Lentivirus preparations

Abstract

This disclosure provides, at least in part, a formulation buffer suitable for lentiviral vectors.

Description

[Technical Field] 【0001】 Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 381,107, filed on 26 October 2022. The contents of the above application are incorporated herein by reference in their entirety. [Background technology] 【0002】 Viruses often evade detection by the immune system of the infected host, yet are highly efficient at delivering nucleic acids to specific cell types. These characteristics make certain viruses attractive candidates as gene delivery vehicles used in gene therapy. 【0003】 Lentiviral vectors are viral vectors that can be used for gene therapy. Such vectors include reconstituted viral vector systems derived from human immunodeficiency virus-1 (HIV-1), and can introduce target genes into animal and human primary cells or cell lines. Lentiviral vector-mediated gene expression allows for continuous and stable protein production because the target gene is integrated into the host cell genome and replicated during cell division. Lentiviral vectors can effectively transduce non-dividing cells and cells actively progressing through the cell cycle. Tissues and cells that can undergo long-term lentiviral vector-mediated expression of target genes include, in particular, the brain, liver, muscle cells, retina, hematopoietic stem cells, bone marrow mesenchymal stem cells, and macrophages. [Overview of the Initiative] [Problems that the invention aims to solve] 【0004】 Large-scale production of lentiviral vectors has been hampered by several challenges, including low viral titers and poor vector stability. Furthermore, lentiviral vectors are susceptible to inactivation during the purification process, which can contribute to reduced final quality and efficacy of vector preparations, presenting yet another obstacle to large-scale production of purified lentiviral vectors. Thus, formulation buffers that maintain vector stability remain necessary. [Means for solving the problem] 【0005】 This disclosure provides, at least in part, a method for producing a high-titer lentiviral vector carrying a target transgene under satisfactory safety conditions. This disclosure also provides, at least in part, a method for purifying such lentiviral particles from, for example, cell cultures. This disclosure also provides formulations for lentiviral preparations that maintain the structural integrity of the viral vector during purification, storage, and gene transfer events (e.g., ex vivo gene transfer). 【0006】 In some aspects, this disclosure is, a) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), b) One or both of the following: a free positively charged amino acid (e.g., arginine, lysine, or histidine) and a free nonpolar amino acid (e.g., proline, methionine, or tryptophan) The present invention provides an aqueous composition containing the following: 【0007】 In some embodiments, the aqueous composition is substantially free of inorganic salts (e.g., none). 【0008】 In some embodiments, the aqueous composition is substantially free of one or both of NaCl and MgCl2 (e.g., not included). 【0009】 In some embodiments, the aqueous composition contains inorganic salts in a total concentration of less than 20, 10, 5, 2, or 1 mM. 【0010】 In some embodiments, HEPES is concentrated at concentrations of 10-200, 10-150, 10-100, 10-50, 10-40, 10-30, 15-25, or 20 mM. 【0011】 In some embodiments, HEPES is present at a concentration of approximately 20 mM. 【0012】 In some embodiments, the aqueous composition comprises a positively charged amino acid, the positively charged amino acid comprising arginine (e.g., L-arginine). 【0013】 In some embodiments, the arginine concentration is at least 25, 50, 75, 100, or 150 mM. 【0014】 In some embodiments, the arginine concentration is 25-50, 50-75, 75-100, 75-125, 100-200, 125-175, or 150 mM. 【0015】 In some embodiments, the arginine concentration is approximately 150 mM. 【0016】 In some embodiments, the aqueous composition comprises a nonpolar amino acid, the nonpolar amino acid comprising proline (e.g., L-proline). 【0017】 In some embodiments, the proline concentration is 25–200, 50–200, 100–200, 125–175, or 150 mM. 【0018】 In some embodiments, the proline concentration is approximately 150 mM. 【0019】 In some embodiments, the aqueous composition further comprises a cryoprotectant, such as a carbohydrate, such as a non-reducing carbohydrate, such as sucrose. 【0020】 In some embodiments, the aqueous composition further comprises sucrose. 【0021】 In some embodiments, the sucrose concentration is 25–200, 50–200, 100–200, 125–175, or 150 mM. 【0022】 In some embodiments, the sucrose concentration is approximately 150 mM. 【0023】 In some embodiments, the aqueous composition further comprises a stabilizer (e.g., HSA). 【0024】 In some embodiments, the aqueous composition includes HSA. 【0025】 In some embodiments, HSA includes recombinant HSA (rHSA) or human-derived HSA (e.g., HSA isolated from human serum). 【0026】 In some embodiments, HSA is present at concentrations of 0.5–3%, 0.5–2%, 0.5–1%, 1–2%, 1.5–2.5%, or 2% w / v. 【0027】 In some embodiments, HSA is present at approximately 2% w / v. 【0028】 In some embodiments, the aqueous composition is substantially HSA-free (e.g., HSA-free). 【0029】 In some embodiments, the aqueous composition has a pH of 6.0-7.5, 6.0-7.0, 6.0-6.5, 6.5-7.0, 6.2-6.8, 6.4-6.6, or 6.5. 【0030】 In some embodiments, the aqueous composition has a pH of about 6.5. 【0031】 In some embodiments, the aqueous composition substantially does not contain one, two, or three of the PEG lipids, F108, and cholesterol (for example, does not contain any). 【0032】 In some embodiments, The composition contains a positively charged amino acid, and the positively charged amino acid contains L-arginine. The composition contains nonpolar amino acids, and the nonpolar amino acids include L-proline. The composition further comprises sucrose, and The composition is substantially free of inorganic salts. 【0033】 In some embodiments, L-arginine is present at a concentration of 100-200 mM. L-proline is present in concentrations of 25-200 mM, and Sucrose is present in concentrations of 25-200 mM. 【0034】 In some embodiments, the HEPES formulation and / or preservation buffer contains L-arginine at a concentration of 100–200 mM, L-proline at a concentration of 25–200 mM, and sucrose at a concentration of 25–200 mM. 【0035】 In some embodiments, the HEPES formulation and / or preservation buffer comprises 20 mHEPES, 150 mM L-arginine, 150 mM L-proline, and 150 mM sucrose. In some embodiments, the HEPES formulation and / or preservation buffer further comprises 2% w / v HSA. 【0036】 In some embodiments, the osmotic pressure of the aqueous composition is approximately 400 mOsm / kg to approximately 700 mOsm / kg, for example, approximately 415 mOsm / kg to approximately 689 mOsm / kg. 【0037】 In some embodiments, the present disclosure provides compositions comprising a lentiviral vector and an aqueous composition of any of the prior embodiments. 【0038】 In some embodiments, the composition is at least 1 × 10 6 , 1 x 10 7 , 5×10 7 , 1 x 108 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 or 6×10 9 and contains a lentiviral vector at a transduction unit / milliliter (TU / mL). 【0039】 In some embodiments, the lentiviral vector contains a transgene. 【0040】 In some embodiments, the transgene encodes a chimeric antigen receptor (CAR). 【0041】 In some embodiments, CAR is TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, CD20, Folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, Tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, Regmine, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivor and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut It contains an antigen-binding domain that binds to a tumor antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and any combination thereof. 【0042】 In some embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, one or more primary signaling domains, and / or one or more co-stimulatory signaling domains. 【0043】 In some embodiments, one or more primary signaling domains include a CD3-ζ stimulating domain. 【0044】 In some embodiments, one or more co-stimulatory signaling domains are, (a)OX40, CD27, CD28, ICAM-1, LFA-1(CD11a / CD18), ICOS(CD278), 4-1BB(CD137), ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4 , CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C D49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 A co-stimulatory protein selected from the group consisting of CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and (b) Ligands that specifically bind to CD83, e.g., the 4-1BB (CD137) costimulatory domain or the CD28 costimulatory domain Includes intracellular domains selected from. 【0045】 In some embodiments, the CAR is a transmembrane domain, (a) comprising the alpha, beta, or zeta chain of a T cell receptor, or a transmembrane domain of a protein selected from CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, (b) containing the transmembrane domain of CD8, (c) containing the amino acid sequence of Sequence ID No. 6 or an amino acid sequence having at least about 90% sequence identity thereto, (d) A transmembrane domain comprising a nucleic acid sequence of Sequence ID No. 17 or a nucleic acid sequence having at least about 90% sequence identity thereto. 【0046】 In some embodiments, the composition exhibits a level of subvisible particles of 25 μm or larger, with a concentration of approximately 50-100 particles / mL or less. 【0047】 In some embodiments, the composition exhibits a level of subvisible particles of 10 μm or larger, with a concentration of approximately 1100 to 2000 particles / mL or less. 【0048】 In some embodiments, the lentiviral vector in the composition maintains a hydrodynamic diameter of approximately 85–200, 90–200, or 85–130 nm. 【0049】 In some embodiments, the present disclosure provides a kit comprising a lentiviral vector and an aqueous composition of any of the prior embodiments. 【0050】 In some embodiments, the present disclosure is a method for producing CAR-expressing cells, The steps include providing a composition of any of the prior embodiments, The steps include bringing the composition into contact with immune effector cells under conditions that enable transduction of one or more immune effector cells, and This invention provides a method for producing CAR-expressing cells, which includes [a specific component]. 【0051】 In some embodiments, the method is either in vitro or ex vivo. 【0052】 In some embodiments, the population of immune effector cells includes either or both T cells and NK cells. 【0053】 In some embodiments, the composition contains HSA at a concentration of, for example, about 0.5% to 3% w / v. 【0054】 In some embodiments, HSA includes recombinant HSA (rHSA) or human-derived HSA (e.g., HSA isolated from human serum). 【0055】 In some embodiments, the present disclosure provides a method for delivering a transgene to a target, comprising the step of administering a composition of any of the prior embodiments, wherein the lentiviral vector of the composition comprises the transgene. 【0056】 In some embodiments, the transgene includes a CAR, for example, the CAR described herein. 【0057】 In some embodiments, the CAR is a CD19 CAR, for example, a CAR having a CDR as shown in Table 1. 【0058】 In some embodiments, the composition further comprises particles, such as silica particles, such as mesoporous particles (MSPs), and optionally, the mesoporous silica particles are mesoporous silica rods. 【0059】 In some embodiments, (i) The retroviral vector associates with mesoporous silica particles non-covalently, e.g., electrostatically or covalently, and / or (ii) The cell activator associates with mesoporous silica particles non-covalently or covalently. 【0060】 In some embodiments, the composition further comprises a cell activator. 【0061】 In some embodiments, the cell activator is (a) Agents that stimulate the CD3 / TCR complex and / or agents that stimulate co-stimulatory molecules and / or growth factor receptors, (b) A multispecific binding molecule comprising (i) an anti-CD3 binding domain and (ii) a costimulatory molecule binding domain (e.g., an anti-CD2 binding domain or an anti-CD28 binding domain), and / or (c) Particles, such as mesoporous silica particles, are conjugated or adsorbed thereon. 【0062】 In some embodiments, the present disclosure provides a method for producing a lentiviral vector, and this method is a) A step of providing multiple mammalian (e.g., human) cells, b) Multiple mammalian cells, i) FectoVIR®-AAV transfection reagent, and ii) Nucleic acids encoding therapeutic effectors, such as therapeutic proteins (e.g., CARs) and sufficient LTR sequences for packaging into viral particles, and optionally, nucleic acids encoding lentiviral packaging proteins and lentiviral envelope proteins. The steps include bringing the cells into contact under conditions that allow nucleic acids to be introduced into at least a subset of cells, c) A step of culturing cells under conditions suitable for lentiviral vector production. Includes. 【0063】 In some embodiments, when multiple mammalian cells are in a 50 L culture, this results in a number of transduction units per ml of culture that is 50%, 60%, 70%, or 80% or more of the number of transduction units per ml of culture in a similar 100 ml culture. 【0064】 In some embodiments, when the method is used under the conditions described in Example 5, it yields at least 1 × 10 7 Or 3 x 10 7 or at least 1 × 10 8 It provides a unit for introducing traits. 【0065】 In some embodiments, this method yields PP (physical particles):IP (infectious particles) ratios of 1188:1, 953:1, and 1800:1 or less. 【0066】 In some embodiments, the mammalian cells are 293 cells, for example, Expi293F cells. 【0067】 In some embodiments, FectoVIR®-AAV is used at a concentration of 0.3 to 0.6 μl of FectoVIR®-AAV per million cells, for example, about 0.4 μl per million cells. 【0068】 In some embodiments, nucleic acids are used at a concentration of 0.3–0.6 μg of nucleic acid per million cells, for example, about 0.4 μg per million cells. 【0069】 In some embodiments, the ratio of FectoVIR®-AAV:DNA for transfection is 1:0.5 to 1:2, for example, about 1:1 (wherein optionally, the DNA for transfection includes DNA encoding a therapeutic effector, DNA encoding one or more retroviral packaging proteins, and DNA encoding a retroviral envelope protein). 【0070】 In some embodiments, the FectoVIR®-AAV transfection reagent is complexed with a nucleic acid. 【0071】 In some embodiments, the method further includes the step of mixing the FectoVIR®-AAV transfection reagent with the nucleic acid prior to step b). 【0072】 In some embodiments, the amount of complex formation between the transfection reagent and nucleic acid is about 1% to about 15%, for example, about 1% to about 10% (for example, about 5 to 7.5% or 7.5 to 10%). 【0073】 In some embodiments, the amount of composite formation is 3-7%, 4-6%, or about 5%. 【0074】 In some embodiments, the FectoVIR®-AAV transfection reagent and nucleic acid are incubated for a sufficient amount of time to allow complex formation to occur, for example, about 10–90 minutes, for example 15–60 minutes, for example 15–30 minutes, 30–45 minutes, or 45–60 minutes. 【0075】 In some embodiments, the present disclosure provides a method for producing a lentiviral vector, and this method is a) A step of culturing multiple mammalian (e.g., human) cells at a pH greater than approximately 6.9 or approximately 6.9 to 7.3, for example, approximately 7.0 to 7.1. b) Following step a), adjust the pH of the culture to approximately 6.0-6.8, for example 6.6-6.8, for example approximately 6.7. c) Following step b), the step of contacting the culture with the transfection reagent and DNA. Includes. 【0076】 In some embodiments, the transfection reagent includes FectoVIR®-AAV transfection reagent. 【0077】 In some embodiments, the DNA encodes one or more retroviral packaging proteins, retroviral envelope proteins, and therapeutic effectors, such as therapeutic proteins (e.g., CARs). 【0078】 In some embodiments, a) includes the step of culturing the cells for about 2 to 4 days, for example, about 3 days. 【0079】 In some embodiments, the method further includes an additional step of culturing cells between step b) and step c). 【0080】 In some embodiments, the method further includes an additional step of culturing cells after step c). 【0081】 In some embodiments, step b) includes lowering the pH by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1. 【0082】 In some embodiments, before step a), multiple mammalian cells are 0.1 × 10 6 cells / mL~0.3×10 6 cells / mL (e.g., approximately 0.15 × 10⁻⁶) 6 Cells / mL or approximately 0.2 × 10⁶ 6 The cells are inoculated into the final volume of culture medium (e.g., FreeStyle® medium) at a rate of (cells / mL). 【0083】 In some embodiments, multiple mammalian cells are inoculated 50 to 80 hours before step a) (e.g., about 55 hours, about 60 hours, about 65 hours, about 70 hours, about 72 hours, about 75 hours, or about 80 hours). 【0084】 In some embodiments, multiple mammalian cells are arranged to achieve a suitable cell density for transfection (e.g., about 1.0 × 10⁶). 6 cells / mL ~ approx. 3.0×10 6 cells / mL (e.g., 1.5 × 10⁻⁶ cells) 6 cells / mL~2.5×10 6 They are cultured under conditions suitable for enabling cell proliferation and amplification to cells / mL. 【0085】 In some embodiments, the present disclosure provides a method for producing a lentiviral vector, and this method is a) A step of providing a composition comprising a lentiviral vector and at least one impurity (for example, wherein the composition comprises a clarified cell collection or filtrate), b) The step of contacting the composition with arginine or a salt thereof. Includes. 【0086】 In some embodiments, one or more of the following: i) The arginine concentration should be approximately 25-50 mM (approximately 50 mM), 50-100 mM (e.g., approximately 75 mM), 100-200 mM (e.g., approximately 150 mM), or 200-400 mM (e.g., approximately 300 mM), or ii) The arginine is at a concentration sufficient to increase the level of transduction units in the lentiviral vector by approximately 10% to 300%, approximately 20% to 180%, approximately 30% to 160%, approximately 50% to 150%, approximately 75% to 125%, or approximately 100% in the assay according to Example 7, compared to other similar compositions. iii) After step b), the composition, when measured by microflow imaging, exhibits a total particle concentration of less than 400,000, less than 300,000, less than 200,000, or less than 100,000 per ml, and optionally, the particles contain agglutinated lentivirus. iv) After step b), the composition, when measured by microflow imaging, exhibits concentrations of particles ≥10 μm of approximately 5,000, approximately 4,500, approximately 4,000, approximately 3,500, approximately 3,000, or less than approximately 2,500 per 1 ml, and optionally, the particles contain agglutinated lentivirus. v) After step b), the composition exhibits a concentration of particles ≥25 μm, less than approximately 500, approximately 400, approximately 300, or approximately 200 per ml, as measured by, for example, microflow imaging, and optionally the particles contain agglutinated lentivirus. vi) After step b), the composition shows reduced aggregation of lentiviral vectors compared to a similar filtrate, except that arginine or its salts are not added. vii) The recovery rate of the transduction units of the lentiviral vector, as measured by the assay according to Example 7, is, for example, at least about 10%, about 20%, about 50%, about 100%, or about 200% higher than a similar control, except without the addition of arginine. 【0087】 In some embodiments, b) includes the step of contacting the composition with a solution containing arginine and a buffer, optionally the buffer being PIPES, and optionally the PIPES being at a concentration of about 10 mM to about 50 mM, for example, about 20 mM, in the solution. In certain embodiments, the solution has a pH of about 6.0 to about 7.0, for example, about 6.5. 【0088】 In some embodiments, the solution further contains a salt, which is optionally selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride, for example, sodium chloride. 【0089】 In some embodiments, the salt is present in the solution at a concentration of about 25–150 mM, for example, about 50–100 mM, for example, about 75 mM. 【0090】 In certain embodiments, the solution has a pH of about 6.5. 【0091】 In some embodiments, the solution further comprises a carbohydrate, such as a non-reducing carbohydrate, such as sucrose or trehalose. 