 59 and 60), R17P1A9 (sequences 61 and 62), R17P1D7 (sequences 63 and 64), R17P1G3 (sequences 65 and 66), R17P2B6 (sequences 67 and 68), R11P3D3KE (sequences 69 and 70), R39P1C12 The TCRα and TCRβ chains may be selected from R39P1F5 (SEQ ID NOs. 71 and 72), R40P1C2 (SEQ ID NOs. 75 and 76), R41P3E6 (SEQ ID NOs. 77 and 78), R43P3G4 (SEQ ID NOs. 79 and 80), R44P3B3 (SEQ ID NOs. 81 and 82), R44P3E7 (SEQ ID NOs. 83 and 84), R49P2B7 (SEQ ID NOs. 85 and 86), R55P1G7 (SEQ ID NOs. 87 and 88), or R59P2A7 (SEQ ID NOs. 89 and 90).

[0277] Table 2 shows examples of peptides to which TCRs bind when the peptide forms a complex with an MHC molecule.

[0278] [Table 2]

[0279] In one embodiment, tumor-associated antigens, TAA peptides that can be used in the methods and embodiments described herein include, for example, those listed in Table 4, and, for example, U.S. Patent Publication No. 20160187351, U.S. Patent Publication No. 20170165335, U.S. Patent Publication No. 20170035807, U.S. Patent Publication No. 20160280759, U.S. Patent Publication No. 20160287687, U.S. Patent Publication No. 20160346371, U.S. Patent Publication No. 20160368965, U.S. Patent Publication No. 20170022251, U.S. Patent Publication No. 20170002055, U.S. Patent Publication No. 20170029486, U.S. Patent Publication No. 20170037089, U.S. Patent Examples of tumor-associated antigen (TAA) peptides described in U.S. Patent Publication No. 20170136108, U.S. Patent Publication No. 20170101473, U.S. Patent Publication No. 20170096461, U.S. Patent Publication No. 20170165337, U.S. Patent Publication No. 20170189505, U.S. Patent Publication No. 20170173132, U.S. Patent Publication No. 20170296640, U.S. Patent Publication No. 20170253633, U.S. Patent Publication No. 20170260249, U.S. Patent Publication No. 20180051080, and U.S. Patent Publication No. 20180164315 include tumor-associated antigen (TAA) peptides described in each of these publications and the sequence lists contained therein, which are incorporated herein by reference in their entirety.

[0280] In another embodiment, dimer Z1Z2 and / or dimer Y1Y2 may be T cell dimeric signaling modules such as CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η; dimers of the TCRα variable region (Vα) and the TCRβ variable region (Vβ); dimers of the immunoglobulin heavy chain variable region (VH) and the immunoglobulin light chain variable region (VL); dimers of Vα and VH; dimers of Vα and VL; dimers of Vβ and VH; or dimers of Vβ and VL.

[0281] In another embodiment, the dimer Z1Z2 and / or dimer Y1Y2 may be TCR coreceptors such as CD8α and CD8β chains, CD4α and CD4β chains, or any other suitable dimeric membrane receptor, preferably expressed in CD8+ T cells and / or CD4+ T cells.

[0282] In some embodiments, the dimer Z1Z2 is a TCR, and the dimer Y1Y2 is a TCR coreceptor.

[0283] Furin is a ubiquitous subtilisin-like proprotein convertase whose native groups include certain serum proteins and growth factor receptors such as insulin-like growth factor receptors. The consensus sequence for furin cleavage is RXXR (SEQ ID NO: 93), but the actual cleavage potential depends on the substrate's tertiary structure and the amino acids immediately surrounding the recognition site. Addition of a linker sequence (such as GSG or SGSG (SEQ ID NO: 5)) to the furin cleavage site may enable highly efficient gene expression.

[0284] In one embodiment, the nutrient sequences of the tandem-placed furin linker and 2A peptide may be positioned between Z1 and Z2, between Z1 and Y1, between Z1 and Y2, between Z2 and Y1, between Z2 and Y2, and / or between Y1 and Y2. The furin may have a consensus sequence of RXXR (SEQ ID NO: 93), such as RAKR (SEQ ID NO: 10). The linker sequence may be 3 to 10 amino acids long, such as 3 to 8 amino acids, 3 to 5 amino acids, or 3 to 4 amino acids. In some embodiments, the linker sequence may be SGS, GGGS (SEQ ID NO: 257), GGGGS (SEQ ID NO: 258), GGSGG (SEQ ID NO: 259), TVAAP (SEQ ID NO: 260), TVLRT (SEQ ID NO: 261), or TVSSAS (SEQ ID NO: 262). In some embodiments, the linker sequence may be GSG or SGSG (SEQ ID NO: 5). The 2A peptide may be selected from P2A (SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5), F2A (SEQ ID NO: 6), or any combination thereof.

[0285] In another embodiment, the nucleotide sequences of the tandem-placed linker and 2A peptide may be positioned between Z1 and Z2, between Z1 and Y1, between Z1 and Y2, between Z2 and Y1, between Z2 and Y2, and / or between Y1 and Y2. The linker sequence may be GSG or SGSG (SEQ ID NO: 5). The 2A peptide may be selected from P2A (SEQ ID NO: 6), T2A (SEQ ID NO: 7), E2A (SEQ ID NO: 8), F2A (SEQ ID NO: 9), or any combination thereof.

[0286] therapeutic composition The present invention also provides a therapeutic composition comprising a population of transduced cells, such as transduced T cells, as described herein.

[0287] The compositions of this disclosure may also include one or more adjuvants. Adjuvants are substances that nonspecifically promote or enhance an immune response (e.g., an immune response to an antigen mediated by CD8-positive T cells and helper T (TH) cells) and are therefore considered useful in the agents of the present invention. Suitable adjuvants include 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod (ALDARA®), reciquimod, ImuFact IMP321; interleukins such as IL-2, IL-13, IL-21, interferon-α or -β, or their PEGylated derivatives; IS patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, montanide IMS 1312, montanide ISA 206, montanide ISA Examples of adjuvants include, but are not limited to, 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector systems, poly(lactidocoglycolide)[PLG]-based and dextran microparticles, talactoferrin SRL172, viromosomes and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, saponin-derived Aquila's QS21 stimulon, mycobacterial extracts and synthetic bacterial cell wall mimics; and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Several immunological adjuvants specific to dendritic cells and their preparations (e.g., MF59) have been previously described (Allison and Krummel, 1995). Cytokines may also be used.Several cytokines have been directly linked to influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, the entirety of which is specifically incorporated herein by reference), and acting as immunostimulants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich et al., 1996).

[0288] CpG immunostimulant oligonucleotides have also been reported to enhance adjuvant effects in the vaccine environment. Without being constrained by theory, CpG oligonucleotides act by activating the intrinsic (maladaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or dead viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced TH1 cell activation and potent cytotoxic T lymphocyte (CTL) generation, even in the absence of CD4 T cell assistance. The TLR9-induced TH1 bias is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote TH2 bias. CpG oligonucleotides exhibit even higher adjuvant activity when compounded with or co-administered with other adjuvants, or in formulations or similar formulations such as microparticles, nanoparticles, or lipid emulsions, which are particularly necessary to induce a potent response when the antigen is relatively weak. They also accelerate the immune response, and in some experiments, have allowed for nearly two-order-of-magnitude reductions in antigen dose with an antibody response equivalent to that of the total vaccine dose without CpG (Krieg, 2006). U.S. Patent No. 6,406,705B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens for inducing an antigen-specific immune response. The CpG TLR9 antagonist is dSLIM (Double Stem-Loop Immunomodulator) manufactured by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR-binding molecules such as RNA-binding TLR7, TLR8, and / or TLR9 may also be used.

[0289] Other examples of useful adjuvants include chemically modified CpGs (e.g., CpR, Idera), dsRNA analogs such as poly(I:C) and their derivatives (e.g., AmpliGen®, Hiltonol®, poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA; as well as immunoactive small molecules and antibodies such as cyclophosphamide and sunitinib; ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and semiprimab, bevacizumab®, Celebrex, NCX-4016, sildenafil, Examples include, but are not limited to, immune checkpoint inhibitors such as tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF trap, ZD2171, AZD2171, and anti-CTLA4; other antibodies that target major structures of the immune system (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptor); and SC58175, which may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of this invention can be readily determined by those skilled in the art without performing excessive experiments.