【0092】 In certain embodiments, carbohydrates are present in the solution at concentrations of about 1 to about 10% by weight / volume of the solution, for example, about 2 to about 5% by weight / volume of the solution, or about 2.5% by weight / volume of the solution. 【0093】 In certain embodiments, carbohydrates are present in the solution at concentrations of about 30–150 mM (about 73 mM) or about 150–300 mM (e.g., about 220 mM). 【0094】 In some embodiments, the solution further comprises one or both of NaCl (e.g., about 25–150 mM, e.g., about 50–100 mM, e.g., about 75 mM) and sucrose (e.g., about 30–150 mM, e.g., about 73 mM or e.g., about 150–300 mM, e.g., about 220 mM or about 2.5% by weight / volume of the solution). 【0095】 In some embodiments, the solution comprises 20 mM PIPES, 75 mM sodium chloride, and 2.5% by weight / volume of sucrose, and the solution has a pH of about 6.5. In certain embodiments, the solution comprises about 20 mM PIPES, about 75 mM sodium chloride, and 2.5% by weight / volume of sucrose, and the solution has a pH of about 6.5. 【0096】 In a particular embodiment, the solution comprises 20 mM PIPES, 75 mM sodium chloride, and 73 mM sucrose, and the solution has a pH of about 6.5. 【0097】 In some embodiments, the solution comprises 20 mM PIPES, 75 mM sodium chloride, and 220 mM sucrose, and the solution has a pH of about 6.5. 【0098】 In some embodiments, the solution further comprises 20 mM PIPES, 75 mM arginine, for example arginine-HCl, and the solution has a pH of about 6.5. 【0099】 In some embodiments, the osmotic pressure of the solution is approximately 270 mOsm / kg to approximately 330 mOsm / kg, for example, approximately 275 mOsm / kg to approximately 300 mOsm / kg, for example, approximately 285 mOsm / kg. 【0100】 In certain embodiments, the method further includes a step of performing a purification step, such as a filtration step, on the composition of c)b) to produce a semi-purified composition containing a lentiviral vector. 【0101】 In certain embodiments, the method further comprises the step of contacting the semi-purified composition with arginine or a salt thereof after step c). 【0102】 In some embodiments, arginine encapsulates the lentiviral vector. 【0103】 In certain embodiments, arginine stabilizes the lentiviral vector. 【0104】 In some embodiments, the impurities include proteins (e.g., host cell proteins), nucleic acids (e.g., host cell nucleic acids), carbohydrates (e.g., host cell carbohydrates), lipids, enzymes, salts, buffers, or any combination thereof. 【0105】 In certain embodiments, the cell density during transfection is approximately 1.0 × 10⁶ 6 cells / mL ~ approx. 3.0×10 6 cells / mL (e.g., 1.5 × 10⁻⁶ cells) 6 cells / mL~2.5×10 6 It is (cells / mL). 【0106】 In certain embodiments, the cell viability is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) at the time of transfection, or is evaluated to be so. 【0107】 In certain embodiments, cell viability is measured at the time of transfection or immediately before or after (for example, within 30 minutes prior to transfection). 【0108】 In some embodiments, this method is used in processes involving two or more nucleic acids (e.g., two or more plasmids, e.g., two plasmids, three plasmids, four plasmids, or five plasmids). 【0109】 In some embodiments, the present disclosure provides a method for producing a lentiviral vector, and this method is a) A step of providing a population of human cells (e.g., 293 cells), b) A step of introducing a retroviral packaging protein, a retroviral envelope protein, and a nucleic acid encoding a therapeutic effector, such as a therapeutic protein (e.g., CAR), into cells, c) A step in which the cells are brought into contact with benzonase approximately 6-40-10-40-10-30 or approximately 20 hours after step b), d) A step of culturing cells under conditions suitable for lentiviral vector production. Includes. 【0110】 In some embodiments, the present disclosure provides a method for producing a lentiviral vector, and this method is a) A step of providing a population of human cells (e.g., 293 cells), b) Steps of introducing retroviral packaging proteins, retroviral envelope proteins, and therapeutic effectors, such as nucleic acids encoding therapeutic proteins (e.g., CARs), into cells. c) A step of bringing cells into contact with benzonase, d) A step of culturing cells under conditions suitable for lentiviral vector production, e) Step c) Collect the lentiviral vector from the cells 6-10 hours, 10-20 hours, 20-30 hours, 30-40 hours, or 40-50 hours later. Includes. 【0111】 In some embodiments, the present disclosure provides a method for producing a lentiviral vector, and this method is a) A step of providing a plurality of mammalian (e.g., human) cells, wherein the plurality of cells (e.g., here the cells are fibroblasts, e.g., embryonic renal fibroblasts, e.g., Expi293F cells) and the cells comprise one or more retroviral packaging proteins, retroviral envelope proteins and therapeutic effectors, e.g., nucleic acids (e.g., DNA) encoding therapeutic proteins (e.g., CARs), b) A step of culturing cells under conditions suitable for lentiviral vector production. Includes. 【0112】 In some embodiments, the present disclosure provides an aqueous composition comprising a lentiviral vector, arginine, 1,4-piperazinediethanesulfonic acid (PIPES) buffer, and a salt. 【0113】 In certain embodiments, the arginine in the aqueous composition is at a concentration of about 25–50 mM (about 50 mM), about 50–100 mM (e.g., about 75 mM), about 100–200 mM (e.g., about 150 mM), or about 200–400 mM (e.g., about 300 mM), and optionally, the PIPES aqueous composition is at a concentration of about 10 mM to about 50 mM, for example, about 20 mM. 【0114】 In some embodiments, the aqueous composition has a pH of about 6.0 to about 7.0, for example, about 6.5. 【0115】 In certain embodiments, the aqueous composition further comprises a salt, optionally selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride. 【0116】 In some embodiments, the salt is sodium chloride (NaCl). 【0117】 In certain embodiments, the salt concentration in the aqueous composition is about 25 mM to about 150 mM, for example, about 50 mM to about 75 mM. 【0118】 In some embodiments, the aqueous composition comprises 20 mM PIPES and 75 mM sodium chloride, and the aqueous composition has a pH of about 6.5. 【0119】 In certain embodiments, the aqueous composition further comprises a carbohydrate, such as a non-reducing carbohydrate, such as sucrose or trehalose. 【0120】 In some embodiments, the carbohydrate is present in the aqueous composition at a concentration of about 1 to about 10% by weight / volume of the solution, for example, about 2 to about 5% by weight / volume of the aqueous composition, or about 2.5% by weight / volume of the aqueous composition. 【0121】 In one embodiment, the carbohydrate is present in the aqueous composition at a concentration of about 30–150 mM (about 73 mM) or about 150–300 mM (for example, about 220 mM). 【0122】 In some embodiments, the aqueous composition comprises one or both of NaCl (e.g., about 25–150 mM, e.g., about 50–100 mM, e.g., about 75 mM) and sucrose (e.g., about 30–150 mM, e.g., about 73 mM or e.g., about 150–300 mM, e.g., about 220 mM or about 2.5% by weight / volume of the aqueous composition). 【0123】 In one embodiment, the aqueous composition comprises 20 mM PIPES, 75 mM sodium chloride, and 2.5% by weight / volume of sucrose, and the aqueous composition has a pH of about 6.5. 【0124】 In certain embodiments, the aqueous composition comprises 20 mM PIPES, 75 mM sodium chloride, and 73 mM sucrose, and the aqueous composition has a pH of about 6.5. 【0125】 In one embodiment, the aqueous composition comprises 20 mM PIPES, 75 mM sodium chloride, and 220 mM sucrose, and the aqueous composition has a pH of about 6.5. 【0126】 In certain embodiments, the osmotic pressure of the aqueous composition is approximately 270 mOsm / kg to approximately 330 mOsm / kg, for example, approximately 275 mOsm / kg to approximately 300 mOsm / kg, for example, approximately 285 mOsm / kg. 【0127】 In one embodiment, the lentiviral vector described in any of the preceding claims is approximately 3 × 10 8 TU / mL ~ approximately 5 x 10 8 It exists at a concentration of TU / mL. 【0128】 In certain embodiments, the aqueous composition does not contain one or more proteins selected from the group consisting of human serum albumin (HSA), recombinant human serum albumin (rHSA), bovine serum albumin (BSA), and lipoproteins. 【0129】 In one embodiment, the lentiviral vector includes a transgene, for example, a protein, such as a protein containing a chimeric antigen receptor (CAR). 【0130】 In certain embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, and one or more signaling domains, arranged from the N-terminus to the C-terminus. 【0131】 In some embodiments, the signaling domain includes one or more primary signaling domains and / or one or more co-stimulatory signaling domains. 【0132】 In certain embodiments, one of the one or more primary signaling domains includes a CD3-ζ stimulating domain. 【0133】 In some embodiments, one or more of the co-stimulus signaling domains are OX40, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD 19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LF The protein includes an intracellular domain selected from a costimulatory protein selected from the group consisting of ligands that specifically bind to A-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83, such as a 4-1BB (CD137) costimulatory domain or a CD28 costimulatory domain. 【0134】 In some embodiments, one or more co-stimulatory signaling domains are ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11 b. Contains an intracellular domain selected from a costimulatory protein selected from the group consisting of ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, or NKG2D. 【0135】 In certain embodiments, the antigen-binding domain is scFv. 【0136】 In some embodiments, the antigen-binding domain is CD19; CD123; CD22; CD30; CD171; CS-1; C-type lectin-like molecule-1; CD33; epidermal growth factor receptor variant III (EGFRvlll); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member B-cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD1 17); Interleukin-13 receptor subunit α-2; Mesothelin; Interleukin-11 receptor α (IL-11Ra); Prostate stem cell antigen (PSCA); Protease serine 21; Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific fetal antigen-4 (SSEA-4); CD20; Folate receptor α; Receptor tyrosine-protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface-related (MUC1); Epidermal growth factor receptor (EGFR); Neuronal cell adhesion molecule (NCAM); Prostase; Prostatic acid phosphatase (PAP); Elongation factor 2 variant (ELF2M); Ephrin B2; Fibroblast-activating protein α (FAP); Insulin-like compound Long factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, β type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of cleavage cluster region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R);Claudin 6 (CLDN6); Thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); Chromosome X open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexasaccharide moiety of globoH glycoceramide (GloboH); Mammary gland differentiation antigen (NY-BR-1); Uloplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenergic receptor β3 (ADRB3); Panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K9 (LY6K); Olfactory receptor 51 E2 (OR51) E2); TCRγ Alternate Reading Frame Protein (TARP); Wilms Tumor Protein (WT1); Cancer / Testicular Antigen 1 (NY-ESO-1); Cancer / Testicular Antigen 2 (LAGE-1a); Melanoma-Associated Antigen 1 (MAGE-A1); ETS Translocation Mutation Gene 6 (ETV6-AML) located on chromosome 12p; Sperm Protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); Angiopoietin-Binding Cell Surface Receptor 2 (Tie 2); Melanoma carcinoma testicular antigen-1 (MAD-CT-1); Melanoma carcinoma testicular antigen-2 (MAD-CT-2); Fos-related antigen 1; Tumor protein p53 (p53); p53 variant; Prostain; Survivin; Telomerase; Prostate cancer tumor antigen-1, Melanoma antigen 1 recognized by T cells; Rat sarcoma (Ras) variant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Melanoma apoptosis inhibitor (ML-IAP); ERG (Transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); Paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc Tri-myelocyte tomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 (SART3) recognized by T cells;Paired box protein Pax-5 (PAX5); Proacrosin-binding protein sp32 (OY-TES1); Lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); Synovial sarcoma, X-cleavage 2 (SSX2); Late glycation end product receptor (RAGE-1); Renal ubiquitous protein 1 (RU1); Renal ubiquitous protein 2 (RU2); Regmine; Human papillomavirus E6 (HPV E6); Human papillomavirus E7 (HPV E7); Enteral carboxylesterase; Heat shock protein 70-2 variant (mut) It binds to antigens selected from the group consisting of hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA receptor Fc fragment (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like receptor 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like receptor 5 (FCRL5); and immunoglobulin λ-like polypeptide 1 (IGLL1), such as CD19, CD22, mesothelin, or CD123. 【0137】 In certain embodiments, the CAR comprises an anti-CD19 antibody or a fragment thereof, a 4-1BB(CD137) transmembrane domain, and a CD3-ζ signaling domain. 【0138】 In some embodiments, the lentiviral vector includes a second transgene encoding a second protein, such as a second protein containing a second chimeric antigen receptor (CAR). 【0139】 In one embodiment, the present disclosure provides a method for producing a lentiviral vector, the method being: a) A step of providing a population of human cells (e.g., 293 cells), b) A step of introducing a retroviral packaging protein, a retroviral envelope protein, and a nucleic acid encoding a therapeutic effector, such as a therapeutic protein (e.g., CAR), into cells, c) A step of bringing the cells into contact with benzonase approximately 2-6 (e.g., approximately 3), 4-10 (e.g., 6), 6-40, 10-40, 10-30 (e.g., approximately 24), or approximately 20 hours after step b), d) A step of culturing cells under conditions suitable for lentiviral vector production. Includes. 【0140】 In certain embodiments, benzonase is added 6–10 hours, 10–20 hours, 20–30 hours, 30–40 hours, or 40–50 hours before the lentiviral vector is collected from the cells. 【0141】 In one embodiment, the present disclosure provides a method for producing a lentiviral vector, the method being: a) A step of providing a population of human cells (e.g., 293 cells), b) Steps of introducing retroviral packaging proteins, retroviral envelope proteins, and therapeutic effectors, such as nucleic acids encoding therapeutic proteins (e.g., CARs), into cells. c) A step in which the cells are brought into contact with benzonase (for example, 3 to 24 hours after step b), d) A step of culturing cells under conditions suitable for lentiviral vector production, e) Step c) Collect the lentiviral vector from the cells 6-10 hours, 10-20 hours, 20-30 hours, 30-40 hours, or 40-50 hours later. Includes. 【0142】 In some embodiments, the benzonase concentration is about 10–40 U / mL, for example 20–30 U / mL, for example about 25 U / mL. 【0143】 In some embodiments, the benzonase concentration is approximately 3–60 U / mL, 3–10 U / mL, 3–7 U / mL, 4–6 U / mL, or approximately 5 U / mL. 【0144】 In one embodiment, the benzonase concentration is 5-50, 5-15, 15-25, or 25-50 U / mL. 【0145】 In certain embodiments, the method further includes the step of contacting benzonase with, for example, about 1–5 mM, about 1–3 mM, or about 2 mM MgCl2 prior to step c). 【0146】 In one embodiment, the present disclosure provides a method for producing a lentiviral vector, the method being: a) A step of providing a plurality of mammalian (e.g., human) cells, wherein the plurality of mammalian cells do not contain the SV40 large T antigen (for example, here the cells are fibroblasts, e.g., embryonic renal fibroblasts, e.g., Expi293F cells), and the plurality of mammalian cells contain one or more retroviral packaging proteins, retroviral envelope proteins and nucleic acids (e.g., DNA) encoding therapeutic effectors, e.g., therapeutic proteins (e.g., CARs), b) A step of culturing cells under conditions suitable for lentiviral vector production. Includes. 【0147】 In certain embodiments, a) includes the step of introducing nucleic acids into multiple mammalian cells. 【0148】 In some embodiments, the method further includes the step of at least partially isolating a lentiviral vector from multiple mammalian cells. 【0149】 In one embodiment, one or more retroviral packaging proteins include lentivirus gag, lentivirus pol, or lentivirus rev, or any combination thereof. 【0150】 In certain embodiments, the retroviral envelope protein includes VSV-G. 【0151】 In some embodiments, the present disclosure provides preparations of lentiviral vectors, which preparations are a) A therapeutic effector, for example, a lentiviral genome encoding a therapeutic protein (e.g., CAR), b) Envelope enclosing the lentiviral genome (where the envelope optionally includes VSV-G) It includes multiple lentiviral vectors, The prepared material should be at least 5 × 10 7 , 1 x 10 8 , 1 x 10 9 or 1 × 10 10 Includes trait introduction units, The preparation contains less than 90% SV40 large T antigen or less than 10 μg / ml or 1 μg / ml of nucleic acid (e.g., DNA) encoding the SV40 large T antigen. 【0152】 In some embodiments, multiple lentiviral vectors are provided, at least 1 × 10⁶ 9 , at least 2 × 10 9 , at least 5 × 10 9 , at least 1 × 10 10 , at least 2 × 10 10 , at least 5 × 10 10 , at least 1 × 10 11 , at least 2 × 10 11 , at least 5 × 10 1 or at least 1 × 10 12 It contains cells. 【0153】 In certain embodiments, the mammalian cells are in a culture medium volume of at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, or at least 500 L. 【0154】 One embodiment includes the step of culturing multiple mammalian cells in serum-free medium. 【0155】 In certain embodiments, multiple mammalian cells are grown in a suspension. 【0156】 In some embodiments, the CAR includes a CD19 CAR (for example, a humanized CD19 CAR, as described in International Publication No. 2014153270A1). 【0157】 In certain embodiments, the CAR includes a dual CAR (for example, a humanized CD19-CD22 CAR, as described in International Publication No. 2016164731A2). 【0158】 In some embodiments, the nucleic acid encoding the CAR further encodes shRNA (as described, for example, in International Publication No. 2017049166A). 【0159】 In one embodiment, the lentiviral vector is produced in cells cultured in the absence of serum. 【0160】 In certain embodiments, the lentiviral vector is characterized by a hydrodynamic radius of 100 ± 25 nm, as measured by dynamic light scattering (DLS). 【0161】 In certain embodiments, the lentiviral vector maintains a hydrodynamic radius of 100 ± 25 nm within a temperature range of 25°C to 55°C (e.g., 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C). 【0162】 In some embodiments, the lentiviral vector is characterized by polydispersity of 10% to 25% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). 【0163】 In one embodiment, the lentiviral vector maintains polydispersity of 10% to 25% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%) within a temperature range of 25°C to 55°C (e.g., 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C). 【0164】 In certain embodiments, the lentiviral vector maintains a concentration of about 70% to about 100% (e.g., about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 100%) after three freeze / thaw cycles compared to the concentration of the lentiviral vector in the aqueous composition before the freeze / thaw cycles, and each freeze / thaw cycle comprises freezing the aqueous composition and subsequently thawing the aqueous composition at room temperature. 【0165】 In some embodiments, the lentiviral vector maintains a concentration of about 70% to about 100% (e.g., about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 100%) after 6 to 10 freeze / thaw cycles, for example, after 6 to 9 freeze / thaw cycles. 【0166】 In some embodiments, the present disclosure provides an aqueous composition comprising a lentiviral vector, a buffer selected from the group consisting of phosphate buffer, sodium citrate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, and 3-morpholinopropane-1-sulfonic acid (MOPS) buffer, and a salt. 【0167】 In some embodiments, the salt is selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride. 【0168】 In one embodiment, the aqueous composition further comprises a non-reducing carbohydrate selected from the group consisting of sucrose and trehalose. 【0169】 In some embodiments, the present disclosure provides a scalable process for producing large quantities of viral vectors (e.g., lentiviral vectors) for, for example, prophylactic, diagnostic, immunotherapy, or therapeutic applications. This process can be carried out using suspension cells (e.g., HEK293 cells, e.g., Expi293F cells). In some embodiments, substantially all suspension cells do not express large T antigens, e.g., SV40 T antigen. In some embodiments, the process can be carried out using a bioreactor. 【0170】 In some embodiments, the disclosure provides a highly reproducible, efficient, and scalable process for producing large quantities of viral vectors (e.g., lentiviral vectors) having either high viral titer or high viral yield, or both. 【0171】 In some embodiments, the present disclosure provides a highly reproducible, efficient, and scalable process for purifying large quantities of viral (e.g., lentivirus) vectors having either high viral titer or high viral yield, or both. 【0172】 In another aspect, the disclosure provides compositions and methods for stabilizing viral vectors, such as lentiviral vectors, during a purification process. [Brief explanation of the drawing] 【0173】 [Figure 1] This is a schematic diagram showing the overall test design. [Figure 2] This graph shows the particle size distribution (PSD; Dh) of LVV products before and after incubation at 37°C for 12 hours using HEPES or XV15 with added sucrose. [Figure 3]Graph showing the particle concentration of the LVV product before and after 12-hour incubation at 37°C using HEPES or XV15 supplemented with sucrose. [Figure 4] Graph showing the TU titer of the LVV product before and after 12-hour incubation at 37°C using HEPES or XV15 supplemented with sucrose. [Figure 5] Graph showing the overall p24 titer of the LVV product before and after 12-hour incubation at 37°C using HEPES or XV15 supplemented with sucrose. [Figure 6] Graph showing the zeta potential (ZP) distribution of LVV in HEPES and PIPES buffers. [Figure 7] Graph showing the phase plot of LVV in HEPES and PIPES buffers. [Figure 8] Graph showing the relationship between pH and the vector size (left y-axis) or PDI (right y-axis). [Figure 9] Graph showing the relationship between pH and the zeta potential of the vector. The dashed line (-30 mV) indicates the potential minimum ZP for stable particles in suspension. [Figure 10] Pareto chart of TU titer by the effect of individual excipients in descending order. The statistically significant effect starts with the dashed vertical line (α = 0.15). The bars with a mottled pattern are non-significant conditions excluded from the model. The bars with a striped pattern are conditions with a major effect on the response. [Figure 11] Pareto chart of MADLS by the effect of individual excipients in descending order. The statistically significant effect starts with the dashed vertical line (α = 0.15). The bars with a mottled pattern are non-significant conditions excluded from the model. The bars with a striped pattern are conditions with a major effect on the response. [Figure 12] Pareto chart of p24 titer by the effect of individual excipients in descending order. The statistically significant effect starts with the dashed vertical line (α = 0.15). The bars with a mottled pattern are non-significant conditions excluded from the model. [Figure 13]This is a Pareto chart of the TU titer obtained from combination 1. [Figure 14] This graph shows the relationship between arginine and the TU titer obtained from combination 1. [Figure 15] This graph shows the relationship between recombinant HSA (rHSA) and the TU titer obtained from combination 1. [Figure 16] This graph shows the relationship between proline and the TU titer obtained from combination 1. [Figure 17] This graph shows the relationship between lactose and the TU titer obtained from combination 1. [Figure 18] This is a Pareto chart of the p24 titer obtained from combination 1. [Figure 19] This graph shows the relationship between glycerol and the p24 titer obtained from combination 1. [Figure 20] This graph shows the relationship between recombinant HSA (rHSA) and the p24 titer obtained from combination 1. [Figure 21] This graph shows the relationship between proline and the p24 titer obtained from combination 1. [Figure 22] This graph shows the relationship between lactose and the p24 titer obtained from combination 1. [Figure 23] This is a Pareto chart of TU titers obtained from combination 2. [Figure 24] This graph shows the relationship between arginine and the TU titer obtained from combination 2. [Figure 25] This graph shows the relationship between glutamic acid and the TU titer obtained from combination 2. [Figure 26] This graph shows the relationship between recombinant HSA (HSA) and the TU titer obtained from combination 2. [Figure 27] This graph shows the relationship between arginine and the TU titer obtained from combination 2. [Figure 28] This graph shows the relationship between MgC2 and the TU titer obtained from combination 2. [Figure 29] This is a Pareto chart of the p24 titer obtained from combination 2. [Figure 30] This figure shows the particle analysis of the factor design of LVV products formulated in PIPES buffer. The concentration of particles larger than 10 μm is plotted against the Z mean. The dashed line represents the LVV particle size distribution (85 nm to 130 nm) and the number of subvisible particles larger than 10 μm (1300). [Figure 31] This figure shows the particle analysis of the factor design of LVV products formulated in PIPES buffer. The concentration of particles larger than 25 μm is plotted against the Z mean. The dashed line represents the LVV particle size distribution (85 nm to 130 nm) and the number of subvisible particles larger than 25 μm (150). [Figure 32] This graph shows the TU potency of LVV products formulated with HEPES-F1 after buffer exchange (ABE), with different HSA sources added, and using 2.0% HSA in the formulation, based on the number of freeze-thaw cycles (FT; white, 0FT; striped, 3FT). [Figure 33] This graph shows the TU potency of LVV products formulated with HEPES-F1 after buffer exchange (ABE), with different HSA sources added, and using 0.5% HSA in the formulation, based on the number of freeze-thaw cycles (FT; white, 0FT; striped, 3FT). [Figure 34] This graph shows the p24 titer of LVV products formulated with HEPES-F1 after buffer exchange (ABE), with different HSA sources added, and using 2.0% HSA in the formulation, based on the number of freeze-thaw cycles (FT; white, 0FT; striped, 3FT). [Figure 35] This graph shows the p24 titer of LVV products formulated with HEPES-F1 after buffer exchange (ABE), with different HSA sources added, and using 0.5% HSA in the formulation, based on the number of freeze-thaw cycles (FT; white, 0FT; striped, 3FT). [Figure 36] This graph shows the ratio of the total number of subvisible particles to the excipient range based on the freeze-thaw cycle (FT; white, 0FT; striped, 3FT). [Figure 37] Graph showing the TU titers for three batches of LVV products formulated with HEPES-F1 after buffer exchange (ABE), HSA addition (+HSA), and three freeze-thaw cycles (+3FT). [Figure 38] Graph showing the p24 ELISA results for three batches of LVV products formulated with HEPES-F1 after buffer exchange (ABE), HSA addition (+HSA), and three freeze-thaw cycles (+3FT). [Figure 39] Graph showing the overall results of p24 titers for three batches of LVV products formulated with HEPES-F1 after buffer exchange (ABE), HSA addition (+HSA), and three freeze-thaw cycles (+3FT). [Figure 40] Graph showing the total number of subvisible particles larger than 10 μm observed in three batches of LVV products formulated with HEPES-F1 after buffer exchange (ABE), HSA addition (+HSA), and three freeze-thaw cycles (+3FT). [Figure 41] Graph showing the total number of subvisible particles larger than 25 μm observed in three batches of LVV products formulated with HEPES-F1 after buffer exchange (ABE), HSA addition (+HSA), and three freeze-thaw cycles (+3FT). [Figure 42] Graph showing the TU titers for three batches of LVV products formulated with HEPES-F2 after buffer exchange (ABE), HSA addition (+HSA), and three freeze-thaw cycles (+3FT). [Figure 43] Graph showing the overall results of p24 titers for three batches of LVV products formulated with HEPES-F2 after buffer exchange (ABE), HSA addition (+HSA), and three freeze-thaw cycles (+3FT). [Figure 44]This graph shows the total number of subvisible particles larger than 10 μm observed in three batches of LVV products formulated with HEPES-F2 after buffer exchange (ABE), HSA addition (+HSA), and three freeze-thaw cycles (+3FT). [Figure 45] This graph shows the total number of subvisible particles larger than 25 μm observed in three batches of LVV products formulated with HEPES-F2 after buffer exchange (ABE), HSA addition (+HSA), and three freeze-thaw cycles (+3FT). [Figure 46] This graph shows the particle size distribution by intensity for three batches of LVV products in PIPES and HEPES buffers. [Figure 47] This graph shows the particle concentrations measured by MADLS in sterile filtered LVV products with one (VS) and two (VS-2SF) HSA formulations, comparing their effectiveness. [Figure 48] This graph compares the effectiveness of HSA preparations, showing the TU titer of LVV products after sterile filtration, with one (VS) and two (VS-2SF) formulations. [Figure 49] This graph shows the TU titer of LVV products formulated in PIPES buffer after storage at different temperatures (25°C, 4°C, -20°C, and -80°C) over a period of up to 9 months. [Figure 50] This graph shows the TU potency of LVV products formulated in HEPES(F1) buffer after storage at different temperatures (25°C, 4°C, -20°C, and -80°C) over a period of up to 9 months. [Figure 51] This graph shows the TU potency of LVV products formulated in HEPES(F2) buffer after storage at different temperatures (25°C, 4°C, -20°C, and -80°C) over a period of up to 9 months. [Modes for carrying out the invention] 【0174】 This disclosure is based at least in part on a method for producing a high-titer lentiviral vector carrying a target transgene under satisfactory safety conditions. This disclosure also provides at least in part a method for purifying such lentiviral particles from, for example, cell cultures. This disclosure also provides formulations for lentiviral preparations that maintain the structural integrity of the viral vector during purification, storage, and ex vivo gene transfer events. 【0175】 definition Unless otherwise stated, the following terms and phrases used herein are intended to have the meanings set forth below. 【0176】 As used herein, the singular forms "a" or "an" include multiple referents unless otherwise specified. 【0177】 The term "or" in this specification means and is used interchangeably with the term "and / or" unless the context explicitly indicates a different interpretation. 【0178】 "Approximately" and "about" generally refer to the degree of acceptable error in a measured quantity, taking into account the nature or precision of the measurement. Exemplary degrees of error are within 10 percent (%), 5 percent, or 2 percent of a given value or range of values. 【0179】 The term "amino acid" refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimes that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, namely, an α-carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimes" are compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids. 【0180】 As used herein, the term “buffer solution” refers to a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid. For example, as used herein, “N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution” refers to a mixture containing N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and the N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid anion. Because a chemical equilibrium is established between the weak acid and its conjugate base, a solution containing a buffer solution resists rapid changes in pH when a small amount of acid or base is added to the solution. 【0181】 As used herein, the term "inorganic salts" refers to chemical components (e.g., components of a solution) that lack carbon-hydrogen bonds, consist of positively charged cations and uncharged anions, and have no net charge. Examples of inorganic salts include sodium chloride (NaCl) and magnesium chloride (MgCl2). In some embodiments, the salts exist in a dissociated form in aqueous solution. 【0182】 As used herein, the term “freeze / thaw cycle” refers to the process of exposing a liquid mixture, such as an aqueous solution or suspension, to a temperature below its freezing point until the mixture freezes, and then thawing the mixture at a temperature above its freezing point. The freezing step can be carried out, for example, by placing the mixture in an environment where the temperature is approximately -80°C to approximately -20°C. The mixture may be kept frozen for a period of time, for example, one or several days, weeks, months, or years before thawing. The thawing step can be carried out by exposing the mixture to conditions where the temperature is approximately 2°C to approximately 8°C, or by storing the mixture at room temperature (e.g., ambient laboratory temperature or approximately 25°C). Alternatively, thawing may be carried out using a water bath (e.g., 37°C). 【0183】 As used herein, the term “hydrodynamic radius” refers to the apparent radius (Rh in nm) of a particle in solution, inferred from the particle’s diffusion properties. The hydrodynamic radius of a virus particle is one factor determining the diffusion rate of the virus particle in aqueous solution and the particle’s ability to move through a polymer gel. The hydrodynamic radius of a virus particle is partially determined by the mass and molecular structure of each component of the particle and its hydration state. Methods for determining the hydrodynamic radius of a virus particle are known in the art and include the use of dynamic light scattering and size exclusion chromatography. 【0184】 As used herein, the term “non-reducing carbohydrate” refers to a carbohydrate that does not exist in chemical equilibrium with an aldehyde and therefore lacks the ability to be oxidized to a carboxylic acid by transition metal cations such as silver (Ag+) and copper (Cu2+). Examples of non-reducing carbohydrates include, but are not limited to, disaccharides such as sucrose, trehalose, and palatinitol, trisaccharides such as raffinose and melegitose, and tetrasaccharides such as stachyose. Non-reducing carbohydrates include monosaccharide derivatives such as sorbitol, mannitol, erythritol, and xylitol; disaccharide derivatives such as racitol and maltitol; aldonic acid and its lactones such as gluconic acid and gluconic acid γ-lactone; aldalic acid and its lactones such as rivalaic acid, arabinaric acid, and galactaric acid; uronic acids such as glucuronic acid, galacuronic acid, and ianthanuronic acid; ester derivatives such as trehalose octaacetate, sucrose octaacetate, and cellobiose octaacetate; and ether derivatives in which the hydroxyl group is O-alkylated. Non-reducing carbohydrates include those having D or L stereochemical orientation. 【0185】 As used herein, the term “osmotic pressure” refers to a measure of the osmotic pressure of solute particles dissolved in an aqueous solution. Solute particles include both ions and non-ionized molecules. Osmotic pressure is expressed as the concentration (i.e., osmoles) of osmotically active particles dissolved in 1 kg of solvent (i.e., water). Osmotic pressure is expressed herein in units of milliosmoles per kg of water (mOsm / kg). 【0186】 As used herein, the terms “weight / volume percentage” or “%w / v” indicate the weight percentage (in grams) of a single component relative to the total volume (in milliliters) of a mixture containing that component. For example, 500 mg of a component in a total volume of 8 ml is 6.25 w / v%, and 500 mg of a component in a total volume of 5 ml is 10 w / v%. 【0187】 As used herein, the term “polydispersibility” refers to the degree of homogeneity of particle sizes within a sample, such as lentiviral particles. A higher degree of polydispersity indicates lower homogeneity, and a lower degree of polydispersity indicates higher homogeneity. For example, a high level of homogeneity is monodisperse, meaning that lentiviral particles can be considered to be similar in size. As will be understood by those skilled in the art, as polydispersity decreases, the level of homogeneity increases. Therefore, a lower degree of polydispersity indicates a higher level of homogeneity. For example, a formulation with a polydispersity of 15% is less homogeneous than a formulation with a polydispersity of 10%. A low level of homogeneity means that the particle population can be considered polydisperse, meaning that it contains significantly different sizes. 【0188】 As used herein, the terms “binding domain” or “antibody molecule” refer to a protein, such as an immunoglobulin chain or a fragment thereof, that contains at least one immunoglobulin variable domain sequence. The terms “binding domain” or “antibody molecule” encompass antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule, for example, comprising multiple immunoglobulin variable domain sequences, where a first sequence of multiple immunoglobulin variable domain sequences has binding specificity to a first epitope, and a second sequence of multiple immunoglobulin variable domain sequences has binding specificity to a second epitope. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity to only two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. 【0189】 The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in naturally occurring antibody molecules, and this usually determines the class to which the antibody belongs. 【0190】 The term "antibody light chain" refers to the smaller of two polypeptide chains found in naturally occurring antibody molecules in their conformations. Kappa (κ) light chains and lambda (λ) light chains refer to the two main antibody light chain isotypes. 【0191】 The term “antigen-binding fragment,” as used herein, refers to one or more portions of an antibody that possess the ability to specifically interact with an antigen epitope (e.g., by binding, steric hindrance, stabilization / destabilization, or spatial distribution). Examples of binding fragments are, but are not limited to, single-strand Fvs(scFv), camel antibodies, disulfide-bound Fvs(sdFv), Fab fragments, F(ab') fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, i.e., bivalent fragments containing two Fab fragments linked by disulfide crosslinking in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; dAb fragments consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs) or other epitope-binding fragments of an antibody. 【0192】 The portion of the CAR described, which includes an antibody or an antibody fragment, may exist in various forms in which the antigen-binding domain is expressed as part of a continuous polypeptide chain containing, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR composition includes an antibody fragment. In other embodiments, the CAR includes an antibody fragment containing an scFv. The precise amino acid sequence boundaries of a given CDR can be determined using one or a combination of several known schemes, including Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme) and Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme). 【0193】 Furthermore, the two domains of the Fv fragment, namely VL and VH, are encoded by separate genes, but they can be joined by a synthetic linker using recombination methods, which allows them to be combined into a single protein chain. In this single protein chain, the VL and VH regions pair up to form a monovalent molecule (known as "single-chain Fv" ("scFv"); see, e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment." These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and these fragments are screened for utility in the same manner as intact antibodies. 【0194】 The term “chimeric antigen receptor” or, instead, “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the “intracellular signaling domain”) comprising a functional signaling domain derived from a stimulating molecule as defined below. In some embodiments, the domains of the CAR polypeptide construct are on the same polypeptide chain, e.g., including a chimeric fusion protein. In some embodiments, the domains of the CAR polypeptide construct are not contiguous with each other, but are on different polypeptide chains, e.g., as provided for RCAR. 