[0290] Preferred adjuvants include anti-CD40, imiquimod, reciquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-α, interferon-β, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, and particulate formulations, virosoms, and / or interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

[0291] In preferred embodiments of the drug composition according to the present invention, the adjuvant is selected from the group consisting of colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, salglamostim), cyclophosphamide, imiquimod, reximod, and interferon-α.

[0292] In preferred embodiments of the pharmaceutical composition according to the present invention, the adjuvant is selected from the group consisting of colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, salglamostim), cyclophosphamide, imiquimod, and reciquimod. In preferred embodiments of the pharmaceutical composition according to the present invention, the adjuvant is cyclophosphamide, imiquimod, or reciquimod. More preferred adjuvants are Montanide IMS 1312, Montanide ISA 20, Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®), and anti-CD40mAB or a combination thereof.

[0293] Polypeptide manufacturing method The present invention also relates to a method for producing recombinant host cells that express a protein such as a therapeutic protein, the method comprising the steps of (i) introducing a vector of the present invention into competent host cells in vitro or in vitro; (ii) culturing the resulting recombinant host cells in vitro or in vitro; and (iii) optionally selecting cells that express and / or secrete the protein.

[0294] The present invention also provides a method for producing polypeptides using a vector, such as a lentiviral transduction vector disclosed herein, and a product of such a method. The method may comprise one or more of the following steps: transducing a host cell with a lentiviral transduction vector to form a transduced host cell, wherein the vector comprises an expressible heterologous polynucleotide encoding a heterologous polypeptide of interest; culturing the transduced host cell under conditions effective for producing the polypeptide of interest; and isolating the polypeptide from the host, for example, from the culture medium, when the polypeptide has been secreted into the culture medium. The heterologous polynucleotide sequence encoding the polypeptide may comprise any further sequences (e.g., secretion sequences) necessary for transcription, translation, and / or secretion into the culture medium. Any cell line, including, for example, CHO (e.g., CHO DG44) and HEK293 (e.g., HEK293F), can be transduced by the present invention.

[0295] Transduction vectors can be prepared routinely, including by the methods described herein. For example, a producing cell line can be transformed with a helper plasmid (containing a suitable envelope and gag / pol precursor) and a transduction vector containing a heterologous NOI under conditions effective for producing a functional transduction vector. The envelope protein may be selected for its ability to transform the target host cell from which the polypeptide is to be produced.

[0296] Examples of host cells include mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established cells (e.g., those produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Other examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"). Since the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in YB2 / 0 cells, YB2 / 0 cells may be preferred for some therapeutic antibodies.

[0297] In one embodiment, the host cells may include T cells such as CD4+ T cells, CD8+ T cells, γδ T cells, and / or natural killer T cells.

[0298] In another embodiment, the host cells may include natural killer (NK) cells, dendritic cells, macrophages, and / or cancer cells.

[0299] In another embodiment, the host cells do not need to contain NK cells.

[0300] In another embodiment, the host cells do not have to include cancer cells.

[0301] In particular, for the expression of therapeutic proteins such as dimeric therapeutic proteins, the expression vector may be of the type in which the gene (chain) encoding one polypeptide and the gene (chain) encoding the other polypeptide are located on separate vectors, or of the type in which both genes are located on the same vector (tandem type). Tandem expression vectors are preferred in terms of ease of construction of the antibody or TCR expression vector, ease of introduction into animal cells, and balance of the expression levels of antibody H chains and L chains, and α chains and β chains in animal cells (Shitara K et al. J Immunol Methods. 1994 Jan. 3; 167(1-2): 271-8).

[0302] For the manufacture of influenza vaccines, cell lines such as HEK293 or CHO;VSV-G, ampho, Mokola, and Paramyxoviridae, along with their corresponding envelope proteins, are preferred (see, for example, ncbi.nlm.nih.gov / ICTVdb / Ictv / fs_param.htm).

[0303] For example, any suitable or desired heterologous sequences may be expressed, including vaccines, interferons (α, β, γ, ε), erythropoietin, factor VIII, coagulation factors, antibodies and their fragments (e.g., including single-chain, Fab, and humanized), insulin, chemokines, cytokines, growth factors, angiogenesis regulators, apoptosis regulators, and so on. Single-chain antibodies (e.g., single-chain variable fragments or "scFv") can be produced routinely.

[0304] In certain embodiments of the present invention, an antigenic preparation for use as a vaccine may be prepared using a lentiviral transduction vector. Any suitable antigen according to the present invention may be prepared, including antigens obtained from prions, viruses, Mycobacterium, protozoa (e.g., Plasmodium falciparum (malaria)), Trypanosoma, bacteria (e.g., Streptococcus, Neisseria, etc.).

[0305] Host cells can be transduced with a single lentiviral vector containing one or more heterologous NOIs, or with multiple lentiviral vectors, each containing the same or different heterologous NOIs. For example, a multi-subunit antigen (including intracellular and cell surface multi-subunit components) can be prepared by expressing individual subunits on separate vectors, but all vectors are used to infect the same host cells so that assembly occurs within the host cells.

[0306] Vaccines often contain multiple antigenic components, for example, derived from different proteins and / or different epitope regions of the same protein. For instance, a vaccine against a viral disease may consist of one or more polypeptide sequences derived from a virus that, when administered to a host, induce an immunogenic or protective response to viral challenge.

[0307] As mentioned, the present invention can also be used to prepare polypeptide multimers, for example, to produce antigen preparations composed of two or more polypeptides. For example, a viral capsid may be composed of two or more polypeptide subunits. By transducing host cells using vectors possessing different viral envelope sequences, proteins can self-assemble into three-dimensional structures containing two or more protein subunits (e.g., in their native configuration) when expressed in cells. The structures may possess functional activities, including antigenic activity, enzymatic activity, and cell-binding activity. Furthermore, when expressed in a suitable cell line, they are secreted into the cell culture medium and are easily purified. For example, when influenza N and H capsid proteins, and optionally M protein (see below), are introduced into a producing cell line using a lentiviral transduction vector, empty capsids or virus-like particles (VLPs) are formed in cells and then secreted into the culture medium. Such VLPs can be routinely isolated and purified and administered as influenza vaccines. VLPs are, for example, self-assembling capsids that contain virtually no viral RNA (e.g., are empty). VLPs can preferably evoke an effective immune response to provide at least some degree of protection against challenge by naturally occurring infectious viral particles, or at least to induce antibodies against them.

[0308] Currently, there are many available viral vaccines, including those for diseases such as measles, mumps, hepatitis (types A and B), rubella, influenza, polio, smallpox, varicella, adenovirus, Japanese encephalitis, rabies, and Ebola. The present invention may be used for the preparation of vaccines for any of the above diseases.

[0309] Examples of viruses for which a vaccine can be produced according to the present invention include, for example, orthomyxoviruses, influenza A (including all strains with different HA and NA proteins, such as H1N1, H1N2, H2N2, H3N2, H7N7, and H3N8, in non-limiting examples); influenza B, influenza C, Togotovirus (including Dori, Batken virus, and SiAR126 virus); and Isavirus (e.g., infectious salmon anemia virus). These include influenza isolated or infected from all species types, including isolated strains from invertebrates, vertebrates, mammals, humans, non-human primates, monkeys, pigs, cattle, and other livestock, birds, poultry such as turkeys, chickens, quail, and ducks, wild birds (including waterfowl and terrestrial birds), and reptiles. These also include existing strains that have been altered, for example, through mutation, antigen drift, antigen shift, recombination, etc., particularly strains with increased pathogenicity and / or interspecies transmission (e.g., human to human).

[0310] Of particular interest are panzoan and / or transspecies influenza viruses, due to their broad host range, recombination in infected hosts, and / or spontaneous or directional mutations. For example, H5N1 (based on the subtype of surface antigens present on the virus, hemagglutinin type 5 and neuraminidase type 1) is a subtype of avian influenza A that caused a major influenza epidemic in poultry in Asia. As of November 2005, more than 120 million birds had died from the infection or been culled to prevent further spread. This virus also infects humans ("avian influenza") and is associated with a high mortality rate.

[0311] Influenza antigenic preparations (such as vaccines) may contain one or more polypeptides that spontaneously occur in the influenza virion. However, they preferably do not contain all polypeptide genes that would give rise to the naturally occurring pathogenic virus. These include, for example, hemagglutinin (encoded by the HA gene), neuraminidase (encoded by the NA gene), nucleoprotein (encoded by the NA gene), matrix (M1) protein (encoded by the M gene), M2 (encoded by the M gene), non-structural protein (encoded by the NS gene), and polymerase. Naturally occurring virions are covered by a lipid bilayer in which endogenous proteins H and N ("capsid layer") are "interspersed." Matrix protein (M1) forms a protein layer ("matrix layer") beneath the viral membrane and is involved in the construction, stability, and integrity of the virus. See, for example, Harris et al., Virol. 289:34-44, 2001. The M2 protein is a membrane protein ion channel. The VLP of the present invention may comprise H, N, and optionally M1 and M2 proteins. The sequences of the said proteins are known in the art and / or can be identified in GenBank. For the M1 and M2 sequences, see, for example, Widjaja et al. J. Virol., 78:8771-8779, 2004.

[0312] These can be cloned into transfer vectors individually or on the same plasmid and used to produce transduction vectors. In one embodiment of the present invention, multiple transduction vectors can be prepared, each containing a unique influenza gene sequence (e.g., encoding H, N, and M1, resulting in three different transduction vectors). When such vectors are co-expressed in the same host cell (e.g., CHO or HEK293), self-organizing VLPs are generated, secreted into the culture medium, recovered by centrifugation, and then administered as a vaccine.

[0313] The transduction vectors of the present invention can result in high levels of heterologous protein production, for example, about 0.1 to 0.3 mg / ml to about 5 to 10 mg / ml or more of recombinant heterologous protein per ml of untreated culture medium, when such proteins are secreted into the culture medium.

[0314] This application also provides a method for producing antibodies. For example, a method for producing monoclonal antibodies (e.g., human, mouse, and other mammalian types) without the need for hybridomas or animal models is provided. In one non-limiting example, a lentiviral vector expressing an oncogenic protein is transduced into peripheral blood B cells of mice pre-stimulated with the antigen. These vectors efficiently transduce the mouse cells into antibody-producing cells. In a second non-limiting example, two lentiviral vectors are manipulated, one expressing a heavy antibody chain and the second vector expressing a light antibody chain. The invariant region of the gene is derived from a human (or other species as needed) immunoglobulin gene (e.g., IgG, IgM, or other types of Ig). The variable region of the gene is modified or degenerated to produce diversity. The degenerate sequence can be obtained by any suitable technique known in the art and cloned into a lentiviral vector to create a library of lentiviral vectors expressing either heavy immunoglobulin molecules or light immunoglobulin molecules. Antibodies can be produced by transducing both vectors into cells and producing functional antibodies containing both heavy and light chains. Transduced expression cells can be selected and screened for binding to the antigen, and then positive clones can be isolated and subjected to multiple affinity maturation cycles.

[0315] The advantage of this method is that antibodies are produced in an unbiased manner. Other methods, such as conventional hybridoma and xenomouse techniques, rely on B cells that have undergone clonal selection and deletion of specific antibody clones, as they react with endogenous, for example, mouse tissue. Some of these deleted clones may have value as antibodies because they can cross-react with human antigens. The advantage of the described method is that there are no deletions of molecular antibody clones, they are all analyzed in an unbiased manner, and they are also fully humanized (where humanization is desired) antibody molecules. Another advantage of lentiviral vectors is that genes are transduced into cells with high multiplicity, and various types of antibodies can be produced in a single cell. This reduces the number of cells that need to be generated to create a library containing a very diverse range of antigen-binding sites. A second advantage is that additional diversity can be generated by transducing cells with a higher infection multiplicity than 1 by placing heavy and light genes into different lentiviral vectors. For example, if an MOI of 10 is used to transduce cells with lentiviral vectors expressing each heavy and light chain, the number of antibody combinations produced by each cell will be 100. Therefore, in a 96-well plate with approximately 10,000 cells per well, the number of possible mutants that can be produced by this method is 1 million per well of the 96-well plate. Thus, by scaling up this method, a large number of antibody mutants can be produced. The method is not limited to using an MOI of 10 per cell for each construct; higher MOIs can also be used as needed. For example, if an MOI of 100 is used, each cell may produce 10,000 mutant antibodies, and each well of a 96-well plate may generate 10 billion mutants. Thus, each 96-well plate can generate 1 × 10⁶ 12Numerous mutant antibody molecules can be generated, which can be used for screening against target antigens using many methods known in the art (e.g., ELISA). Once specific wells producing the desired antibody response are identified, the cells are subjected to limiting dilution, and cell clones expressing the correct antibody can be found. Once this clone is identified, PCR can be used to clone a vector expressing the heavy and light chains of the antibody. The vector DNA can then be transfused using a helper construct to produce the vector. Alternatively, this cell clone can be directly transfused using a helper construct (PEI, calcium phosphate, lipotransfection, or other transfusion methods known in the art) to produce a mutant lentiviral vector. The resulting vector is then titered and transfused into cells with a low MOI but high number, and clones producing the desired antibody are isolated. Once cell clones are isolated, antibodies can be produced with higher titers by transducing the cells with a higher infection multiplicity. This method is not limited to whole antibody molecules but can also be applied to single-chain antibodies, antibody fragments, phage displays, and other antibody-like molecules, all of which are publicly known in the art. In addition to antibody expression, vectors can express other genes to increase the production of monoclonal antibodies or increase their yield. Such genes may be oncogenes such as ras and myc, but other genes such as anti-apoptotic genes such as Bcl-2 can also be used. Furthermore, monoclonal antibodies can be produced from B cells in the blood of animals exposed to an antigen using such vectors. For example, B cells from mice exposed to an antigen can be transformed into myeloma cells using a combination of oncogenes or gene-silencing RNA.Examples of such genes include growth factors, such as amphiregulin, B-lymphocyte stimulating factor, interleukin-16 (IL-16), thymopoietin, TRAIL, Apo-2, pre-B cell colony enhancing factor, endothelial differentiation-related factor 1 (EDF-1), endothelial monocyte-activating polypeptide II, macrophage migration inhibitor MIF, natural killer cell enhancing factor (NKEFA), osteomorphogenetic protein 8 (osteogenesis protein 2), osteomorphogenetic protein 6, connective tissue growth factor (CTGF), and CGI-149 protein (neuronomorphogenetic protein). (Distribution factors), cytokine A3 (macrophage inflammatory protein 1-α), glioblastoma cell differentiation-related protein (GBDR1), hepatocellular carcinoma-derived growth factor, neuromedin U-25 precursor, any oncogene, oncogene, proto-oncogene or cell regulatory gene (may be found at condor.bcm.tmc.edu / oncogene), vascular endothelial growth factor (VEGF), vascular endothelial growth factor B (VEGF-B), T cell-specific RANTES precursor, thymic dendritic cell-derived factor 1; type II activin A receptor (ACVR) 2) Receptors such as β-signal sequence receptor (SSR2), CD14 monocyte LPS receptor, CD36 (Type I collagen / thrombospondin receptor)-like receptor 2, CD44R (Hermes antigen gp90 homing receptor), G protein-coupled receptor 9, chemokine CxC receptor 4, colony-stimulating factor 2 receptor β (CSF2RB), FLT-3 receptor tyrosine kinase, transient receptor potential C precursor, killer cell lectin-like receptor subfamily B, low-density lipoprotein receptor gene, low-affinity Fc-gamma receptor IIC, MCP-1 Examples include receptors, monocyte chemotactic protein 1 receptor (CCR2); nuclear receptor subfamily 4, group A, member 1; orphan G protein-coupled receptor GPRC5D; peroxisome proliferation-activated receptor γ pheromone-related receptor (rat); vasopressin-activated calcium mobilization putative receptor; retinoin x receptor; Toll-like receptor 6; transmembrane activator and CAML interactor (TACI); B cell maturation peptide (BCMA); CSF-1 receptor; and interferon (α, β, and γ) receptor 1 (IFNAR1).