【0195】 In some embodiments, the cytoplasmic signaling domain includes a primary signaling domain (e.g., a CD3-ζ primary signaling domain). In some embodiments, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In some embodiments, the co-stimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino terminus (N terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N terminus of the extracellular antigen-recognition domain, the leader sequence being optionally cleaved from the antigen-recognition domain (e.g., scFv) during cell processing and cell membrane localization of the CAR. 【0196】 A CAR containing an antigen-binding domain (e.g., scFv, single-domain antibody, or TCR (e.g., TCR alpha-binding domain or TCR beta-binding domain)) that targets a specific tumor marker X (where X may be a tumor marker as described herein) is also referred to as an X CAR. For example, a CAR containing an antigen-binding domain that targets BCMA is referred to as a BCMA CAR. CARs can be expressed in any cell, for example, immune effector cells as described herein (e.g., T cells or NK cells). 【0197】 The term “signaling domain” refers to a portion of the functionality of a protein that functions by transmitting information within a cell to modulate cellular activity via a signaling pathway defined by generating secondary messengers or acting as effectors in response to such messengers. The term “stimulating molecule” refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides a cytoplasmic signaling sequence that stimulates the activation of immune cells in at least some aspects of immune cell signaling pathways. In some aspects, the signal is a primary signal, e.g., triggered by the binding of a TCR / CD3 complex to a peptide-loaded MHC molecule, which leads to the mediation of T cell responses, including, but not limited to, proliferation, activation, and differentiation. Primary cytoplasmic signaling sequences that function in a stimulating manner (also referred to as “primary signaling domains”) may include signaling motifs known as immunoreceptor-activated tyrosine motifs or ITAMs. Examples of ITAM-containing cytoplasmic signaling sequences include, but are not limited to, those derived from CD3ζ, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R16), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In certain CARs, the intracellular signaling domain in any one or more CARs described herein includes an intracellular signaling sequence, such as the primary signaling sequence of CD3ζ. In some CARs of this disclosure, the primary signaling sequence of CD3ζ is a human sequence or equal residues from non-human species, such as mice, rodents, monkeys, and apes. 【0198】 The term "intracellular signaling domain" refers to the intracellular portion of a molecule when used herein. The intracellular signaling domain generates signals that promote the immune effector functions of CAR-containing cells, such as CAR T cells. Examples of immune effector functions (e.g., in CAR T cells) include cytolytic activity and helper activity, including cytokine secretion. 【0199】 In some embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains may include those derived from molecules involved in the primary stimulus or antigen-dependent stimulus. In some embodiments, the intracellular signaling domain may include a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains may include those derived from molecules involved in the co-stimulatory signal or antigen-independent stimulus. For example, in the case of CAR T cells, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from the co-receptor or co-stimulatory molecule. 【0200】 The primary intracellular signaling domain may contain signaling motifs known as immunoreceptor-activating tyrosine motifs or ITAMs. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3ζ, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 beta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. 【0201】 The terms "ζ" or alternatively "ζ chain," "CD3ζ," or "TCRζ" refer to CD247. Swiss-Prot accession number P20963 provides an exemplary human CD3ζ amino acid sequence. "ζ stimulating domain" or alternatively "CD3ζ stimulating domain" or "TCRζ stimulating domain" refer to the stimulating domain of CD3ζ or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions). In some embodiments, the cytoplasmic domain of ζ includes residues 52-164 of GenBank accession number BAG36664.1 or its variants (e.g., molecules having mutations, e.g., point mutations, fragments, insertions, or deletions). Alternatively, or further, the terms “ζ” or alternatively “ζ chain,” “CD3-ζ” (or “CD3ζ, CD3 ζ or CD3z”) or “TCR-ζ” are defined as equal residues from the protein provided as GenBank accession number BAG36664.1 or from non-human species, e.g., mice, rodents, monkeys, apes, etc., and the “ζ stimulating domain” or alternatively “CD3-ζ stimulating domain” or “TCR-ζ stimulating domain” are defined as amino acid residues from the cytoplasmic domain of the ζ chain or its functional derivatives sufficient to functionally transmit the initial signals necessary for T cell activation. In some embodiments, the cytoplasmic domain of ζ includes equal residues from residues 52-164 of GenBank accession number BAG36664.1 or their functional orthologues from non-human species, e.g., mice, rodents, monkeys, apes, etc. 【0202】 The term "costimulatory molecule" refers to a congenital binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by T cells, such as proliferation, but is not limited to these. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, and CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12 contribute to efficient immune responses. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such co-stimulatory molecules include ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, I It contains ligands that specifically bind to TGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83. 【0203】 The co-stimulatory intracellular signaling domain can be the intracellular portion of a co-stimulatory molecule. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activators (SLAM proteins), and activated NK cell receptors. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and CD83. 【0204】 An intracellular signaling domain may include the entire intracellular portion of its originating molecule, the entire innate intracellular signaling domain, or a functional fragment or derivative thereof. 【0205】 The "complementarity-determining domain" or "complementarity-determining region" ("CDR") interchangeably refers to the hypervariable regions of the VL and VH. The CDR is the target protein binding site of the antibody chain, and it is specific to that target protein. Each human VL or VH has three CDRs (CDR1-3, numbered sequentially from the N-terminus), which constitute approximately 15-20% of the variable domain. The CDR is directly responsible for binding specificity because it is structurally complementary to the epitope of the target protein. The remaining section of the VL or VH, the so-called framework region, exhibits lower amino acid sequence variability (Kuby, Immunology, 4th ed., Chapter 4. WH Freeman & Co., New York, 2000). 【0206】 The location of the CDR and framework area can be determined using various known definitions in the art, such as Kabat, Chothia, the International ImMunoGeneTics Database (IMGT) (World Wide Web: imgt.org) and AbM (see, for example, Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J.Mol.Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J.Mol.Biol., 227:799-817 (1992); Al-Lazikani et al., J.Mol.Biol., 273:927-748 (1997)). The definition of antigen-binding sites is also described in the following literature: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J.Mol.Biol., 262:732-745 (1996); Martin et al., Proc.Natl.Acad.Sci.USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). 【0207】 As used herein, the term “contaminating polynucleotide” refers to polynucleotides not derived from the lentiviral vector. Contaminating polynucleotides may include, for example, non-lentiviral polynucleotides derived from the cell from which the lentiviral vector was produced, such as chromosomal mammalian DNA (e.g., human DNA) not present in the lentiviral vector’s transgene or other components. 【0208】 "Derived from," as used herein, refers to the relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first and second molecules and does not imply or include limiting methods or sources relating to the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3ζ molecule, the intracellular signaling domain retains a sufficient CD3ζ structure to have the desired function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include limiting to a specific method of constructing an intracellular signaling domain, and does not mean, for example, that one must start with a CD3ζ sequence and delete unwanted sequences or confer mutations to arrive at an intracellular signaling domain. 【0209】 As used herein, the terms “prevent,” “prevent,” or “prevention” of any disease or disorder mean preventive measures for the disease or disorder; or delaying the onset or progression of the disease or disorder. 【0210】 As used herein, the term “recognizes” refers to an antibody or its antigen-binding fragment that finds and interacts with (e.g., binds to) an epitope, whether the epitope is linear or conformated. The term “epitope” refers to a site on an antigen to which the antibody or antigen-binding fragment of this disclosure specifically binds. Epitopes can be formed from both continuous or discontinuous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from continuous amino acids are typically retained upon exposure to a denaturing solvent, while epitopes formed by tertiary folding are typically lost upon treatment with a denaturing solvent. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include techniques in this field, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)). 【0211】 As used herein, the term “retroviral packaging protein” refers to a protein derived from a retrovirus or a variant thereof that assists in the packaging of nucleic acids (e.g., viral genomes) into an envelope. Examples of retroviral packaging proteins include gag, pol, and rev, e.g., lentiviral gag, pol, and rev, e.g., wild-type proteins or variants thereof, e.g., sequences having at least 80%, 90%, or 95% sequence identity to them. In some embodiments, one or more retroviral packaging proteins are provided as polyproteins. 【0212】 As used herein, the term “retroviral envelope protein” refers to a protein derived from a retrovirus or a variant thereof that can be assembled to form an envelope around a nucleic acid (e.g., a viral genome). An exemplary retroviral envelope protein is env, e.g., wild-type or a variant thereof. In some embodiments, the retroviral envelope protein is a lentiviral envelope protein, e.g., wild-type or a variant thereof. In some embodiments, the retroviral envelope protein is VSV-G, e.g., wild-type or a variant thereof. In some embodiments, the retroviral envelope protein is a pseudotype. In some embodiments, the retroviral envelope protein is derived from a virus different from one or more LTRs of the retroviral packaging protein or nucleic acid to be packaged. 【0213】 As used herein, the terms “specifically bind” and “bind” refer to a binding reaction that determines the presence of a particular protein recognized by a specific ligand, for example, in a heterogeneous population of proteins and other biomolecules. Ligands that specifically bind to proteins (e.g., proteins, proteoglycans, glycosaminoglycans, etc.) bind to proteins with a KD of less than 500 nM. For example, ligands that specifically bind to proteins bind to proteins with a KD of up to 500 nM (e.g., 1 pM to 500 nM). Ligands that do not show specific binding to a protein or its domain show a KD greater than 500 nM (e.g., greater than 600 nm, 700 nM, 800 nM, 900 nM, 1 μM, 100 μM, 500 μM, or 1 mM) to that particular protein or its domain. Various assay formats can be used to determine the affinity of a ligand to a particular protein. For example, solid-phase ELISA assays are commonly used to identify ligands that specifically bind to target proteins. For a description of assay formats and conditions that can be used to determine specific protein binding, see, for example, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999). 【0214】 The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise noted, the terms "patient" and "subject" are used interchangeably herein. 【0215】 The term "therapeutic effector" as used herein refers to a molecule (e.g., RNA or polypeptide) that can exert a therapeutic effect on a target at an effective level. 【0216】 The terms “therapeutically acceptable dose” or “therapeutic effective dose” are interchangeable and refer to a dose sufficient to produce the desired outcome (i.e., reduction in tumor size, inhibition of tumor growth, prevention of metastasis, or inhibition or prevention of viral, bacterial, fungal, or parasitic infection). In some embodiments, a therapeutically acceptable dose does not induce or trigger any undesirable side effects. In some embodiments, a therapeutically acceptable dose may induce or trigger side effects, but only to the extent that they are acceptable to the healthcare provider in light of the patient’s condition. A therapeutically acceptable dose can be determined by administering a low dose initially and then gradually increasing the dose until the desired effect is achieved. “Prophylactically effective dose” and “therapeutically effective dose” can, in some embodiments, prevent the onset of symptoms of a disease, such as cancer-related symptoms, or reduce their severity, respectively. 【0217】 As used herein, the term “transfection” refers to the introduction of DNA into eukaryotic cells. Transfection can be achieved by a variety of means, but are not limited to, calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybren-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and bioristics. 【0218】 As used herein, the terms “to treat,” “to treat,” or “treatment” any disease or disorder mean, in one embodiment, improving the disease or disorder (i.e., delaying, preventing, or reducing the onset of at least one of the disease or its clinical symptoms). In another embodiment, “to treat,” “to treat,” or “treatment” means mitigating or improving at least one physical parameter, including those that are not perceptible to the patient. In yet another embodiment, “to treat,” “to treat,” or “treatment” means modifying the disease or disorder physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing physical parameters), or both. 【0219】 As used herein, the term “viral titer” refers to the number of infectious vector particles or “transduction units” that result in the transcription of a given nucleic acid sequence from a particle to a target cell. Viral titer can be measured by functional assays such as those described in Xiao et al., Exp. Neurobiol 144:1 13-124, 1997 or Fisher et al., J. Virol. 70:520-532, 1996, and the disclosures in either of these documents are incorporated by reference in their entirety. Alternatively, viral titer can also be measured by determining the amount of viral DNA incorporated into the host cell genome, for example, using polymerase chain reaction (PCR) techniques known in the art. 【0220】 As used herein, the term “viral vector” refers to a viral particle capable of introducing nucleic acid molecules into a host. Exogenous gene-carrying viral vectors are typically packaged into infectious viral particles via a viral package by packaging a plasmid using a specific cell line. The infectious viral particle infects a cell and achieves the expression of the exogenous gene. A “recombinant” viral vector refers to a viral vector constructed by genetic engineering techniques. Recombinant viral vectors can be constructed by any suitable method, for example, by transfecting or transfecting a packaging cell line with nucleic acids encoding a viral genome, and then isolating the newly packaged viral particles. It is understood that recombinant engineering techniques can be carried out at an upstream stage in the production of the viral vector itself. For example, plasmids can be produced using recombinant engineering techniques, then the plasmids can be produced on a large scale, and finally the plasmids can be introduced into a cell line for packaging to produce a viral vector. 【0221】 The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, such as the self-inactivating lentiviral vector provided in Milone et al., Mol.Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in medical facilities include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology and Lentigen's LENTIMAX® vector system. Non-clinical lentiviral vectors are also available and will be known to those skilled in the art. 【0222】 Lentivirus production method This disclosure provides, in particular, an improved method for producing lentiviral vectors. The following general procedure can be used. First, host cells can be cultured. Exemplary types of host cells, such as human cells lacking the large T antigen, are described in more detail in the section entitled “Host Cells” herein. While we do not wish to be bound by theory, it is thought that host cells lacking the large T antigen may offer manufacturing advantages compared to host cells containing the large T antigen (see, for example, Example 1 of PCT / IB2022 / 053880, which is incorporated herein by reference in its entirety). 【0223】 In some embodiments, in order to produce a large number of cells, host cells are cultured sequentially in larger containers (e.g., bioreactors) until a sufficiently large number of cells are produced. 【0224】 Once a sufficient number of host cells are obtained, the desired nucleic acid can be introduced into the host cells. The nucleic acid may be introduced by transfection, for example, using the FectoVIR®-AAV transfection reagent, as described in the section titled “Transfection” herein. The transfected nucleic acid may contain the viral genome to be packaged, which contains the therapeutic gene of interest and sufficient LTR sequences for packaging into viral particles. Additional nucleic acids that may be introduced into the host cells include plasmids that facilitate packaging, such as plasmids encoding viral gag, pol, env, and rev. In some embodiments, the pH of the culture medium may be changed downward, for example, from about 7.1 to about 6.7, before transfection, as described in the section titled “Culture Conditions and Transfection Conditions” herein. The cells then begin to produce lentiviruses. 【0225】 After transfection, nucleases such as benzonase may be added to the culture medium, for example, as described in the section titled "Culture Medium." While we do not wish to be bound by theory, in some embodiments, the cell culture medium is a source of contaminating nucleic acids for the final lentiviral preparation; for example, the culture medium may contain host cell DNA derived from lysed host cells. Therefore, adding benzonase to the cell culture medium allows for the degradation of contaminating nucleic acids, thereby improving the purification of the lentivirus. 【0226】 Next, to begin lentivirus purification, the lentivirus can be collected from the host cell culture. In some embodiments, lentivirus collection involves separating the supernatant or cell culture medium from the cells. In some embodiments, the cells are not lysed before clarification. In some embodiments, the cells may be lysed and the lysate clarified. 【0227】 The purification of lentiviruses from cell culture media or cell lysates typically involves several sequential purification steps. These purification steps may include filtration (e.g., ultrafiltration) and chromatography. In some embodiments, arginine may be added during the purification process, for example, before or after the filtration or chromatography step. The addition of arginine is described, for example, in a section titled "Purification." While we do not wish to be bound by theory, in some embodiments, arginine stabilizes lentiviral vectors and / or reduces their aggregation. 【0228】 Purified lentiviruses can be used for a variety of applications. For example, lentiviruses can be used to deliver genes to cells ex vivo, for instance, to generate CART cells from immune effector cells derived from apheresis samples. Another example is that lentiviruses can be administered to a target subject to deliver genes in situ. For example, lentiviruses can be used for in vivo CART production. In some embodiments, lentiviruses are suitable for administration in human subjects, and for example, a lentivirus encoding CAR can be administered to a subject to enable the introduction of CAR-coding nucleic acids into immune effector cells in the subject's body. 