[0316] treatment The vectors provided herein can be used in a wide variety of therapeutic applications.

[0317] In some embodiments, lentiviral vectors for therapeutic use are provided that express a native or fusion polypeptide comprising any individual human chemokine and a viral or bacterial antigen (e.g., HIV, diphtheria toxin antigen), a chemokine (e.g., IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP1, RANTES, SDF-1, MIG and / or MDC), or an apoptosis-promoting protein, a suicide gene protein, or a protein that promotes an inflammatory response, or a combination thereof.

[0318] Furthermore, the present invention provides a method for inducing an immune response in a subject, comprising administering to a subject any of the individual or fusion polypeptides of the present invention, such as chemokines and human immunodeficiency virus (HIV) antigens, or chemokines, pro-apoptotic genes, suicide genes, and tumor antigens, as either proteins or nucleic acids encoding individual or fusion polypeptides expressed from a lentiviral vector. Also provided is a method for treating cancer in a subject, comprising administering to a subject a lentiviral vector expressing any of the individual or fusion polypeptides of the present invention, such as chemokines and tumor antigens, as either proteins or nucleic acids encoding fusion polypeptides.

[0319] Further provided are methods for treating or preventing HIV infection in a subject, comprising administering to the subject any combination of the following peptides derived from the following proteins: chemokines, suicide genes, HIV proteins, cytokines, cell surface proteins, tumor antigens, or any cellular genes that affect the production of HIV from cells (either by overexpression of the cellular gene or inhibition of its expression by RNAi), all supplied and expressed from lentiviral vectors.

[0320] In some embodiments, compositions containing engineered immune cells, such as T cells (e.g., γδT cells), as described herein may be administered for prophylactic and / or therapeutic purposes. In therapeutic use, the pharmaceutical composition may be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially prevent the symptoms of the disease or condition. Engineered immune cells may also be administered to reduce the likelihood of onset, illness, or exacerbation of the condition. The effective dose of a population of engineered immune cells for therapeutic use may vary based on the severity and course of the disease or condition, previous treatments, the subject's health status, weight, and / or response to the drug, and / or the judgment of the treating physician.

[0321] Using the manipulated T cells and other immune cells described herein, subjects requiring treatment for medical conditions such as cancer as described herein may be treated.

[0322] A method of treating a disease (e.g., illness) in a subject using engineered immune cells, such as engineered T cells, may include administering a therapeutically effective amount of engineered immune cells, such as engineered T cells, to the subject. Engineered immune cells, such as engineered T cells, as disclosed herein may be administered in various regimens (e.g., timing, concentration, dose, treatment interval, and / or formulation). The subject may also be pre-conditioned, for example, with chemotherapy, radiation, or a combination of both, before being administered engineered immune cells, such as engineered T cells, as disclosed herein. Populations of engineered immune cells, such as engineered T cells, may also be frozen or cryopreserved before being administered to the subject. Populations of engineered immune cells, such as engineered T cells, may include two or more cells expressing identical, different, or identical and different tumor-recognizing moieties. For example, populations of engineered immune cells, such as engineered T cells, may include several different engineered immune cells, such as engineered T cells, designed to recognize different antigens or different epitopes of the same antigen.

[0323] Manipulated immune cells, such as manipulated T cells, may be used to treat a variety of medical conditions. In one embodiment, manipulated immune cells, such as manipulated T cells, may be used to treat cancers, including solid tumors and hematological malignancies.Non-specific examples of cancer include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, neuroblastoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer; brain tumors such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primordial neuroectodermal tumor, visual tract and hypothalamic glioma; breast cancer, bronchial adenoma, Burkitt lymphoma, cancer of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, and chronic lymphocytic leukemia. Chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, fibroplastic round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer including non-small cell and small cell lung cancer, lymphoma, leukemia Macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin, oral cancer, multiple endocrine adenoma syndrome, myelodysplastic syndrome, myeloid leukemia, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell cancer, paranasal and nasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal These include gastroblastoma, pituitary adenoma, pleuroblastoma, plasma cell neoplasm, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, Merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, pharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, gestational trophoblastoma, cancer of unknown primary origin, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0324] In one embodiment, an infectious disease may be treated using engineered immune cells, such as engineered T cells, according to the Disclosure. In another embodiment, an infectious disease may be treated using engineered immune cells, such as engineered T cells, according to the Disclosure, and the infectious disease may be caused by a virus. In yet another embodiment, an immune disease, such as an autoimmune disease, may be treated using engineered immune cells, such as engineered T cells, according to the Disclosure.

[0325] Therapies with engineered immune cells, such as engineered T cells, as disclosed herein may be provided to subjects before, during, and after the clinical onset of a disease. Therapies may be provided to subjects one day, one week, six months, twelve months, or two years after the clinical onset. Therapies may be provided to subjects for more than one day, one week, one month, six months, twelve months, two years, three years, four years, five years, six years, seven years, eight years, nine years, or ten years after the clinical onset of the disease. Therapies may also include treating humans in clinical trials. Therapies may include administering to subjects a pharmaceutical composition comprising engineered immune cells, such as engineered T cells, as disclosed herein.

[0326] In another embodiment, administration of engineered immune cells, such as engineered T cells of the Disclosure, to a subject may modulate the activity of endogenous lymphocytes in the subject's body. In another embodiment, administration of engineered immune cells, such as engineered T cells, to a subject may provide antigens to endogenous T cells and enhance the immune response. In another embodiment, the memory T cells may be CD4+ T cells. In another embodiment, the memory T cells may be CD8+ T cells. In another embodiment, administration of modified immune cells, such as modified T cells of the Disclosure, to a subject may activate the cytotoxicity of other immune cells. In another embodiment, the other immune cells may be CD8+ T cells. In another embodiment, the other immune cells may be natural killer T cells. In another embodiment, administration of engineered immune cells, such as engineered T cells of the Disclosure, to a subject may suppress regulatory T cells. In another embodiment, the regulatory T cells may be FOX3+ Treg cells. In another embodiment, the regulatory T cells may be FOX3-Treg cells. Non-limiting examples of cells whose activity can be regulated by manipulated immune cells such as manipulated T cells in this disclosure include hematopoietic stem cells; B cells; CD4; CD8; erythrocytes; white blood cells; dendritic cells, including dendritic antigen-presenting cells; leukocytes; macrophages; memory B cells; memory T cells; monocytes; natural killer cells; neutrophil granulocytes; T helper cells; and T killer cells.

[0327] During most bone marrow transplants, a combination of cyclophosphamide and total body irradiation may be conventionally used to prevent rejection of hematopoietic stem cells (HSCs) by the target immune system during transplantation. In one embodiment, in vitro incubation of donor bone marrow with interleukin-2 (IL-2) may be performed to enhance the production of killer lymphocytes in the donor bone marrow. Interleukin-2 (IL-2) is a cytokine that may be necessary for the growth, proliferation, and differentiation of wild-type lymphocytes. Current research on adoptive immunization of γδT cells to humans may require co-administration of γδT cells and interleukin-2. However, both low and high doses of IL-2 can have highly toxic side effects. IL-2 toxicity can manifest in multiple organs / systems, most notably the heart, lungs, kidneys, and central nervous system. In another embodiment, the disclosure provides a method for administering engineered γδT cells to a subject without co-administration of naive cytokines or modified versions thereof such as IL-2, IL-15, IL-12, or IL-21. In another embodiment, engineered γδT cells may be administered to a subject without co-administration with IL-2. In another embodiment, engineered γδT cells may be administered to a subject during a procedure such as bone marrow transplantation without co-administration with IL-2.

[0328] Method of administration One or more populations of engineered immune cells, such as engineered T cells, may be administered to a subject in any order or simultaneously. If administered simultaneously, multiple engineered immune cells, such as engineered T cells, may be provided in a single, unified form, such as intravenous injection, or in multiple forms, such as multiple intravenous infusions, subcutaneous injections, injections, or pills. Engineered immune cells, such as engineered T cells, may be packaged together or separately in a single package or multiple packages. Engineered immune cells, such as engineered T cells, or all of them, may be administered multiple times. If not administered simultaneously, the timing of multiple doses may vary, such as approximately one week, one month, two months, three months, four months, five months, six months, or approximately one year. In another embodiment, engineered immune cells, such as engineered T cells, may proliferate in vivo within the subject's body after administration to the subject. Engineered immune cells, such as engineered T cells, may be frozen, and multiple therapeutic cells may be provided in the same cell preparation. The engineered immune cells, such as engineered T cells, and pharmaceutical compositions comprising them, as disclosed herein, may be packaged as kits. The kits may include instructions (e.g., written instructions) on the use of the engineered immune cells, such as engineered T cells, and compositions comprising them.

[0329] In another embodiment, a method for treating cancer comprises administering a therapeutically effective dose of engineered immune cells, such as engineered T cells, to a target, the administration of which treats cancer. In another embodiment, the therapeutically effective dose of engineered immune cells, such as engineered T cells, may be administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In another embodiment, the therapeutically effective dose of engineered immune cells, such as engineered T cells, may be administered for at least 1 week. In another embodiment, the therapeutically effective dose of engineered immune cells, such as engineered T cells, may be administered for at least 1 week.

[0330] The engineered immune cells, such as engineered T cells, described herein may be administered before, during, or after the onset of a disease or condition, and the timing of administration of a pharmaceutical composition containing engineered immune cells, such as engineered T cells, may vary. For example, engineered immune cells, such as engineered T cells, may be used prophylactically and administered sequentially to subjects with a condition or predisposition to reduce the likelihood of the onset of a disease or condition. Engineered immune cells, such as engineered T cells, may be administered to a subject during or as soon as possible after the onset of symptoms. Administration of engineered immune cells, such as engineered T cells, may be initiated immediately after the onset of symptoms, within the first three hours of symptom onset, within the first six hours of symptom onset, within the first 24 hours of symptom onset, within 48 hours of symptom onset, or within any period after symptom onset. The initial administration may be via any practical route, such as any route described herein, using any formulation described herein. In another embodiment, the administration of engineered immune cells, such as engineered T cells, of the Disclosed may be intravenous. One or more doses of immune cells, such as engineered T cells, may be administered as soon as possible after the onset of cancer, infectious disease, immune disorder, or sepsis, or via bone marrow transplantation, for periods necessary to treat the immune disorder, such as approximately 24 to 48 hours, approximately 48 hours to approximately 1 week, approximately 1 week to approximately 2 weeks, approximately 2 weeks to approximately 1 month, or approximately 1 month to approximately 3 months. For the treatment of cancer, one or more doses of immune cells, such as engineered T cells, may be administered several years after the onset of cancer, before or after other treatments.In another embodiment, engineered immune cells, such as engineered T cells, may be administered for at least approximately 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The duration of treatment may vary depending on the subject.

[0331] keep In one embodiment, engineered immune cells, such as engineered T cells, may be prepared in a freezing medium and placed in a cryogenic storage unit such as a liquid nitrogen freezer (-196°C) or an ultra-low temperature freezer (-65°C, -80°C, -120°C, or -150°C) for long-term storage for at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The freezing medium may contain dimethyl sulfoxide (DMSO) and / or sodium chloride (NaCl) and / or dextrose and / or dextran sulfate and / or hydroxyethyl starch (HES), which have physiological pH buffers to maintain the pH between about 6.0 and about 6.5, about 6.5 and about 7.0, about 7.0 and about 7.5, about 7.5 and about 8.0, or about 6.5 and about 7.5. Cryopreserved engineered immune cells, such as engineered T cells, may be thawed and further treated by stimulation with antibodies, proteins, peptides, and / or cytokines as described herein. Cryopreserved immune cells, such as T cells, may be thawed and genetically modified with viral vectors (including retroviruses, adeno-associated viruses (AAAV), and lentiviral vectors) or nonviral means (including RNA, DNA such as transposons, and proteins) as described herein. Modified immune cells, such as modified T cells, may be further cryopreserved at a rate of at least about 10 per 1 mL of freezing medium. 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or at least about 10 10Cell banks can be prepared using cells in quantities of at least approximately 1, 5, 10, 100, 150, 200, or 500 vials. Cryopreserved cell banks may retain their function and can be thawed, further stimulated, and grown. In another embodiment, thawed cells may be stimulated and grown in a suitable closed container such as a cell culture bag and / or bioreactor to produce a certain amount of cells as an allogeneic cell product. Cryopreserved immune cells, such as T cells, can retain their biological function under cryogenic storage conditions for at least approximately 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 24, 30, 36, 40, 50 months, or at least approximately 60 months. In another embodiment, preservatives cannot be used in the formulation. Cryopreserved immune cells, such as T cells, can be thawed and administered as a ready-made, allogeneic cell product to multiple patients.

[0332] In one embodiment, the manipulated immune cells, such as the manipulated T cells described herein, are at least 1 × 10⁻⁶ 3 Cells / ml, at least 2 × 10⁶ 3 Cells / ml, at least 3 × 10⁶ 3 Cells / ml, at least 4 × 10⁶ 3 Cells / ml, at least 5 × 10 3 Cells / ml, at least 6 × 10⁶ 3 Cells / ml, at least 7 × 10⁶ 3 Cells / ml, at least 8 × 10 3 cells / ml, at least 9 × 10³ cells / ml, at least 1 × 10⁴ cells / ml, at least 2 × 10 4 Cells / ml, at least 3 × 10⁶ 4 Cells / ml, at least 4 × 10⁶ 4 Cells / ml, at least 5 × 10 4 Cells / ml, at least 6 × 10⁶ 4 Cells / ml, at least 7 × 10⁶ 4 Cells / ml, at least 8 × 10 4 Cells / ml, at least 9 × 10 4 Cells / ml, at least 1 × 10⁶ 5Cells / ml, at least 2 × 10⁶ 5 Cells / ml, at least 3 × 10⁶ 5 Cells / ml, at least 4 × 10⁶ 5 Cells / ml, at least 5 × 10 5 Cells / ml, at least 6 × 10⁶ 5 Cells / ml, at least 7 × 10⁶ 5 Cells / ml, at least 8 × 10 5 Cells / ml, at least 9 × 10 5 Cells / ml, at least 1 × 10⁶ 6 Cells / ml, at least 2 × 10⁶ 6 Cells / ml, at least 3 × 10⁶ 6 Cells / ml, at least 4 × 10⁶ 6 Cells / ml, at least 5 × 10 6 Cells / ml, at least 6 × 10⁶ 6 Cells / ml, at least 7 × 10⁶ 6 Cells / ml, at least 8 × 10 6 Cells / ml, at least 9 × 10 6 Cells / ml, at least 1 × 10⁶ 7 Cells / ml, at least 2 × 10⁶ 7 Cells / ml, at least 3 × 10⁶ 7 Cells / ml, at least 4 × 10⁶ 7 Cells / ml, at least 5 × 10 7 Cells / ml, at least 6 × 10⁶ 7 Cells / ml, at least 7 × 10⁶ 7 Cells / ml, at least 8 × 10 7 Cells / ml, at least 9 × 10 7 Cells / ml, at least 1 × 10⁶ 8 Cells / ml, at least 2 × 10⁶ 8 Cells / ml, at least 3 × 10⁶ 8 Cells / ml, at least 4 × 10⁶ 8 Cells / ml, at least 5 × 10 8 Cells / ml, at least 6 × 10⁶ 8 Cells / ml, at least 7 × 10⁶ 8 Cells / ml, at least 8 × 10 8 Cells / ml, at least 9 × 10 8 Cells / ml, at least 1 × 10⁶9 cells / ml or more, about 1×10 3 cells / ml to at least about 1×10 8 cells / ml, about 1×10 5 cells / ml to at least about 1×10 8 cells / ml, or about 1×10 6 cells / ml to at least about 1×10 8 It may be present in the composition in an amount of cells / ml.

[0333] In one aspect, using the methods described herein, autologous or allogeneic products according to aspects of the present disclosure may be produced.