【0229】 Naturally occurring lentiviruses are a genus of viruses belonging to the Retroviridae family and are characterized by long incubation periods. Lentiviruses can generally deliver large amounts of genetic information to the DNA of host cells. Examples of lentiviruses include: HIV (human immunodeficiency virus; including HIV1 and HIV2), the cause of human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep; canine arthritis-encephalitis virus, which causes immunodeficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immunodeficiency in cats; bovine immunodeficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infections in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in great apes. Diseases caused by these viruses are characterized by long incubation periods and prolonged courses. Typically, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T cells). 【0230】 A method for producing lentiviruses is described, for example, in international application PCT / IB2022 / 053880, for example in Examples 1 to 13 therein, and the application, including the examples, is incorporated by reference in its entirety. 【0231】 Transgene In some embodiments, the lentiviruses or lentiviral vectors disclosed herein may include nucleic acids, such as transgenes, for example, transgenes encoding proteins. The nucleic acids may include, for example, transgenes as described in the section titled “Transgenes” herein. The transgenes may be functionally linked to a promoter sequence. The nucleic acids may also include one or more (e.g., two) LTR sequences. While we do not wish to be bound by theory, LTRs may facilitate the insertion of the transgene and promoter into the host cell genome. The LTR sequences may include wild-type lentiviral LTR sequences or variants thereof. For example, a 3' LTR may include a deletion that self-inactivates the virus after integration. Furthermore, a 5' LTR may be a chimeric LTR. In some embodiments, the transgenes may be integrated into the chromosomal DNA of a target cell. 【0232】 Exemplary transgenes include those encoding chimeric antigen receptors (CARs). A CAR may contain several domains, including an antigen-binding domain, a transmembrane domain, and one or more signaling domains. In such cases, the signaling domains may include one or more primary signaling domains (such as a CD3-ζ stimulating domain) and / or one or more co-stimulatory signaling domains (ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, or CD83). 【0233】 In some embodiments, the transgene, such as a CAR-containing transgene, may encode an antigen-binding domain (e.g., scFv) that binds to a specific target protein or carbohydrate. Examples of antigens include: CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvll), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B-cell maturation (BCMA), Tn antigen ((TnAg) or (GalNAca-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG 72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117) Interleukin-13 receptor subunit α-2, mesothelin, interleukin-1 receptor α (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor β (PDGFR-β), stage-specific fetal antigen-4 (SSEA-4), CD20, folate receptor α, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), mucin 1, cell surface-related (MUC1), epidermal growth factor receptor (EGFR), nerve The oncogene fusion consists of cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), ephrin B2, fibroblast-activating protein α (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropain) subunits, β-type, 9 (LMP2), glycoprotein 100 (gp100), cleavage cluster region (BCR), and Abelson mouse leukemia virus oncogene homolog 1 (Abl) (bcr-abl). Protein, tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D) Chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide portion of globoH glycoceramide (GloboH), mammary gland differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cell receptor 1 (HAVCR1), adrenergic receptor β3 (ADRB3), panexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, gene locus K9 (LY6K), olfactory receptor 51 E2 (OR51)E2), TCRγ Alternate Reading Frame Protein (TARP), Wilms Tumor Protein (WT1), Cancer / Testicular Antigen 1 (NY-ESO-1), Cancer / Testicular Antigen 2 (LAGE-1a), Melanoma-Associated Antigen 1 (MAGE-A1), ETS Translocation Mutation Gene 6 located on chromosome 12p (ETV6-AML), Sperm Protein 17 (SPA17), X Antigen Family, Member 1A (XAGE1), Angiopoietin-Binding Cell Surface Receptor 2 (Tie 2) Melanoma carcinoma testicular antigen-1 (MAD-CT-1), Melanoma carcinoma testicular antigen-2 (MAD-CT-2), Fos-related antigen 1, Tumor protein p53 (p53), p53 variant, Prostain, Survivin, Telomerase, Prostate cancer tumor antigen-1, Melanoma antigen 1 recognized by T cells, Rat sarcoma (Ras) variant, Human telomerase reverse transcriptase (hTERT), Sarcoma translocation breakpoint, Apoptosis-induced melanoma inhibitor (ML-IAP), ERG (transmembrane type Protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc trimyelomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 (SART3) recognized by T cells, paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), synovial sarcoma, X-cleavage 2 (SSX2), late glycation end product receptor (RAGE-1), renal ubiquitous protein 1 (RU1), renal ubiquitous protein 2 (RU2), regmine, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 variant (muthsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), and immunoglobulin λ-like polypeptide 1 (IGLL1). 【0234】 In some embodiments, the lentiviral vector described herein comprises two or more transgenes, for example, a first transgene encoding a first CAR, for example, a CD19 CAR, and a second transgene encoding a second CAR, for example, a CD22 CAR. 【0235】 In some embodiments, the dual-CAR lentiviral vector described herein encodes two different CARs, e.g., a CD19 CAR and a CD22 CAR. In some embodiments, the two CARs are part of a single open reading frame, separated by a protease cleavage site, e.g., an autocleavage site, e.g., a P2A site. In some embodiments, the open reading frame encodes, from N-terminus to C-terminus, a first leader sequence, a first scFv (e.g., one that binds to CD22), optionally a first hinge domain, a first transmembrane domain, a first co-stimulatory domain (e.g., 4-1BB), a first primary signaling domain (e.g., CD3-ζ), a protease cleavage site (e.g., P2A), a second leader sequence, a second scFv (e.g., one that binds to CD19), optionally a second hinge domain, a second transmembrane domain, a second co-stimulatory domain (e.g., 4-1BB), and a second primary signaling domain (e.g., CD3-ζ). In some embodiments, the first and second leader sequences have the same sequence. In some embodiments, the first and second hinge domains have the same sequence. In some embodiments, the first and second transmembrane domains have the same sequence. In some embodiments, the first and second co-stimulatory domains have the same sequence. In some embodiments, the first and second primary signaling domains have the same sequence. 【0236】 Additional CARs that may be encoded by the transgenes described herein are provided, for example, in the section titled “CAR Targets” herein. 【0237】 In some embodiments, the lentiviral vectors described herein encode siRNA or shRNA that target nucleic acids in immune effector cells. For example, the siRNA or shRNA may target nucleic acids encoding TCR and / or HLA and / or inhibitory molecules (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFβ) in T cells. The expression systems for siRNA and shRNA, as well as exemplary shRNAs, are described, for example, in paragraphs 649 and 650 of the brochure International Publication No. 2015 / 142675, filed on March 13, 2015, and are incorporated in their entirety by reference. These nucleic acids can also be targeted, for example, using the CRISPER system, zinc finger nucleases, or TALENs. The immune effector cells may be autologous or allogeneic to the target to be treated. 【0238】 In some embodiments, the lentiviral vectors described herein include or encode one or more inhibitors of methylcytosine dioxygenase genes (e.g., Tet1, Tet2, or Tet3). The use of such compositions and methods to enhance the functional activity of engineered cells (e.g., genetically modified antigen-specific T cells such as CAR-T cells) is also intended. Disruption of a single allele of the Tet gene (Tet1, Tet2, or Tet3) reduces the total level of 5-hydroxymethylcytosine, leading to enhanced proliferation, regulation of effector cytokine production, and degranulation, thereby increasing the proliferation and / or function of CAR T cells. In some embodiments, the expression and / or function of Tet2 in cells is reduced or eliminated. 【0239】 In some embodiments, the inhibitors of Tet1, Tet2, and / or Tet3 are siRNA or shRNA specific to the nucleic acid encoding Tet1, Tet2, Tet3, or siRNA or shRNA. In some embodiments, the siRNA or shRNA includes a sequence complementary to the Tet2 mRNA sequence, and includes, for example, the target sequences of shRNA listed in Table 4 of International Publication No. 2017 / 049166, the entirety of which this application, including Table 4, is incorporated herein by reference. In some embodiments, the inhibitors of Tet1, Tet2, and / or Tet3 are (1) a gene editing system that targets one or more sites in the gene encoding Tet1, Tet2, and / or Tet3 or their regulatory elements, for example, Tet2 or its regulatory elements; (2) a nucleic acid encoding one or more components of the gene editing system; or (3) a combination thereof. In some embodiments, the gene editing system is selected from the group consisting of CRISPR / Cas9 systems, zinc finger nuclease systems, TALEN systems, and meganuclease systems. 【0240】 In some embodiments, the lentiviral vectors described herein include a transgene, for example, a transgene encoding a chimeric antigen receptor (CAR), and further include siRNA or shRNA that targets nucleic acids in immune effector cells. 【0241】 Characteristics of lentiviral vectors In some embodiments, the lentiviral vector is characterized by a hydrodynamic radius of 90–200 nm, as measured by dynamic light scattering (DLS). For example, the lentiviral vector may maintain a hydrodynamic radius of 90–200 nm within a temperature range of 25°C–37°C. 【0242】 In some embodiments, lentiviral vectors are characterized by polydispersity of 10% to 25%. For example, lentiviral vectors can maintain polydispersity of 10% to 25% within a temperature range of 25°C to 37°C. 【0243】 In some embodiments, the lentiviral vector maintains a concentration of about 70% to about 100% of the concentration of the lentiviral vector in the aqueous composition prior to the freeze / thaw cycle after 3, 6, or 9 freeze / thaw cycles, each of which comprises freezing the aqueous composition and then thawing the aqueous composition at room temperature. 【0244】 In some embodiments, lentiviruses prepared, purified or stored using any of the methods or formulations disclosed herein may have a lower vector copy number (VCN), for example, at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, or at least 60% lower VCN, compared to lentiviruses not produced, purified or stored by the methods or formulations disclosed herein, when tested at an MOI of 1. 【0245】 Lentiviral vector packaging system A packaging system can be used to package nucleic acids, such as RNA encoding a transgene, into a lentiviral vector. Therefore, the systems and methods described herein may include, for example, a lentiviral packaging system comprising at least one plasmid suitable for the production of a lentiviral vector, such as a lentiviral vector optionally containing a transgene. Various lentiviral components useful for the production of lentiviral vectors are known in the art. See, for example, Zufferey et al., 1997, Nat. Biotechnol. 15:871-875 and Dull et al., 1998, J. Virol. 72(11):8463-8471. A variety of functions suitable for lentiviral vector production can be conferred to host cells in a lentiviral packaging system comprising one or more nucleic acids (e.g., plasmids), for example, at least one, two, three, or four plasmids, where one plasmid encodes a retroviral envelope protein (Env plasmid), one plasmid encodes one or more retroviral packaging proteins, for example, Gag and Pol proteins (packaging plasmid or Gag-Pol plasmid), and one plasmid encodes one or more plasmids comprising a lentiviral Rev protein (Rev plasmid) and at least one transgene of interest (TOI) expression cassette. In some embodiments, the lentiviral packaging system further comprises at least one, two, three, or four plasmids, or the methods described herein include the use of them. In some embodiments, the lentiviral packaging system further comprises a fifth plasmid, or the methods described herein include the use of it. In certain embodiments, the methods described herein include transfecting five plasmids into host cells, where the fifth plasmid does not encode a protein in the lentiviral vector packaging system.In some embodiments, the lentiviral packaging system comprises one or more nucleic acids (e.g., plasmids), e.g., five plasmids, such that, as described in Rout-Pitt et al., J Biol. Methods 5(2):1-9, 2018), one plasmid encodes an expression vector, one plasmid encodes Tat (e.g., pcDNATat), one plasmid encodes a Rev protein (e.g., pHCMV-Rev), one plasmid encodes gagpol (e.g., pHCMV-gagpol), and one plasmid also encodes VSV-G (e.g., pVSVG). In some embodiments, the plasmids may comprise a dual gene expression cassette, e.g., a bisistron cassette, e.g., a bisistron construct encoding two target transgenes. In some embodiments, the first target transgene encodes a first CAR, e.g., CD19 CAR, and the second target transgene encodes a second CAR, e.g., CD22 CAR. In some embodiments, the retroviral packaging protein is derived from a lentivirus, for example, a lentiviral packaging protein, such as lentiviral gag and pol proteins. In some embodiments, the lentiviral gag protein is a wild-type lentiviral gag protein, and in other embodiments, it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral pol protein is a wild-type lentiviral pol protein, and in other embodiments, it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the rev protein is a wild-type rev protein, and in other embodiments, it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral vector may be a pseudotype vector containing a modified envelope protein, for example, an envelope protein derived from a different virus or a chimeric envelope protein, for example, the Env plasmid may encode a VSV-G Env protein, for example, a wild-type VSV-G protein or a modified variant. 【0246】 In some embodiments, the lentiviral vector is prepared using a packaging system containing pMDLgpRRE, pRSV-Rev, and pMD.G plasmids (Dull al., op. cit.), but instead of the ampicillin gene, for example, a kanamycin resistance marker, such as a marker conferring resistance to both kanamycin and neomycin, or neomycin phosphotransferase II is used. 【0247】 In some embodiments, the systems described herein include, for example, a transfer vector containing a kanamycin resistance marker, such as a marker conferring resistance to both kanamycin and neomycin, or neomycin phosphotransferase II, instead of the ampicillin gene. In some embodiments, the transcription vector includes a sequence derived from a pELPS construct, such as disclosed in, for example, International Publication No. 2017087861A or Milone al., Mol. Ther. 17(8):1453-1464, 2009 (each incorporated herein by reference in its entirety). In some embodiments, the therapeutic protein is encoded in a self-inactivating transfer vector containing one or more, for example all, lentivirus 5'LTR (e.g., cleaved lentivirus 5'LTR), lentivirus 3'LTR, cPPT, and WPRE. In some embodiments, the transcription vector is missing one or more, or all, of the genes encoding a bacterial-active promoter (e.g., all of the T7 promoter, T3 promoter, and lac promoter), an M13 primer binding site (e.g., both the M13 forward primer binding site and the M13 reverse primer binding site), a phage origin (e.g., f1 ori), and a fluorescent protein (e.g., GFP, e.g., EGFP). In some embodiments, the transfer vector is missing both the CAP binding site and the lac operator. In some embodiments, the transfer vector is missing the T7 promoter, M13 forward primer binding site, f1 ori, CAP binding site, IPTG-inducible promoter, lac operator, M13 reverse primer binding site, T3 promoter, and EGFP, except that the transfer vector comprises a pELPS construct as disclosed in International Publication No. 2017087861, and the transfer vector encodes a therapeutic protein, e.g., CAR.In some embodiments, the transfer vector has one or more of the following properties: (a) it is more stable than a otherwise similar control transfer vector; (b) it produces less cytotoxicity than a otherwise similar control transfer vector; or (c) it results in a lower vector copy number (VCN) when incorporated into target cells, for example, as described herein. In some embodiments, the control transfer vector comprises a T7 promoter, an M13 forward primer binding site, an f1 ori, a CAP binding site, an IPTG-inducible promoter, a lac operator, an M13 reverse primer binding site, and a T3 promoter. 【0248】 In some embodiments, the gene expression cassette encodes a protein, such as a chimeric antigen receptor (CAR). In some embodiments, the gene expression cassette encodes two proteins, such as a first CAR and a second CAR. Exemplary transgenes suitable for gene expression cassettes are described herein. 【0249】 Transfection In some embodiments, a lentiviral packaging system suitable for lentiviral vector production is transfected to multiple host cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells (e.g., multiple Expi293F cells growing in suspension under serum-free conditions), by transfection, e.g., transient or stable transfection, of a lentiviral packaging system suitable for lentiviral vector production. In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95% of host cells, e.g., HEK293 cells, e.g., Expi293F cells, are transfected. Methods of transfection or infection are well known to those skilled in the art. In some embodiments, for transfection, at least 0.3 μg, at least 0.4 μg, at least 0.5 μg, at least 0.6 μg, at least 0.7 μg, at least 0.8 μg of cells, at least 0.9 μg, or at least 1.0 μg of lentiviral packaging system per million cells are provided. In some embodiments, a transfection reagent is used to transfect host cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells. In some embodiments, a transfection reagent is used. Transfection reagents are known in the art and are available from suppliers. Examples of transfection reagents, but not limited to, include Lipofectamine® (Invitrogen), Polifectamine, LentiTran (Origene), PEIpro® (Polyplus), FectoVIR®-AAV (Polyplus), and ProFection® (Promega). In some embodiments, the transfection reagent, such as FectoVIR®-AAV, is used at levels of 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 0.5 μl, 0.6 μl, 0.7 μl, 0.8 μl, 0.9 μl, or 1.0 μl per million cells.In some embodiments, the packaging system and transfection reagent, such as FectoVIR®-AAV, are used for transfection in ratios of approximately 1:0.5, 1:0.75, 1:1, 1:1.5, 1:2, or any range in between. 【0250】 In some embodiments, the transfection reagent includes a synthetic transfection reagent, which includes chemically based and / or free of animal components. 【0251】 In some embodiments, the transfection reagent includes the FectoVIR®-AAV transfection reagent. FectoVIR®-AAV can be obtained, for example, from Polyplus (850 bd Sebastien Brant, 67400 Illkirch, FRANCE; 1251 Ave of the Americas; 3rd Fl, New York; NY 10020 USA). FectoVIR®-AAV is a synthetic, chemical-based, animal-free transfection reagent. 【0252】 In some embodiments, during transfection, the cells (e.g., Expi293F cells) are approximately 0.5 × 10⁻⁶. 6 cells / mL~1×10 7 cells / mL, 1×10 6 cells / mL~6×10 6 cells / mL, 1×10 6 cells / mL~5×10 6 cells / mL, 1.50×10 6 cells / mL~2.50×10 6 cells / mL, 2.0×10 6 cells / mL~3.0×10 6 cells / mL, 2.0×10 6 cells / mL~2.5×10 6 This is the cell density per mL. In some embodiments, at the time of transfection, the cell population has a viability of at least about 80%, 90%, or 95%. 【0253】 In some embodiments, the PP / IP (physical particle / infectious particle) ratio is less than 500, 700, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, and 2000 after transfection. 【0254】 host cell The methods disclosed herein can be carried out using a viral vector, such as a lentiviral vector, or any host cell suitable for the expression of a lentiviral vector as disclosed herein. In some embodiments, the suitable host cell is a eukaryotic cell, such as a mammalian cell. In some embodiments, the mammalian cell may be a virus, such as a lentivirus, such as a lentiviral vector, or a genetically modified mammalian cell for expressing the lentivirus of interest. Some mammalian cell lines are suitable host cells for recombinant viral expression. Examples of mammalian host cell lines include COS, PER.C6, TM4, VERO, MDCK, BRL-3A, W138, Hep G2, MMT, MRC 5, FS4, CHO, 293T, A431, 3T3, CV-1, C3H10T1 / 2, Colo205, HEK293, HeLa, L cells, BHK, HL-60, FRhL-2, U937, HaK, Jurkat cells, Rat2, BaF3, 32D, FDCP-1, PC12, M1x, mouse myeloma (e.g., SP2 / 0 and NS0), and C2C12 cells, as well as transformed primate cell lines, hybridomas, normal diploid cells, and cell lines derived from in vitro cultures of primary tissues and primary explants. In some embodiments, the host cells are HEK293 cells, including cells derived from HEK293 cells, such as 293F cells, such as Expi293F cells. In some embodiments, at least 80%, at least 85%, at least 90%, at least 90%, and at least 95% of the host cells in the culture express large T antigens, such as polyomavirus large T antigens, such as SV40 large T antigens, such as mutant SV40 large T antigens. In some embodiments, at least 99%, at least 98%, at least 97%, at least 96%, and at least 95% of the host cells in the culture do not express large T cell antigens. In some embodiments, the host cells are suitable for growth in suspension. 【0255】 Culture process The cell lines described herein can be cultured under conditions that enable the production of high-titer lentiviral vector particles. Eukaryotic cells, such as mammalian cells, such as HEK293 cells, such as Expi293F cells, can be cultured as non-anchorage-dependent cells that grow freely in suspension for most of the culture; or as anchorage-dependent cells that require attachment to a solid substrate for their growth (e.g., as a monolayer). 【0256】 In some embodiments, a microcarrier system may be used to adapt to cell proliferation. In some embodiments, the microcarrier system may include suspension culture, e.g., large-scale suspension culture. The suspension culture may be operated in an open or closed system, e.g., a batch or fed-batch closed system. In some embodiments, no nutrients are added, and waste is not removed throughout the culture period. In some embodiments, waste, including cells, products, by-products, and toxic metabolites, is not removed, but nutrients are continuously supplied to the system to extend the growth cycle. In some embodiments, the culture system may be open, e.g., a continuous system, e.g., a perfusion system or a chemostat system. In some embodiments, the system may comprise one or more cell retention devices. Cell retention devices may include, for example, microcarriers, fine mesh spin filters, hollow fibers, plate membrane filters, sedimentation tubes, ultrasonic cell retention devices, etc. In some embodiments, the cell concentration in the bioreactor is higher than the cell concentration present in the supernatant collected from the bioreactor. In some embodiments, the cell concentration in the bioreactor is substantially the same as that in the supernatant collected from the bioreactor. 【0257】 In continuous fermentation processes, a prescribed culture medium is often continuously added to a bioreactor while an equal volume of culture medium is simultaneously removed to recover the product. Continuous culture generally maintains cells at a constant cell density during the logarithmic phase of growth. In continuous or semi-continuous culture methods, it is possible to modulate one or any number of factors that affect cell growth or final product concentration. For example, one approach may allow restricting the carbon source and allowing all other parameters to mitigate metabolism. In some systems, many factors affecting growth may change continuously, but the cell concentration, measured by the turbidity of the medium, is kept constant. Continuous systems often maintain steady-state growth, so the cell growth rate is often balanced against cell loss due to the removal of medium from the culture. Methods for modulating nutrients and growth factors for continuous culture processes are known, and various methods are known in the art. 【0258】 In some embodiments, the suspension cell culture contains only cells in the suspension. In some embodiments, the suspension cell culture may contain, for example, a small number of cells (e.g., less than 1%) that transiently adhere to the surface. 【0259】 "Cell culture" can refer to any in vitro culture of cells. This term includes serial cell lines (e.g., those with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and other cell populations maintained in vitro. 【0260】 In some embodiments, the systems or methods described herein utilize packaging cells or packaging cell lines for the production of viral vectors. The cell lines can be stably transfected with elements for lentiviral vector production, such as retroviral packaging proteins and retroviral envelope proteins. Typically, such packaging cells include one or more expression cassettes capable of expressing viral proteins (e.g., gag, pol, and env), but the expression cassettes do not contain packaging signals. The packaging cells may be cells cultured in vitro. Using a packaging cell line, a producer cell line for lentiviral particle production can be constructed by providing, for example, at least one plasmid containing at least one transgene-of-interest (TOI) expression cassette. In some embodiments, the producer cells transiently express a plasmid (e.g., a transfer plasmid) that encodes a therapeutic effector and contains sufficient LTR sequences to enable the packaging of RNA containing the LTR into a viral vector. In some embodiments, the producer cell line stably expresses an expression cassette that encodes a therapeutic effector and contains sufficient LTR sequences to enable the packaging of RNA containing the LTR into a viral vector. 【0261】 Culture medium The methods of the present disclosure may be carried out using any medium suitable for culturing eukaryotic cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells (e.g., supporting cell proliferation and maintenance under the conditions of the present disclosure). The terms “cell culture medium” and “culture medium” (or simply “medium”) refer to eukaryotic cells, e.g., mammalian cells, which refer to a nutrient solution used for the proliferation of, e.g., HEK293 cells, e.g., Expi293F cells, and typically provide at least one component from one or more of the following categories: (1) salts that contribute to the osmotic pressure of the medium (e.g., sodium, potassium, magnesium, calcium, etc.); (2) energy sources, usually in the form of carbohydrates such as glucose; (3) all essential amino acids and usually a basic set of 20 amino acids; (4) vitamins and / or other organic compounds required in low concentrations; and (5) trace elements (trace elements are usually defined as inorganic compounds that are usually required in very low concentrations, usually in the micromolar range). Such culture medium compositions are known in the art (see, for example, Mather, JP, et al. (1999) “Culture media, animal cells, large scale production,” Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, Vol. 2: 777-785, which is incorporated herein by reference in its entirety). The nutrient solution may optionally be supplemented with one or more components from any of the following categories: (a) animal serum; (b) hormones and other growth factors, e.g., insulin, transferrin, and epidermal growth factor; and (c) hydrolysates of plants, yeast, and / or tissues (including their protein hydrolysates). 【0262】 In some embodiments, the culture medium may contain serum, such as fetal bovine serum (FBS). In some embodiments, the culture medium is serum-free. In some embodiments, the medium is of a known composition and does not contain, for example, animal-derived components. As used herein, “animal-derived” components are any components produced by intact animals (e.g., proteins isolated and purified from serum) or any components produced using components produced by intact animals (e.g., amino acids produced using enzymes isolated and purified from animals to hydrolyze plant-derived material). In contrast, proteins produced in vitro in cell cultures (e.g., in recombinant yeast or bacterial cells or in established serial eukaryotic cell lines, whether recombinant or non-recombinant) using a medium that has the sequence of an animal protein (i.e., has genomic origin in animals) but does not contain components produced by intact animals or a medium isolated and purified from intact animals are not “animal-derived” components. 【0263】 A culture medium of known composition is a medium in which all components have known chemical structures. Culture media of known composition are available from suppliers such as Sigma, ThermoFisher, Invitrogen, JRH Biosciences, and Gibco. In some embodiments, the medium is FreeStyle® 293 Expression Medium. In some embodiments, a medium containing concentrated serum, for example, nutrients at concentrations higher than those typically required and normally provided to growth cultures, may be used. In some embodiments, the medium may contain amino acids obtained from any source or method known in the art. 【0264】 In some embodiments, an enzyme, such as a nuclease, such as an endonuclease, such as a recombinant endonuclease, such as benzonase®, may be added to the culture medium. In some embodiments, at least 2 U / ml, at least 5 U / ml, at least 7 U / ml, at least 10 U / ml, at least 15 U / ml, at least 20 U / ml, at least 25 U / ml, at least 25 U / ml, at least 30 U / ml, at least 35 U / ml, at least 40 U / ml, at least 45 U / ml, at least 50 U / ml, at least 55 U / ml, or at least 60 U / ml of benzonase® is added. In some embodiments, 2 U / mL to 10 U / mL, 10 U / mL to 20 U / mL, 20 U / mL to 30 U / mL, 30 U / mL to 40 U / mL, 40 U / mL to 50 U / mL, or 50 U / mL to 60 U / mL of benzonase® is added. In some embodiments, benzonase® is added at approximately 5–40, 10–40, 10–30, 20–30, or approximately 20 or 24 hours after transfecting host cells, e.g., Expi293F cells. In some embodiments, benzonase is added at a concentration of 3–7 U / mL (e.g., approximately 5 U / mL) 20–30 hours (e.g., approximately 24 hours) after transfecting host cells. In some embodiments, benzonase is added at a concentration of 3–7 U / mL (e.g., approximately 5 U / mL) 1–5 hours (e.g., approximately 3 hours) after transfecting host cells. In some embodiments, benzonase is added at a concentration of 3–7 U / mL (e.g., approximately 5 U / mL) 4–8 hours (e.g., approximately 6 hours) after transfecting host cells. In some embodiments, benzonase is added at a concentration of 12–18 U / mL (e.g., about 15 U / mL) 1–5 hours (e.g., about 3 hours) after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 12–18 U / mL (e.g., about 15 U / mL) 4–8 hours (e.g., about 6 hours) after transfecting the host cells.In some embodiments, benzonase is added at a concentration of 12–18 U / mL (e.g., about 15 U / mL) 20–30 hours (e.g., about 24 hours) after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 20–30 U / mL (e.g., about 25 U / mL) 1–5 hours (e.g., about 3 hours) after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 20–30 U / mL (e.g., about 25 U / mL) 4–8 hours (e.g., about 6 hours) after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 20–30 U / mL (e.g., about 25 U / mL) 20–30 hours (e.g., about 24 hours) after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 40–60 U / mL (e.g., about 50 U / mL) 1–5 hours (e.g., about 3 hours) after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 40–60 U / mL (e.g., about 50 U / mL) 4–8 hours (e.g., about 6 hours) after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 40–60 U / mL (e.g., about 50 U / mL) 20–30 hours (e.g., about 24 hours) after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 5 U / mL about 3 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 15 U / mL about 3 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 25 U / mL about 3 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 50 U / mL approximately 3 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 5 U / mL approximately 6 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 15 U / mL approximately 6 hours after transfecting the host cells.In some embodiments, benzonase is added at a concentration of 25 U / mL about 6 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 50 U / mL about 6 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 5 U / mL about 24 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 15 U / mL about 24 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 25 U / mL about 24 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 50 U / mL about 24 hours after transfecting the host cells. In some embodiments, a salt, such as MgCl2, is added to benzonase® at a concentration of, for example, about 1–5 mM, 1–3 mM, or about 2 mM. In some embodiments, the methods disclosed herein may include the addition of benzonase® during the production and / or purification process. 【0265】 In some embodiments, the compound can be added to the culture medium to affect culture growth, such as inhibition of growth, induction of differentiation, and induction or suppression of gene expression. In some embodiments, the compound is sodium butyrate. In some embodiments, the cell culture medium described herein contains sodium butyrate. 【0266】 Culture conditions and transfection conditions Culture conditions may include any conditions suitable for maintaining cells (e.g., quiescent or growth state). For example, culture conditions may include, but are not limited to, several parameters, including temperature, oxygen content, nutrient content (e.g., glucose content), pH (e.g., increase or decrease in pH), stirring level (e.g., revolutions per minute), gas flow rate (e.g., air, oxygen, nitrogen gas), oxidation-reduction potential, cell density (e.g., optical density), and cell viability. Changes in culture conditions may include altering, modifying, or changing one or more culture parameters. For example, culture conditions can be changed by increasing or decreasing temperature, increasing or decreasing pH (e.g., adding or removing acid, base, or carbon dioxide), increasing or decreasing oxygen content (e.g., introducing air, oxygen, carbon dioxide, or nitrogen), increasing or decreasing air pressure (e.g., by introducing air, oxygen, carbon dioxide, or nitrogen), increasing or decreasing stirring and / or adding or removing nutrients (e.g., one or more sugars or sugar sources, biomass, vitamins, etc.), increasing or decreasing the ratio of culture to flask volume, or a combination thereof. In some embodiments, changes in culture conditions, such as an increase or decrease in pH, are introduced at a specific point in time during culture, for example, before transfection. In some embodiments, the pH is modified before transfection by the lentiviral packaging system, adjusted to, for example, about 6.0–6.8, for example 6.2–6.8, for example 6.4–6.8, for example 6.7–6.75. 【0267】 Culture volume and culture units The methods disclosed can be carried out in small-scale cell cultures, e.g., laboratory scale, or large-scale cultures, e.g., industrial scale. These methods can be carried out in appropriate culture units, e.g., culture flasks or bioreactors. The bioreactors can be of any size, as long as they are useful for culturing cells, e.g., mammalian cells. In some embodiments, the methods disclosed are highly scalable, for example, multiple mammalian cells may be present in a scaled culture (e.g., at least 1 L, at least 2 L, at least 5 L, at least 10 L, at least 15 L, at least 20 L, resulting in a transduction unit count per ml of culture that is 30%, 40%, 50%, 60%, 70%, or 80% or more of the transduction units per ml of culture in other similar small-scale cultures, e.g., 100 ml, 200 ml, 300 ml, 400 ml, 500 ml). In some embodiments, scale cultures (i.e., those with culture volumes exceeding 50 L) are considered particularly suitable for scaling up from small, laboratory-scale cultures (e.g., 10 L) to production-scale cultures (e.g., 50 L or more) with minimal changes to culture conditions. Internal conditions of the culture unit, including but not limited to pH, pO2, and temperature, are typically controlled during the culture period. A production culture unit refers to the final culture unit used for the production of polypeptides, viruses, and / or other products of interest. The volume of a large-scale production culture unit is generally greater than about 50 L and can be about 100, 200, 300, 500, 800, 1000, 2500, 5000, 8000, 10,000, 12,000 L or more, or any intermediate volume. A suitable culture unit or production culture unit may consist of any material suitable for holding cell cultures suspended in a culture medium under the culture conditions intended herein, and a material that promotes the proliferation and viability of mammalian cells, such as HEK293 cells, such as Expi293F cells. Examples of suitable materials, but not limited to, include glass, plastics, and / or metals. In some embodiments, the material does not interfere with, or significantly or substantially interfere with, the expression and / or stability of the desired product, such as a lentiviral vector. 【0268】 In some embodiments, the cell culture process operates in two or more different culture units, such as using one or more seed culture units followed by a production culture unit. In some embodiments, this process includes transferring the grown seed culture from one or more seed culture units to a larger production unit. In some embodiments, cell growth, production culture unit, and production stage can be achieved in a single physical culture unit, for example, by growing the cells to final production scale and then switching the process to production conditions. Used culture medium is collected at the end of the culture period for downstream processing of lentiviruses or lentiviral vectors. In some embodiments, the collected material may be gathered at 24, 48, 72, 96, or 120 hours after transfection. 【0269】 In some embodiments, downstream processing includes purification, formulation, and / or long-term storage of the lentivirus. In some embodiments, the viral sample collected at the end of the culture period is, for example, about 5 × 10⁻⁶. 6 Transduction units / milliliter (TU / mL) ~ approximately 6 × 10 9 TU / mL (e.g., 5 × 10) 6 TU / mL, 5.5 × 10 6 TU / mL, 6 × 10 6 TU / mL, 6.5 × 10 6 TU / mL, 7×10 6 TU / mL, 7.5 × 10 6 TU / mL, 8 × 10 6 TU / mL, 8.5 × 10 6 TU / mL, 9×10 6 TU / mL, 9.5 × 10 6 TU / mL, 1 × 10 7 TU / mL, 1.5 × 10 7 TU / mL, 2 × 10 7 TU / mL, 2.5 × 10 7 TU / mL, 3 × 10 7 TU / mL, 3.5 × 10 7 TU / mL, 4 × 10 7 TU / mL, 4.5 × 10 7TU / mL, 5×10 7 TU / mL, 5.5×10 7 TU / mL, 6×10 7 TU / mL, 6.5×10 7 TU / mL, 7×10 7 TU / mL, 9×10 7 TU / mL, 1×10 8 TU / mL, 2.5×10 8 TU / mL, 5×10 8 TU / mL, 8×10 8 TU / mL, 1×10 9 TU / mL, 1.5×10 9 TU / mL, 2×10 9 TU / mL, 2.5×10 9 TU / mL, 3×10 9 TU / mL, 4×10 9 TU / mL, 5×10 9 TU / mL or 6×10 9 TU / mL). In some embodiments, the virus harvest collected at the end of the culture period is at least 5×10 6 TU / mL, 5.5×10 6 TU / mL, 6×10 6 TU / mL, 6.5×10 6 TU / mL, 7×10 6 TU / mL, 7.5×10 6 TU / mL, 8×10 6 TU / mL, 8.5×10 6 TU / mL, 9×10 6 TU / mL, 9.5×10 6 TU / mL, 1×10 7 TU / mL, 1.5×10 7 TU / mL, 2×10 7 TU / mL, 2.5×10 7 TU / mL, 3×10 7 TU / mL, 3.5×10 7 TU / mL, 4×10 7 TU / mL, 4.5×10 7 TU / mL, 5×10 7 TU / mL, 5.5×10 7 TU / mL, 6×10 7 TU / mL, 6.5×10 7TU / mL, 7×10 7 TU / mL, 9×10 7 TU / mL, 1×10 8 TU / mL, 2.5×10 8 TU / mL, 5×10 8 TU / mL, 8×10 8 TU / mL, 1×10 9 TU / mL, 1.5×10 9 TU / mL, 2×10 9 TU / mL, 2.5×10 9 TU / mL, 3×10 9 TU / mL, 4×10 9 TU / mL, 5×10 9 TU / mL or 6×10 9 TU / mL at a concentration of, containing lentivirus. In some embodiments, the virus harvest collected at the end of the culture period is 5×10 6 TU / mL to 6×10 6 TU / mL, 6×10 6 TU / mL to 7×10 6 TU / mL, 7×10 6 TU / mL to 8×10 6 TU / mL, 8×y0 6 TU / mL to 9×10 6 TU / m, 9×10 6 TU / mL to 1×10 7 TU / mL, 1×10 7 TU / mL to 2×10 7 TU / mL, 2×10 7 TU / mL to 3×10 7 TU / mL, 3×10 7 TU / mL to 4×10 7 TU / mL, 4×10 7 TU / mL to 5×10 7 TU / mL, 5×10 7 TU / mL to 6×10 7 TU / mL, 6×10 7 TU / mL to 7×10 7 TU / mL, 7×10 7 TU / mL to 9×10 7 TU / mL, 9×10 7 TU / mL to 1×10 8 TU / mL, 1×10 8 It should be noted that there seems to be a "y0" in "8×y0" in the original text which might be a typo. If it's a specific placeholder or a known error, it needs to be further clarified for an accurate translation.TU / mL ~ 2.5 × 10 8 TU / mL, 2.5 × 10 8 TU / mL ~ 5 × 10 8 TU / mL, 5 × 10 8 TU / mL ~ 8 × 10 8 TU / mL, 8 × 10 8 TU / mL ~ 1 × 10 9 TU / mL, 1 × 10 9 TU / mL ~ 1.5 × 10 9 TU / mL, 1.5 × 10 9 TU / mL ~ 2 × 10 9 TU / mL, 2 × 10 9 TU / mL ~ 2.5 × 10 9 TU / mL, 2.5 × 10 9 TU / mL ~ 3 × 10 9 TU / mL, 3 × 10 9 TU / mL ~ 4 × 10 9 TU / mL, 4 × 10 9 ~5×10 9 TU / mL or 5 × 10 9 ~6×10 9 Contains lentivirus at a concentration of TU / mL. 