Examples

[0334] Example 1

[0335]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

[0336] [Table 4-1] [Table 4-2]

[0337] Example 2 Creation of WPRE mutants The wild-type WPRE sequence is used in the lentiviral construct to stabilize and enhance gene transcription. Several reports have concluded that the protein (X protein) contained in WPRE may induce tumorigenesis, leading the U.S. FDA to recommend finding alternatives to using wild-type WPRE for lentiviral constructs used in clinical trials of gene and cell therapies. We believe that by meeting the FDA requirements, our T-cell product can be used in clinical trials and potentially avoid safety concerns in several aspects of lentiviral vector design.

[0338] In an attempt to develop WPRE mutants that do not express the functional X protein while maintaining WPRE-mediated post-transcriptional enhancement of gene expression, we investigated two separate WPRE mutation strategies.

[0339] In this process, we developed one mutant in which both the promoter region and the start codon of the X protein were mutated (SEQ ID NO: 4).

[0340] In this process, we developed another variant in which the X protein promoter and the complete putative sequence are deleted along with any ORF mutation start codon greater than 25aa within the WPRE (SEQ ID NO: 3).

[0341] Example 3 Lentivirus construct A schematic diagram of the expression cassette used in this specification is provided in Figure 3.

[0342] Figure 4 provides a description of the cassette used in the lentiviral construct, which will be used in the experiments detailed below to investigate the efficacy of the WPRE variant.

[0343] The lentiviral vectors used herein include a central polyprint lactate (cPPT) to improve self-renewal and nuclear delivery, a promoter from mouse stem cell virus (MSCV) (SEQIDNO:94) which has been shown to reduce vector silencing in several cell types, and several elements previously shown to enhance vector function, including a deletion-type 3'-LTR self-inactivation (SIN) vector design in which the backbone may improve safety, sustained gene expression, and anti-silencing properties (the entire content of which is referenced by Yang et al. Gene Therapy (2008) 15, 1411-1423).

[0344] The lentiviral vectors used herein encode both the TCRα and TCRβ chains. In particular, the vectors used herein encode the R4P3F9α and β chains (SEQ ID NOs. 25 and 26) and their variants. Vectors described herein beginning with the abbreviation "R4" encode the wild-type R4P3F9α and β chains (SEQ ID NOs. 25 and 26); vectors beginning with the abbreviation "R4-B4" encode the wild-type R4P3F9α chain (SEQ ID NOs. 25) and the variant R4P3F9β chain (SEQ ID NOs. 92); and vectors beginning with the abbreviation "R4-A1B4" encode the variant R4P3F9α chain (SEQ ID NOs. 91) and the variant R4P3F9β chain (SEQ ID NOs. 92) (Figure 4).

[0345] For each of the above TCRαβ dimers, four separate WPRE variations were tested. "Mutant A" is the wild-type WPRE described in SEQ ID NO: 2 (positive control); "Mutant B" does not contain WPRE (negative control); "Mutant C" contains the mutant WPRE described in SEQ ID NO: 4, in which the X protein promoter and start codon are mutated; and "Mutant D" contains the mutant WPRE described in SEQ ID NO: 3, in which the start codon located throughout the WPRE sequence is mutated and the X protein promoter and ORF are deleted.

[0346] Example 4 The effect of WPRE mutations on the efficacy of lentiviral constructs in T cells T cells were obtained from a donor on day 0, activated on day 1, transduced on day 2 with various lentiviral vectors as described in Example 3 above, and collected for testing on day 6. TCR surface expression was determined by flow cytometry, and vector copy number was determined by qPCR.

[0347] Figure 5 shows the HEK-293T titers obtained after transduction using lentiviral constructs according to several embodiments of the present disclosure. The titers obtained using lentiviral constructs containing mutant WPRE (LV-C and LV-D) were similar to those obtained using lentiviral constructs containing wild-type (WT) WPRE (LV-A).

[0348] Figure 6 shows the expression of TCR on the surface of CD8+ cells 6 days after transduction with the R4-B4 lentiviral construct according to several embodiments of this disclosure. Expression was detected by tetramers using lentiviral titer in two separate donors. Panel A is donor #1, and panel B is donor #2. Logarithmic viral dilution factors are presented along the x-axis. Surprisingly, CD8+ cells transduced with lentiviral constructs containing mutant WPRE (mutants C and D) showed higher TCR expression compared to cells transduced with lentiviral constructs containing either WT WPRE (mutant A) or no WPRE (mutant B).

[0349] Figure 7 shows the expression of TCR on the surface of CD8+ cells 4 days after transduction with the R4-A1B4 lentiviral construct according to several embodiments of this disclosure. Expression was detected by tetramers using lentiviral titer in two separate donors. Panel A is donor #1, and panel B is donor #2. Logarithmic viral dilution factors are presented along the x-axis. Similar to the results for the R4-B4 vector shown in Figure 6, TCR expression was highest in CD8+ cells transductioned with the lentiviral construct containing variant D (mutant WPRE described in SEQ ID NO: 3).

[0350] Figure 8 shows TCR expression on the surface of CD8+ cells (A) or CD4+ cells (B) four days after transduction with the R4-B4 lentiviral construct according to several embodiments of the present disclosure. Expression was detected by tetramers using lentiviral titration. Logarithmic viral dilution factors are presented along the X axis. These results further demonstrate that TCR expression was higher in both CD8+ and CD4+ cells transduced with lentiviral constructs containing mutant WPRE (mutant C and D) compared to cells transduced with lentiviral constructs containing either WT WPRE (mutant A) or no WPRE (mutant B).

[0351] Figure 9 shows TCR expression on the surface of CD4+ cells (A) or CD4+ cells (B) four days after transduction with the R4-A1B4 lentiviral construct according to several embodiments of the present disclosure. Expression was detected by tetramers using lentiviral titration. Logarithmic viral dilution factors are presented along the x-axis. Similar to the results shown in Figures 6-8, TCR expression was highest in CD8+ and CD4+ cells transductioned with the lentiviral construct containing mutant D (mutant WPRE described in SEQ ID NO: 3). IMA203 is a lentiviral construct containing WT WPRE that expresses the R11KE TCR and is used as a negative control.

[0352] The replication ratio was not affected by the WPRE mutation (Figure 10). Cell viability exceeded 90% in all lentiviral constructs tested at the optimal MOI (data not shown).

[0353] WPRE variants exhibit equivalent TCR tetramer surface expression normalized to vector copy number (Figure 11) or viral titer (Figure 12).

[0354] Similarly, Figure 13 shows that the WPRE mutant exhibits equivalent TCR tetramer surface expression, as determined by flow cytometry. Panel A shows CD4-CD8+ / tetramer+ data. Panel B shows CD4+CD8- / tetramer+ data.

[0355] Figure 14 shows cytokine production by CD4+ or CD8+ T cells in the presence of target-positive tumor cells. Panel A shows interferon-γ (IFN-γ) production in CD8+ T cells. Panel B shows IFN-γ production in CD4+ T cells. Panel C shows tumor necrosis factor-α (TNF-α) production in CD8+ T cells. Panel D shows TNF-α production in CD4+ T cells. MCF7=negative; SW982=460CpC.

[0356] Example 5 γδT cell production To isolate γδT cells, in one embodiment, γδT cells may be isolated from a subject or from a composite sample of the subject. In one embodiment, the composite sample may be a peripheral blood sample, umbilical cord blood sample, tumor, stem cell precursor, tumor biopsy, tissue, lymph, or from an epithelial site of the subject in direct contact with the external environment, or derived from stem precursor cells. γδT cells may be isolated directly from the composite sample of the subject, for example, by sorting γδT cells expressing one or more cell surface markers using flow cytometry techniques. Wild-type γδT cells may exhibit a number of antigen-recognizing, antigen-presenting, costimulatory, and adhesion molecules that may be associated with γδT cells. Wild-type γδT cells may be isolated from the composite sample using one or more cell surface markers, such as a specific γδTCR, antigen-recognizing, antigen-presenting, ligand, adhesion molecule, or costimulatory molecule. γδT cells may be isolated from a composite sample, for example, by using various molecules associated with or expressed by γδT cells, such as the isolation of a mixed population of Vδ1, Vδ2, Vδ3 cells or any combination thereof.

[0357] For example, PBMCs can be collected from a subject using an apheresis therapy device, such as the Ficol-Paque® PLUS (GE Healthcare) system, or another suitable device / system. γδT cells or a desired subpopulation of γδT cells can be purified from the collected sample, for example, by flow cytometry. Umbilical cord blood cells can also be obtained from umbilical cord blood at the time of the subject's birth.

[0358] By using positive and / or negative selection of cell surface markers expressed on collected γδT cells, γδT cells or γδT cell populations expressing similar cell surface markers can be directly isolated from peripheral blood samples, umbilical cord blood samples, tumors, tumor biopsies, tissues, lymphoids, or epithelial samples of interest. For example, γδT cells can be isolated from composite samples based on the positive or negative expression of CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCRα, TCRβ, TCRα, TCRδ, NKG2D, CD70, CD27, CD30, CD16, CD337 (NKp30), CD336 (NKp46), OX40, CD46, CCR7, and other appropriate cell surface markers.

[0359] Figure 15 shows the production of γδT cells according to one embodiment of the present disclosure. This step may include collecting or obtaining leukocytes or PBMCs from a leukocyte apheresis product. Leukocyte apheresis may include collecting whole blood from a donor and separating components using an apheresis device. The apheresis device separates the desired blood components, and the remainder is returned to the donor's circulation. For example, white blood cells, plasma, and platelets may be collected using an apheresis device, and red blood cells and neutrophils may be returned to the donor's circulation. A commercially available leukocyte apheresis product may be used in this step. Another method for obtaining leukocytes is to obtain them from a buffy coat. To isolate the buffy coat, anticoagulated whole blood is obtained from a donor and centrifuged. After centrifugation, the blood is separated into plasma, red blood cells, and a buffy coat. The buffy coat is a layer located between the plasma layer and the red blood cell layer. Leukocyte apheresis may yield higher purity and a significantly increased mononuclear cell content than that achieved by buffy coat apheresis. The mononuclear cell content achievable by leukocyte apheresis can typically be 20 times that obtained from buffy coat. Further separation using a Ficoll gradient may be necessary to enrich mononuclear cells.

[0360] To deplete αβT cells from PBMCs, for example, αβTCR-expressing cells may be isolated from the PBMCs by magnetic separation using CliniMACS® magnetic beads coated with an anti-αβTCR antibody, followed by cryopreservation of the αβTCR-T cell-depleted PBMCs. To produce "off-the-shelf" T cell products, cryopreserved αβTCR-T cell-depleted PBMCs may be thawed in small / medium-scale containers such as 24-4-6 well plates or T75 / T175 flasks, or in large-scale containers such as 50 ml-100 liter bags, and activated over 1-10 days, for example, 2-7 days, in the presence of aminobisphosphonates such as zoledronate; and / or isopentenyl pyrophosphate (IPP); and / or cytokines such as interleukin-2 (IL-2), interleukin-15 (IL-15), and / or interleukin-18 (IL-18); and / or other activators such as Toll-like receptor 2 (TLR2) ligands.

[0361] Figure 15 shows that activated T cells may be manipulated by transducing isolated γδ T cells with viral vectors, such as lentiviral vectors, that express exogenous genes of interest, such as αβTCRs for specific cancer antigens and CD8. Transduction may be performed once or multiple times over a period of 1 / 2 to 5 days, for example, 1 day, on a small scale, such as in 24 to 4-6 well plates, or on a medium / large scale, to achieve stable transgene expression.

[0362] Figure 16 further illustrates that the proliferation of transduced or engineered γδ T cells may be carried out over a period of 7 to 35 days, for example, 7 to 28 days, in small / medium scale, for example, in a flask / G-Rex, or in large scale, for example, in a 50 ml to 100 liter bag, in the presence of cytokines such as IL-2, IL-15, IL-18. The proliferated transduced T cell product may then be cryopreserved as a “ready-made” T cell product for infusion to patients.

[0363] Example 6 Comparison of γδ T cells transduced with lentiviral vectors (LV) containing different WPREs. Figure 17 shows an example of a γδT cell production process comparing γδT cells transduced from LV expressing TCR (SLLQHLIGL (SEQ ID NO: 148) / MHC complex) with CD8 cells having different WPREs. Briefly, on day 0, γδT cells were activated in the presence of zoledronate and cytokines, and then on day 2, 1 × 10⁶ cells were transduced. 6 Cells were transduced with 3.75 μl, 7.50 μl, 15 μl, 30 μl, 60 μl, or 120 μl of LV per cell, expressing wild-type (WT) WPRE (SEQ ID NO: 2) (A), WPRE-free (B), WPREmut1 (SEQ ID NO: 4) (C), or WPREmut2 (SEQ ID NO: 3) (D) TCRs and CD8. The LV titers for batch #1 and batch #2 are shown in Table 5.

[0364] [Table 5]

[0365] Table 5 shows that the LV from batch #1 had approximately 10 times higher titer than the LV from batch #2. Transduced cells were grown on day 3. On day 9, the cells were counted and analyzed by FACS to determine the copy number of TCR / CD8-expressing γδ T cells and the incorporated transgene.

[0366] LV from Batch #1 In FACS analysis, TCR+CD8α+γδT cells were stained using anti-Vβ8 and anti-CD8α antibodies. Figure 18A shows that %Vβ8+CD8α+γδT cells increase with increasing amounts of LV used for transduction. There was no significant difference in transduction efficiency among γδT cells transduced with LV having wild-type (WT) WPRE (A), no WPRE (B), WPREmut1 (C), and WPREmut2 (D). Non-transduced (NT) cells served as a negative control. SLLQHLIGL (SEQ ID NO: 148) / MHC tetramer and anti-CD8α antibody were used to stain TCR+CD8α+γδT cells. Figure 18B shows that %tetramer+CD8α+γδT cells increase with increasing amounts of LV used for transduction. There was no significant difference in transduction efficiency among γδT cells transduced in LVs with wild-type (WT) WPRE (A), no WPRE (B), WPREmut1 (C), and WPREmut2 (D). Non-transduced (NT) cells served as negative controls. Next, transduction efficiency was normalized relative to the efficiency of WT WPRE. For %Vβ8+CD8α+γδT cells (Figure 19A) and %tetramer+CD8α+γδT cells (Figure 19B), there was no significant difference in normalized transduction efficiency among γδT cells transduced in LVs with wild-type (WT) WPRE (A), no WPRE (B), WPREmut1 (C), and WPREmut2 (D). These results indicate that transduction efficiency is equivalent among γδT cells transduced in LVs with WT WPRE, WPREmut1, WPREmut2, and no WPRE.

[0367] Figure 20 shows that the copy number of integrated transgenes in γδT cells generally increases with increasing amount of LV used for transduction. There is no significant difference in the copy number of integrated transgenes among γδT cells transduced with LV containing wild-type (WT) WPRE (A), no WPRE (B), WPREmut1 (C), and WPREmut2 (D). 120 μl / 1 × 10⁻⁶ 6γδT cells transduced with LV(B) without WPRE appear to have a slightly higher number of integrated transgenes than those transduced with LV having a different WPRE. Next, the transduction efficiency / copy number ratio was determined. Figure 21 shows that the %Vβ8+CD8α+ / copy number ratio is equivalent among γδT cells transduced with WT WPRE, WPREmut1, WPREmut2, and LV without WPRE. Similarly, Figure 22 shows that the %tetramer+CD8α+ / copy number ratio is equivalent among γδT cells transduced with WT WPRE, WPREmut1, WPREmut2, and LV without WPRE.

[0368] LV from Batch #2 As shown in Table 5, the LV from batch #1 has a titer approximately 10 times higher than that from batch #2. In general, transduction using LV from batch #2 resulted in lower transduction efficiency than transduction using LV from batch #1, due to the lower titer of the LV. Figure 23 shows 120 μl LV / 1 × 10⁻⁶. 6 The study shows that γδT cells obtained from donors #4 and #5, transduced with LV without WPRE, yielded higher %Vβ8+CD8α+γδT cells (11.7% and 7.91%, respectively) than those transduced with WT WPRE (6.90% and 4.98%, respectively), WPREmut1 (6.01% and 3.71%, respectively), and WPREmut2 (4.67% and 3.60%, respectively).