【0270】 Purification method including filtration and chromatography In some embodiments, the disclosure provides a process for purifying lentiviral vectors with improved efficiency, such as recovering a larger volume of lentiviral vectors. In some embodiments, at least one step in the purification process includes improving the purification process by adding a drug, such as an amino acid or a salt thereof, such as arginine or a salt thereof, such as arginine-HCl, to the purified intermediate composition (an intermediate composition including the buffer before the completion of purification) before further purification, such as centrifugation, filtration, or chromatography. In some embodiments, filtration may refer to, but is not limited to, flow filtration, depth filtration, or tangential flow filtration. In some embodiments, chromatography may include, but is not limited to, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, or ion exchange chromatography. 【0271】 In some embodiments, lentiviral vectors produced according to the methods described herein have one or more of the following characteristics: compliant with GMP guidelines, sterile, substantially free of contaminants, suitable for pharmaceutical use, suitable for administration to human subjects, or suitable for ex vivo treatment of human cells. 【0272】 In some embodiments of the methods described herein, a solution or suspension is subjected to a semipermeable membrane (filtration) that allows the solvent and small solute molecules to pass through but retains larger particles, such as viral particles. In some embodiments, the methods described herein use a filter to remove and replace salts, sugars, and non-aqueous solvents, separate what is liberated from bound species, remove low molecular weight substances, and / or cause a rapid change in the ionic and / or pH environment. The filtration step can be used to increase the concentration of vectors in the solution or suspension. In some embodiments, the filtration step is used to increase the concentration of lentivirus particles in the sample. In some embodiments, the methods described herein utilize a process, technique, or combination of techniques that sequentially or simultaneously include a filtration step (e.g., one or more of microfiltration, ultrafiltration, nanofiltration, and diafiltration). In some embodiments, filtration is performed using a flat sheet membrane or hollow fiber. In some embodiments, filtration is performed using an average intermembrane pressure differential of about 0.1 to 0.5 bar (e.g., about 0.1, 0.2, 0.3, 0.4, or 0.5 bar). In some embodiments, filtration is performed at a rate of 4-100 L / m³. 2 For example, this is carried out using loads of approximately 4-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, or 80-90. In certain embodiments of the present disclosure, a filtration step is used to exchange various buffers used in connection with the present disclosure, optionally in combination with chromatography or other purification steps, and optionally to remove impurities from the viral sample. 【0273】 Lentiviral preparations can be produced substantially free of the microorganisms and cells (e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells) on which the lentiviral vector is prepared, using filtration techniques such as those described above and known in the art. Furthermore or alternatively, the lentiviral vector preparations of this disclosure may be treated with a nuclease to produce preparations substantially free of contaminating polynucleotides (e.g., non-lentiviral polynucleotides derived from the cells on which the lentiviral vector was produced, e.g., DNA, RNA, or other polynucleotides not contained in the lentiviral transgene). 【0274】 In some embodiments, compositions substantially free of a particular component contain less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.01%, or less than 0.001% by weight of the component in the composition. In some embodiments, the component in a composition substantially free of a particular component is not detectable, for example, by conventional methods known in the art. 【0275】 For example, buffers used for purification. Various buffers, such as aqueous compositions containing buffers used in the purification of viral vectors, are known in the art and include, but are not limited to, sulfonic acid buffers, such as 1,4-piperazinediethansulfonic acid (PIPES) buffers (PIPES buffers), polyol buffers, Tris buffers, phosphate buffers, acetate buffers, and citrate buffers. In some embodiments, the buffer used in connection with the purification process disclosed herein is a sulfonic acid buffer, such as a PIPES buffer. In some embodiments, the PIPES buffer may contain a buffer, such as PIPES at concentrations of about 10 mM to about 50 mM, about 15 mM to about 40 mM, about 20 mM to about 30 mM, for example, about 20 mM. 【0276】 In some embodiments, the purification buffer may further contain salts, such as sodium chloride (NaCl), magnesium chloride (MgCl2), or calcium chloride (CaCl2), or any combination thereof. The salts may be present in aqueous lentivirus preparations in concentrations of about 1 mM to about 1 M (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 2 It may be present at concentrations of 25 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 450 mM, 475 mM, 500 mM, 525 mM, 575 mM, 600 mM, 625 mM, 650 mM, 675 mM, 700 mM, 725 mM, 750 mM, 775 mM, 800 mM, 825 mM, 850 mM, 875 mM, 900 mM, 925 mM, 950 mM, 957 mM, or 1 M. In some embodiments, the salt concentration is about 25 mM to about 250 mM, about 50 mM to about 75 mM, about 50 mM to about 200 mM, or about 100 mM to about 150 mM (e.g., 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 100 mM, 125 mM, or 150 mM). In some embodiments, the salt concentration may be 50 mM or 75 mM, as needed. 【0277】 In some embodiments, the purified buffer may further contain carbohydrates, such as non-reducing carbohydrates, such as sucrose or trehalose. In some embodiments, the carbohydrates, such as sucrose, are present at concentrations of about 30 mM to about 300 mM, about 40 mM to about 275 mM, about 50 mM to about 250 mM, about 60 mM to about 240 mM, about 70 mM to about 220 mM, about 30 mM to about 150 mM, or about 150 to 300 mM. In some embodiments, the purified buffer, such as PIPES buffer, contains sucrose at a concentration of about 50 mM to about 80 mM, for example, about 73 mM. In some embodiments, the purified buffer, such as PIPES buffer, contains sucrose at a concentration of about 200 mM to 250 mM, for example, about 220 mM. 【0278】 In some embodiments, carbohydrates may be present during production at concentrations of, for example, about 1% to about 10%, about 2.5% to about 10%, or about 2.5% to about 5% by weight / volume (w / v). For example, carbohydrates such as the non-reducing carbohydrates described herein may be present in the aqueous lentivirus preparation during production at concentrations of 1 w / v%, 1.5 w / v%, 2 w / v%, 2.5 w / v%, 3 w / v%, 3.5 w / v%, 4 w / v%, 4.5 w / v%, 5 w / v%, 5.5 w / v%, 6 w / v%, 6.5 w / v%, 7 w / v%, 7.5 w / v%, 8 w / v%, 8.5 w / v%, 9 w / v%, 9.5 w / v%, or 10 w / v%. In some embodiments, carbohydrates such as the non-reducing carbohydrates described herein may be present in the lentivirus aqueous preparation during production at concentrations of at least 1 w / v%, 1.5 w / v%, 2 w / v%, 2.5 w / v%, 3 w / v%, 3.5 w / v%, 4 w / v%, 4.5 w / v%, 5 w / v%, 5.5 w / v%, 6 w / v%, 6.5 w / v%, 7 w / v%, 7.5 w / v%, 8 w / v%, 8.5 w / v%, 9 w / v%, 9.5 w / v%, or 10 w / v%. In some embodiments, carbohydrates such as the non-reducing carbohydrates described herein may be present in the lentivirus aqueous preparation during production at concentrations of 1 w / v% to 2 w / v%, 2 w / v% to 3 w / v%, 3 w / v% to 4 w / v%, 4 w / v% to 5 w / v%, 5 w / v% to 6 w / v%, 6 w / v% to 7 w / v%, 7 w / v% to 8 w / v%, 8 w / v% to 9 w / v%, and 9 w / v% to 10 w / v%. 【0279】 In some embodiments, the buffer contains arginine or a salt thereof, e.g., arginine-HCl. In some embodiments, the agent, e.g., arginine or a salt thereof, e.g., arginine monohydrochloride (arginine-HCl), is added at concentrations of about 25–50 mM (e.g., about 50 mM), 50–100 mM (e.g., about 75 mM), 100–200 mM (e.g., about 150 mM), or 200–400 mM (e.g., about 300 mM). In some embodiments, at least one of the buffers, e.g., the PIPES buffer used for virus purification (e.g., lentivirus purification using the processes disclosed herein), contains arginine, e.g., arginine-HCl. 【0280】 In some embodiments, the pH of the buffer used in the purification process disclosed herein is about 5.0 to about 8.0, for example 6.0 to about 7.0 (for example 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0), for example about 6.5. 【0281】 In some embodiments, the PIPES buffer can be used as one or more of the replacement buffer and / or filtration buffers. In some embodiments, the ratio of concentrations of PIPES, NaCl, and sucrose differs between the PIPES filtration buffer and the PIPES replacement buffer. In some embodiments, the ratio of concentrations of PIPES, NaCl, and sucrose is the same between the PIPES filtration buffer and the PIPES replacement buffer. In some embodiments, the ratio of concentrations of PIPES, NaCl, and sucrose is the same between the PIPES replacement buffer and the PIPES filtration buffer. 【0282】 Arginine added In some embodiments, arginine, for example, arginine-HCl, is added to the cell culture sample during purification. In some embodiments, arginine, for example, arginine-HCl, is added to a buffer, for example, PIPES buffer or a purification intermediate composition containing PIPES buffer, during purification. In some embodiments, arginine, for example, arginine-HCl, is added to a PIPES buffer that does not contain arginine. In some embodiments, arginine, for example, arginine-HCl, is added to a PIPES buffer that contains arginine. In some embodiments, the agent, for example, arginine or its salt, for example, monosodium arginine (arginine-HCl), is added at concentrations of about 25-50 mM (e.g., about 50 mM), 50-100 mM (e.g., about 75 mM), 100-200 mM (e.g., about 150 mM), or 200-400 mM (e.g., about 300 mM). 【0283】 In some embodiments, the vector recovery rate, the amount of lentiviral transduction units, is about 10% to 300%, about 20% to 180%, about 30% to 160%, about 50% to 150%, about 75% to 125%, or about 100% higher in a purification process that includes a purification step of adding arginine to the purified intermediate composition, for example, compared to a purification process that does not include a purification step of adding arginine to the purified intermediate composition. In some embodiments, the addition of arginine reduces the purification processing time. In some embodiments, when purification includes adding, for example, arginine or a salt thereof, such as arginine-HCl, to the purified intermediate composition, the purification processing time is improved by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to a similar purification method that does not include the addition of arginine to the purified intermediate composition. In some embodiments, the purified intermediate composition after the addition of arginine or its salt, e.g., arginine-HCl, and the subsequent purification step, exhibits total particle concentrations of less than 400,000, less than 300,000, less than 200,000, or less than 100,000 per ml, as measured by microflow imaging. While we do not wish to be bound by theory, in some embodiments, individual lentiviral particles (e.g., infectious virus particles) are substantially undetectable by microflow imaging, but larger particles, including aggregates of non-functional viruses, are detectable. In some embodiments, the purified intermediate composition after the addition of arginine or its salt, e.g., arginine-HCl, and the subsequent purification step, exhibits concentrations of ≥10 μm particles of less than approximately 5,000, approximately 4,500, approximately 4,000, approximately 3,500, approximately 3,000, or approximately 2,500 per ml, as measured by microflow imaging.In some embodiments, the purified intermediate composition after the addition of arginine or a salt thereof, such as arginine-HCl, and the subsequent purification step, exhibits concentrations of less than ≥25 μm, approximately 500, 400, 300, or 200 per ml, as measured by microflow imaging. In some embodiments, a reduction in aggregates reduces clogging of the filtration membrane at a given time point. In some embodiments, arginine stabilizes lentivirus particles. 【0284】 In some embodiments, the purified lentivirus composition is, for example, about 1 × 10⁶ per milliliter. 7 Transduction units (TU / mL) ~ approximately 7 × 10 7 TU / mL (e.g., 1 × 10) 7 TU / mL, 1.5 × 10 7 TU / mL, 2 × 10 7 TU / mL, 2.5 × 10 7 TU / mL, 3 × 10 7 TU / mL, 3.5 × 10 7 TU / mL, 4 × 10 7 TU / mL, 4.5 × 10 7 TU / mL, 5 × 10 7 TU / mL, 5.5 × 10 7 TU / mL, 6 × 10 7 TU / mL, 6.5 × 10 7 TU / mL or 7 × 10 7 Contains a lentiviral vector at a concentration of TU / mL. 【0285】 For example, aqueous compositions for the formulation and preservation of lentiviruses In some embodiments, the present disclosure provides preparations, such as aqueous mixtures, aqueous solutions or suspensions, or aqueous compositions, comprising the lentiviral vector and buffer disclosed herein, for example, formulation buffers or preservation buffers, for example, HEPES buffers, for example, free positively charged amino acids (e.g., arginine (e.g., L-arginine), lysine, or histidine) and free nonpolar amino acids (e.g., proline (e.g., L-proline), methionine, or tryptophan). In some embodiments, lentiviral preparations comprising HEPES buffers, for example, formulation buffers or preservation buffers, for example, HEPES buffers, for example, free positively charged amino acids (e.g., arginine (e.g., L-arginine), lysine, or histidine) and free nonpolar amino acids (e.g., proline (e.g., L-proline), methionine, or tryptophan), or both, exhibit improved biological properties compared to lentiviral preparations containing conventional lentiviral formulation buffers such as PIPES. These improved biological properties include increased resistance to agglutination over a wide range of temperatures as specified herein. In some embodiments, HEPES buffer exhibits increased resistance to loss of infectivity during multiple freeze / thaw cycles, along with improved transduction ability at physiological temperatures. Other buffers useful in conjunction with the lentivirus preparations of this disclosure include histidine buffer, phosphate buffer, sodium citrate buffer, MES buffer, MOPS buffer, and PIPES buffer. The lentivirus preparations of this disclosure may optionally contain salts such as sodium chloride, and may optionally contain carbohydrates such as non-reducing carbohydrates. 【0286】 In some embodiments, the buffer for formulating and / or preserving HEPES may contain a buffer, such as HEPES, at concentrations of about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 100 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, or about 15 mM to about 25 mM, for example, about 20 mM. 【0287】 The lentiviral vector preparations of this disclosure may include amino acids or salts thereof; for example, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or salts thereof. “Amino acid” is understood to be synonymous with “free amino acid” (compared, for example, to a reference to a polypeptide molecule consisting of a set of amino acids) unless otherwise specifically indicated in the context. Therefore, in some embodiments, the HEPES formulation and / or preservation buffer may include one or both of the free positively charged amino acids (e.g., arginine, lysine, or histidine) or salts thereof and the free nonpolar amino acids (e.g., proline, methionine, or tryptophan) or salts thereof. 【0288】 In some embodiments, the HEPES formulation and / or preservation buffer contains free arginine (e.g., L-arginine) or a salt thereof. In some embodiments, the free arginine or salt thereof is at a concentration of at least 25, 50, 75, 100, or 150 mM. In some embodiments, the free arginine or salt thereof is at a concentration of about 150 mM. 【0289】 In some embodiments, the HEPES formulation and / or preservation buffer contains free proline (e.g., L-proline) or a salt thereof. In some embodiments, the free proline or salt thereof is at a concentration of 25–200, 50–200, 100–200, 125–175, or 150 mM. In some embodiments, the free proline or salt thereof is at a concentration of about 150 mM. 【0290】 In some embodiments, the HEPES formulation and / or preservation buffer comprises either free arginine and free proline or their salts (e.g., arginine-HCl and proline-HCl). 【0291】 In some embodiments, the HEPES formulation and / or preservation buffer contains less than 20 mM, less than 10 mM, less than 5 mM, less than 2 mM, or less than 1 mM of total inorganic salts. In some embodiments, the HEPES formulation and / or preservation buffer contains about 0.1 mM to about 20 mM of total inorganic salts. In some embodiments, the HEPES formulation and / or preservation buffer is substantially free of inorganic salts (e.g., none). In some embodiments, the HEPES formulation and / or preservation buffer is free of inorganic salts. In some embodiments, the HEPES formulation and / or preservation buffer is substantially free of either or both NaCl and MgCl2 (e.g., none). In some embodiments, the HEPES formulation and / or preservation buffer is free of either or both NaCl and MgCl2. 【0292】 The lentiviral vector preparations of this disclosure may further contain carbohydrates, such as non-reducing carbohydrates described herein, for example, as cryoprotectants. Examples of non-reducing carbohydrates include, in particular, sucrose and trehalose. When included in the lentiviral vector preparation, the carbohydrate (e.g., non-reducing carbohydrate, e.g., sucrose) may be present at concentrations of, for example, about 25–200 mM, about 50–200 mM, about 100–200 mM, about 125–175 mM, or about 150 mM. For example, a carbohydrate such as a non-reducing carbohydrate described herein, e.g., sucrose, may be present in an aqueous lentiviral preparation at a concentration of about 150 mM. 【0293】 In some embodiments, the HEPES formulation and / or preservation buffer further comprises a stabilizer. In some embodiments, the HEPES formulation and / or preservation buffer further comprises human serum albumin (HSA). In some embodiments, the HSA is human-derived HSA (e.g., HSA isolated from human serum). Human-derived HSA can be obtained, for example, by isolating HSA from human serum according to a common method known in the art. In some embodiments, the HSA is recombinant HSA (rHSA). In some embodiments, the HSA is present at 0.5–3%, 0.5–2%, 0.5–1%, 1–2%, 1.5–2.5%, or 2% w / v. In some embodiments, the HSA is present at about 2% w / v. In other embodiments, the HEPES formulation and / or preservation buffer is substantially HSA-free (e.g., does not contain HSA). In other embodiments, the HEPES formulation and / or preservation buffer does not contain HSA. 【0294】 The lentiviral vector preparations described herein may exhibit a pH of, for example, about 6.0 to about 7.5, such as 6.0 to 7.5, 6.0 to 7.0, 6.0 to 6.5, 6.5 to 7.0, 6.2 to 6.8, 6.4 to 6.6, or 6.5. In some embodiments, the pH of the lentiviral vector preparation is 6.5. 【0295】 In some embodiments, the HEPES formulation and / or preservation buffer is substantially free of one, two, or three of PEG lipids, F108, and cholesterol (e.g., not included). 【0296】 In some embodiments, the HEPES formulation and / or preservation buffer contains L-arginine, L-proline, and sucrose, and is substantially free of inorganic salts. 【0297】 In some embodiments, the HEPES formulation and / or preservation buffer comprises L-arginine at a concentration of 100–200 mM, L-proline at a concentration of 25–200 mM, and sucrose at a concentration of 25–200 mM. 【0298】 In some embodiments, the HEPES formulation and / or preservation buffer comprises 20 mM HEPES, 150 mM L-arginine, 150 mM L-proline, and 150 mM sucrose. In some embodiments, the HEPES formulation and / or preservation buffer further comprises 2% w / v HSA. 