[0369] Table 6 shows the copy numbers of incorporated genes in γδT cells obtained from donors #4 and #5 transduced with WT WPRE, WPRE-free, WPREmut1, and WPREmut2 LV. Overall, the copy numbers of incorporated transgenes are lower than in batch #1 due to the lower LV titers.

[0370] [Table 6-1]

[0371]

Table 6-2

[0372] Figure 24 shows that the % tetramer + CD8α+ / copy number ratio is equivalent among γδ T cells obtained from donors #4 and #5 and transduced with LV having WT WPRE (A), WPREmut1 (C), WPREmut2 (D), and without WPRE (B) in 120 μl LV / 1×10 6 cells. Figure 25 shows the combined data in 120 μl LV / 1×10 6 cells obtained from donors #3, #4, and #5. These combined results show that the % tetramer + CD8α+ / copy number ratio is equivalent among γδ T cells transduced with LV having WT WPRE (A), WPREmut1 (C), WPREmut2 (D), and without WPRE (B). γδ T cells transduced with LV without WPRE appear to have less variation in the % tetramer + CD8α+ / copy number ratio than those transduced with LV having different WPREs.

[0373] All references mentioned in this specification are incorporated herein by reference as if each reference was specifically and individually indicated to be incorporated by reference. Any citation of appropriate references is for its disclosure prior to the filing date, and the present disclosure should not be construed as admitting any right to anticipate such references by virtue of the nature of the prior inventions.

[0374] It will be understood that useful applications may be found in various other ways, different from those described above, by each or more of the elements described above. Without further analysis, the foregoing fully illustrates the essence of this disclosure, so that others may readily adapt it to various applications by applying their current knowledge without omitting features that fairly constitute the essential features of the general or specific aspects of this disclosure as described in the appended claims, from the perspective of the prior art. The embodiments described above are shown for illustrative purposes only; the scope of this disclosure is limited only by the following claims.

Claims

1. A pharmaceutical agent for the treatment of cancer, comprising as an active ingredient a vector, T cells or a population of T cells transduced by the vector, or a pharmaceutical composition containing the vector or the T cells or population of T cells, The vector comprises a nucleotide sequence encoding a protein and a variant woodchuck post-transcriptional regulatory element (WPRE) of SEQ ID NO: 1, The aforementioned mutant WPR contains mutations in five or six start codons at positions selected from the group consisting of nucleotide positions 106-108, 152-154, 245-247, 272-274, 283-285, and 362-364 of SEQ ID NO: 1, The aforementioned mutant WPR comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 3, The aforementioned mutant WPR does not contain the X protein promoter. The aforementioned mutant WPR does not contain an open reading frame (ORF) of the X protein, and The mutant WPR can enhance the expression of the protein. A drug used to treat cancer.

2. The cancer treatment drug according to claim 1, wherein the five or six start codons are mutated at one, two, or all three positions within the start codon.

3. The cancer treatment drug according to claim 1, wherein the five or six start codons are mutated from ATG to TTG.

4. The cancer treatment pharmaceutical according to any one of claims 1 to 3, wherein the protein is selected from the group consisting of enzymes, cytokines, chemokines, antibodies, engineered immunoglobulin-like molecules, single-chain antibodies, fusion proteins, immune costimulatory molecules, immunomodulatory molecules, transdominant-negative variants of target proteins, toxins, conditional toxins, antigens, antigen receptors, chimeric antigen receptors, T cell receptors (TCRs), tumor suppressor proteins, growth factors, membrane proteins, pro-angiogenic and anti-angiogenic proteins and peptides, vasoactive proteins and peptides, antiviral proteins, and derivatives thereof.

5. The cancer treatment drug according to any one of claims 1 to 4, wherein the vector comprises a first nucleotide sequence S1 encoding protein Z1 and a second nucleotide sequence S2 encoding protein Z2, and Z1 and Z2 form a first dimer.

6. The cancer treatment drug according to claim 5, wherein the first dimer Z1Z2 is a T cell dimer signaling module, a TCR, an antibody, an antigen receptor, or a chimeric antigen receptor.

7. The cancer treatment pharmaceutical according to claim 5, wherein the first dimer Z1Z2 is a TCR that binds to a target antigen (TA) peptide, and the target antigen (TA) peptide is a viral peptide, a bacterial peptide, or a tumor-associated antigen (TAA) antigenic peptide.

8. The cancer treatment drug according to claim 5, wherein the first dimer Z1Z2 is sequence numbers 13 and 14.

9. The cancer treatment drug according to claim 5, wherein the vector further comprises a third nucleotide sequence S3 encoding protein Y1 and a fourth nucleotide sequence S4 encoding protein Y2, and Y1 and Y2 form a second dimer, and the first dimer Z1Z2 is structurally different from the second dimer Y1Y2.

10. The cancer treatment drug according to claim 9, wherein the second dimer Y1Y2 is a TCR coreceptor.

11. The cancer treatment drug according to claim 9, wherein the second dimer Y1Y2 is sequence numbers 11 and 12.

12. The cancer treatment drug according to any one of claims 1 to 3, wherein the vector further comprises a nucleotide sequence encoding a 2A peptide and a nucleotide sequence encoding a linker peptide.

13. The cancer treatment drug according to any one of claims 1 to 3, wherein the vector further comprises a nucleotide sequence encoding a furin cleavage site (SEQ ID NO: 10).

14. The cancer treatment pharmaceutical according to any one of claims 1 to 3, wherein the vector further comprises a promoter sequence selected from a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, a myelin basic protein (MBP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a modified MoMuLV LTR (MNDU3) containing a myeloproliferative sarcoma virus enhancer, a ubiquitin C promoter, an EF-1α promoter, and a mouse stem cell virus (MSCV) promoter.

15. The cancer treatment pharmaceutical according to claim 1, wherein the vector is a viral vector selected from adenovirus, poxvirus, alphavirus, arenavirus, flavivirus, rhabdovirus, retrovirus, lentivirus, herpesvirus, paramyxovirus, and picornavirus.

16. The cancer treatment pharmaceutical according to claim 15, wherein the vector is pseudotyped with the envelope protein of a virus selected from natural feline endogenous virus (RD114), a chimeric version of RD114 (RD114TR), gibbon leukemia virus (GALV), a chimeric version of GALV (GALV-TR), bitrophozoic mouse leukemia virus (MLV4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), avian plague virus (FPV), Ebola virus (EboV), and lymphocytic choriomeningitis virus (LCMV), or baby retrovirus envelope glycoprotein (BaEV).

17. The cancer treatment drug according to claim 1, wherein the vector further comprises a nucleotide sequence encoding RNA selected from the group consisting of antisense RNA, small interfering RNA (siRNA), microRNA, shRNA, RNAi, and ribozymes.

18. The cancer treatment drug according to claim 1, wherein the mutant WPRE includes mutations in the start codons at nucleotide positions 106-108, 152-154, 245-247, 272-274, 283-285, and 362-364 of SEQ ID NO:

1.

19. The cancer treatment drug according to claim 1, wherein the mutant WPRE comprises a nucleotide sequence that is at least 100% identical to SEQ ID NO:

3.

20. The cancer treatment drug according to claim 19, wherein the vector further comprises nucleotide sequences encoding the TCRα chain of SEQ ID NO: 13 and the TCRβ chain of SEQ ID NO:

14.

21. The cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer, as described in any one of claims 1 to 20.

22. Use of a vector, a T cell or T cell population transduced by the vector, or a pharmaceutical composition comprising the vector or the T cell or T cell population in the manufacture of a pharmaceutical for the treatment of cancer, The vector comprises a nucleotide sequence encoding a protein and a variant woodchuck post-transcriptional regulatory element (WPRE) of Sequence ID No. 1, The aforementioned mutant WPR contains mutations in five or six start codons at positions selected from the group consisting of nucleotide positions 106-108, 152-154, 245-247, 272-274, 283-285, and 362-364 of SEQ ID NO: 1, The aforementioned mutant WPR comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 3, The aforementioned mutant WPR does not contain the X protein promoter. The aforementioned mutant WPR does not contain an open reading frame (ORF) of the X protein, and The mutant WPR can enhance the expression of the protein. use.

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