【0299】 In some embodiments, the HEPES formulation buffer and the HEPES storage buffer have the same composition. In some embodiments, the HEPES formulation buffer and the HEPES storage buffer have different compositions. 【0300】 Lentiviral vectors may be present in the lentiviral preparations of this disclosure at various concentrations. For example, lentiviral vectors may be present in the lentiviral preparations at concentrations of, for example, about 1 × 10⁻⁶. 7 Transduction units / milliliter (TU / mL) ~ approximately 1 × 10⁻⁶ 9 TU / mL (e.g., 1 × 10) 7 TU / mL, 2 × 10 7 TU / mL, 3 × 10 7 TU / mL, 4 × 10 7 TU / mL, 5 × 10 7 TU / mL, 6 × 10 7 TU / mL, 7×10 7 TU / mL, 8 × 10 7 TU / mL, 9×10 7 TU / mL, 1 × 10 8 TU / mL, 1.5 × 10 8 TU / mL, 2 × 10 8 TU / mL, 2.5 × 10 8 TU / mL, 3 × 10 8 TU / mL, 3.5 × 10 8 TU / mL, 4 × 10 8TU / mL, 4.5 × 10 8 TU / mL, 5 × 10 8 TU / mL, 5.5 × 10 8 TU / mL, 6 × 10 8 TU / mL, 6.5 × 10 8 TU / mL, 7×10 8 TU / mL, 7.5 × 10 8 TU / mL, 8 × 10 8 TU / mL, 8.5 × 10 8 TU / mL, 9×10 8 TU / mL, 9.5 × 10 8 TU / mL, 1 × 10 9 TU / mL, 2 × 10 9 TU / mL, 3 × 10 9 TU / mL, 4 × 10 9 TU / mL, 5 × 10 9 TU / mL or 6 × 10 9 It may be present at a concentration of TU / mL. If necessary, the lentivirus preparation should be approximately 3 × 10⁻⁶. 8 TU / mL ~ approximately 5 x 10 8 TU / mL (e.g., 3 × 10) 8 TU / mL, 3.5 × 10 8 TU / mL, 4 × 10 8 TU / mL, 4.5 × 10 8 TU / mL or 5 × 10 8 It may contain a lentiviral vector at a concentration of TU / mL. 【0301】 In some embodiments, the aqueous composition contains cholesterol. In some embodiments, the aqueous composition contains MgCl2. In some embodiments, the aqueous composition contains lysine. In some embodiments, the aqueous composition contains lactose. In some embodiments, the aqueous composition contains sorbitol. In some embodiments, the aqueous composition contains glycerol. In some embodiments, the aqueous composition contains PEG lipids. In some embodiments, the aqueous composition contains F108. In some embodiments, the aqueous composition contains glutamic acid. 【0302】 In some embodiments, aqueous compositions, such as aqueous compositions comprising the lentiviral vector described herein, can be stored at low temperatures, for example, 10°C, 6°C, 4°C, 0°C, -10°C, -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, or 90°C for a certain period of time, for example, about 20 minutes, 40 minutes, 60 minutes, 1.5 hours, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days, 25 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or longer. In some embodiments, aqueous compositions are stored at 10°C, 6°C, 4°C, 0°C, -10°C, -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, or below 90°C. In some embodiments, purified lentivirus samples stored in HEPES storage buffer are stored frozen at -80°C immediately after purification. In some embodiments, lentivirus preparations thus stored can be thawed and refrozen before use (e.g., freeze-thaw cycles). In some embodiments, lentivirus preparations prepared and stored as disclosed herein can undergo at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, and at least nine freeze-thaw cycles without significant loss of stability and / or infectivity. In some embodiments, the preparation exhibits a loss of stability and / or infectivity of 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% or less compared to a lentivirus preparation that has not undergone any freeze-thaw cycles. 【0303】 In some embodiments, the lentivirus preparations disclosed herein can be stored at 4°C for a certain period of time, for example, about 20 minutes, 40 minutes, 60 minutes, 1.5 hours, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days, 25 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or longer. In some embodiments, the lentivirus preparations disclosed herein can be stored at -80°C for a certain period of time, for example, about 20 minutes, 40 minutes, 60 minutes, 1.5 hours, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days, 25 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or longer. In some embodiments, lentivirus preparations stored as disclosed herein (e.g., stored in a frozen state) exhibit at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% infectivity compared to lentivirus that has never been frozen. In some embodiments, lentiviral preparations retain at least 0.5%, 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 80% infectivity after undergoing two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) freeze-thaw cycles. In some embodiments, lentiviral preparations stored as disclosed herein (e.g., stored in a frozen state) are at least 30%, 40%, 50%, 60%, 70%, 70%, 80%, or 100% stable compared to lentiviruses that have never been frozen.In some embodiments, the lentiviral preparation is used after being frozen for at least 5 hours, at least 12 hours, at least 18 hours, at least 1 day, at least 2 days, at least 3 days, at least 5 days, or at least 7 days to improve vector integrity. 【0304】 This disclosure further includes dried or lyophilized compositions prepared by drying or lyophilizing the aqueous compositions described herein, and aqueous compositions prepared by reconstituting such dried or lyophilized compositions in the buffers described herein (or other standard vehicles for administration). 【0305】 The HEPES buffer disclosed in this section is suitable for the formulation and storage of lentiviruses, and can also be used in other steps of the lentivirus manufacturing and purification process, such as during filtration (e.g., ultrafiltration before sterile filtration). 【0306】 This disclosure provides, in particular, formulations comprising lentiviral vectors and aqueous compositions described herein. As used herein, “formulation” is synonymous with “composition.” In some embodiments, the composition is a pharmaceutical composition. 【0307】 CAR target This specification describes a viral vector for transducing immune effector cells (e.g., T cells, NK cells) that have been engineered to contain one or more chimeric antigen receptors (CARs) that direct immune effector cells toward undesirable cells (e.g., cancer cells). This is achieved through an antigen-binding domain on the CAR that is specific to cancer-associated antigens. Two classes of cancer-associated antigens (tumor antigens) that can be targeted by CARs are: (1) cancer-associated antigens expressed on the surface of cancer cells; and (2) cancer-associated antigens that are intracellular themselves, but fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC (major histocompatibility complex). 【0308】 In some embodiments, tumor antigens include CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); type C lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation ( BCMA); Tn antigen ((TnAg) or (GalNAcα-Ser / Thr)); Prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin-11 receptor α IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testicin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific fetal antigen-4 (SSEA-4); CD20; folate receptor α; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-related (MUC1); epidermal growth factor receptor (EGFR); nerve cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP) ;Elongation factor 2 mutant (ELF2M);Ephrin B2;Fibroblast-activating protein α (FAP);Insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (prosome, macropine) subunit, beta type, 9 (LMP2);Glycoprotein 100 (gp100);Oncogene fusion protein (bcr-abl) consisting of a cleavage cluster region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl);Tyrosinase;Ephrin type A receptor 2 (EphA2);Fucosyl GM1;Sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globo H glycoceramide (Glo boH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor β3 (ADRB3); panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, gene locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative leading frame protein (TARP); Wilms tumor protein (WT1); cancer / testicular antigen 1 (NY-ESO-1); cancer / testicular antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); Melanoma carcinoma testicular antigen-1 (MAD-CT-1); Melanoma carcinoma testicular antigen-2 (MAD-CT-2); Fos-related antigen 1; Tumor protein p53 (p53); p53 variant; Prostain; Survivin; Telomerase; Prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), Melanoma antigen 1 recognized by T cells (MelanA or MART1); Rat sarcoma (Ras) mutant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Melanoma apoptosis inhibitor (ML-IAP); ERG (Transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17);Paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc (myelocytoma virus oncogene neuroblastoma-derived homolog (MYCN)); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (a brother of BORIS or imprinting site regulators), squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-section 2 (SSX2); late glycation end product receptor (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); regmine; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); enteric carboxylesterase; heat shock protein 70-2 mutant (mut One or more of the following are selected: hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA receptor Fc fragment (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin λ-like polypeptide 1 (IGLL1). 【0309】 The CARs described herein may include an antigen-binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., the tumor-supporting antigen described herein). In some embodiments, the tumor-supporting antigen is an antigen present on stromal cells or myeloid-derived suppressor cells (MDSCs). Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation. While we do not wish to be bound by theory, in some embodiments, CAR-expressing cells destroy tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival. 【0310】 In several embodiments, the stromal cell antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast-activating protein (FAP), and tenascin. In one embodiment, the FAP-specific antibody is cibrotuzumab, competes with cibrotuzumab for binding, or has the same CDR as cibrotuzumab. In several embodiments, the MDSC antigen is selected from one or more of CD33, CD11b, C14, CD15, and CD66b. Thus, in some embodiments, the tumor-supporting antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast-activating protein (FAP), or tenascin, CD33, CD11b, C14, CD15, and CD66b. 【0311】 CD19 A non-exclusive exemplary tumor antigen is CD19. CARs that bind to CD19 are known in the art. For example, those disclosed in International Publication No. 2012 / 079000 and International Publication No. 2014 / 153270 may be used in accordance with this disclosure. In the art, any known CD19 CAR, for example, the CD19 antigen-binding domain of any known CD19 CAR, may be used in accordance with this disclosure. For example, LG-740; CD19 CAR described in U.S. Patent No. 8,399,645; U.S. Patent No. 7,446,190; 122(17):2965-2973(2013);Brentjens et al.,Blood,118(18):4817-4828(2011);Kochenderfer et al.,Blood 116(20):4099-102(2010);Kochenderfer et al.,Blood 122(25):4129-39(2013); and 16th Annu Meet Am Soc Gen Cell Ther(ASGCT)(May 15-18,Salt Lake City)2013,Abst 10. 【0312】 Non-exclusive exemplary CD19 CARs include, for example, the CD19 CARs described herein or Xu et al. Blood 123.24(2014):3750-9; Kochenderfer et al. Blood 122.25(2013):4129-39, Cruz et al. Blood 122.25(2013):4129-39, Cruz et al.17(2013):2965-73, NCT00586391, NCT01087294, NCT02456350, NCT00840853, NCT02659943, NCT02650999, NCT02640209, NCT01747486, N CT02546739, NCT02656147, NCT02772198, NCT00709033, NCT02081937, NCT00924326, NCT02735083, NCT02794246, NCT02746952, NCT01593 696, NCT02134262, NCT01853631, NCT02443831, NCT02277522, NCT02348216, NCT02614066, NCT02030834, NCT02624258, NCT02625480, NC T02030847, NCT02644655, NCT02349698, NCT02813837, NCT02050347, NCT01683279, NCT02529813, NCT02537977, NCT02799550, NCT026725 01, NCT02819583, NCT02028455, NCT01840566, NCT01318317, NCT01864889, NCT02706405, NCT01475058, NCT01430390, NCT02146924, NCT 02051257, NCT02431988, NCT01815749, NCT02153580, NCT01865617, NCT02208362, NCT02685670, NCT02535364, NCT02631044, NCT0272888 2. Includes anti-CD19 CARs as described in NCT02735291, NCT01860937, NCT02822326, NCT02737085, NCT02465983, NCT02132624, NCT02782351, NCT01493453, NCT02652910, NCT02247609, NCT01029366, NCT01626495, NCT02721407, NCT01044069, NCT00422383, NCT01680991, NCT02794961, or NCT02456207, each of which is incorporated herein by reference in its entirety. 【0313】 In some embodiments, the antigen-binding domain binds to CD19 and has the same or similar binding specificity as the FMC63 scFv fragment described in Nicholson et al. Mol.Immun. 34(16-17):1157-1165 (1997). In some embodiments, the antigen-binding domain binds to CD19 and contains the scFv fragment described in Nicholson et al. Mol.Immun. 34(16-17):1157-1165 (1997). 【0314】 In some embodiments, the antigen-binding domain (e.g., a humanized antigen-binding domain) binds to CD19 and contains a sequence from Table 3 of International Publication No. 2014 / 153270 (incorporated herein by reference). International Publication No. 2014 / 153270 also describes methods for assaying the binding and efficacy of various CAR constructs. 【0315】 Humanization of mouse CD19 antibodies is desired in clinical practice in patients undergoing CART19 therapy, i.e., therapy with T cells transduced with CAR19 constructs, where mouse-specific residues can induce a human anti-mouse antigen (HAMA) response. The production, characterization, and efficacy of humanized CD19 CAR sequences are described in their entirety in International Publication No. 2014 / 153270, which is incorporated herein by reference, including Examples 1-5 (pp. 115-159). 【0316】 In some embodiments, the antigen-binding domain comprises a parental mouse scFv sequence of the CAR19 construct provided in International Publication No. 2012 / 079000 (incorporated herein by reference). In some embodiments, the antigen-binding domain binds to CD19 and comprises the scFv described in International Publication No. 2012 / 079000. 【0317】 In some embodiments, the CD19 CAR comprises a fusion polypeptide sequence provided as Sequence ID No. 12 in International Publication No. 2012 / 079000, which provides a mouse-derived scFv fragment that specifically binds to human CD19. 【0318】 In some embodiments, the CD19 CAR comprises an amino acid sequence provided as Sequence ID No. 12 in International Publication No. 2012 / 079000. 【0319】 In some embodiments, the CD19 CAR is [ka] It contains the amino acid sequence of or a sequence substantially homologous thereto. 【0320】 In some embodiments, the CD19 CAR is [ka] It contains the amino acid sequence. 【0321】 In some embodiments, the CD19 CAR has the amino acid sequence: [ka] It is a humanized CD19 CAR that contains [unclear]. 【0322】 In some embodiments, the CD19 CAR includes sequences, for example, the CDR, VH, VL, scFv, or full-length CAR sequences disclosed in Table 1 below, or sequences having at least 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the CD19 CAR includes sequences disclosed in Table 1 below, for example, CDR, VH, VL, scFv, or complete CAR sequences, or sequences having at least one amino acid substitution therefor, for example, substitutions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. 【0323】 [Table 1] 【0324】 [Table 2] 【0325】 [Table 3] 【0326】 [Table 4] 【0327】 [Table 5] 【0328】 [Table 6] 【0329】 [Table 7] 【0330】 [Table 8] 【0331】 Exemplary CD19 CAR A In some embodiments, the CD19 CAR includes a binding domain for a single-strand variable fragment (scFv) derived from an FMC63 monoclonal antibody, an IgG4 hinge region, a CD28 transmembrane domain, a 4-1BB (CD137) costimulatory domain, and a CD3ζ activating domain. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence from Table 25 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions to the nucleotide sequences from Table 25. In some embodiments, the CD19 CAR includes a polypeptide encoded by a nucleotide sequence from Table 25 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions relative to the nucleic acid sequence in Table 25. In some embodiments, the CD19 CAR comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to the polypeptide sequence in Table 25. In some embodiments, the CD19 CAR comprises a polypeptide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer amino acid substitutions relative to the amino acid sequence in Table 25. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence in Table 25. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence in Table 25 by Kabat. In some embodiments, the CD19 CAR includes heavy chain CDR1-3 and light chain CDR1-3 in the sequence of Table 25 by Chothia. In some embodiments, the CD19 CAR includes heavy chain CDR1-3 in the sequence of Table 25. In some embodiments, the CD19 CAR includes heavy chain CDR1-3 in the sequence of Table 25 by Kabat.In some embodiments, the CD19 CAR includes heavy chains CDR1-3 of the sequence shown in Table 25 by Chothia. In some embodiments, the CD19 CAR includes light chains CDR1-3 of the sequence shown in Table 25. In some embodiments, the CD19 CAR includes light chains CDR1-3 of the sequence shown in Table 25 by Kabat. In some embodiments, the CD19 CAR includes light chains CDR1-3 of the sequence shown in Table 25 by Chothia. 【0332】 [Table 9] 【0333】 [Table 10] 【0334】 [Table 11] 【0335】 [Table 12] 【0336】 Example CD19 CAR B In some embodiments, the CD19 CAR comprises a mouse anti-CD19 single-strand variable fragment (scFv) linked to CD28 and CD3-ζ costimulatory domains. In certain embodiments, the anti-CD19 single-strand variable fragment comprises an FMC63 antibody (e.g., the antibody described in Nicholson et al., Molecular Immunology, 34(16-17):1157-1165, 1997; the entire content thereof is incorporated herein by reference). In some embodiments, the CD19 CAR is encoded by a nucleotide sequence of Table 26 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions relative to the nucleotide sequences of Table 26. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence from Table 26 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions to the nucleic acid sequence from Table 26. In some embodiments, the CD19 CAR comprises a polypeptide sequence from Table 26 or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer amino acid substitutions to the amino acid sequence from Table 25. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence from Table 26. In some embodiments, the CD19 CAR includes heavy chain CDR1-3 and light chain CDR1-3 in the sequence of Table 26 by Kabat. In some embodiments, the CD19 CAR includes heavy chain CDR1-3 and light chain CDR1-3 in the sequence of Table 26 by Chothia. In some embodiments, the CD19 CAR includes heavy chain CDR1-3 in the sequence of Table 26.In some embodiments, the CD19 CAR includes heavy chains CDR1-3 of the sequence shown in Table 26 by Kabat. In some embodiments, the CD19 CAR includes heavy chains CDR1-3 of the sequence shown in Table 26 by Chothia. In some embodiments, the CD19 CAR includes light chains CDR1-3 of the sequence shown in Table 26. In some embodiments, the CD19 CAR includes light chains CDR1-3 of the sequence shown in Table 26 by Kabat. In some embodiments, the CD19 CAR includes light chains CDR1-3 of the sequence shown in Table 26 by Chothia. 【0337】 [Table 13] 【0338】 [Table 14] 【0339】 Example CD19 CAR C In some embodiments, the CD19 CAR comprises a mouse anti-CD19 single-stranded variable fragment (scFv) linked to CD28 and CD3-ζ costimulatory domains. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence from Table 27 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions to the nucleotide sequences from Table 27. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence from Table 27 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions to the nucleic acid sequence in Table 27. In some embodiments, the CD19 CAR comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to the polypeptide sequence in Table 27. In some embodiments, the CD19 CAR comprises a polypeptide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions to the amino acid sequence in Table 27. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence in Table 27. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence in Table 27 by Kabat. In some embodiments, the CD19 CAR includes heavy chain CDR1-3 and light chain CDR1-3 in the sequence of Table 27 by Chothia. In some embodiments, the CD19 CAR includes heavy chain CDR1-3 in the sequence of Table 27 by Kabat. In some embodiments, the CD19 CAR includes heavy chain CDR1-3 in the sequence of Table 27 by Chothia. In some embodiments, the CD19 CAR includes light chain CDR1-3 in the sequence of Table 27 by Chothia.In some embodiments, the CD19 CAR includes light chains CDR1-3 with the sequences shown in Table 27 by Kabat. In some embodiments, the CD19 CAR includes light chains CDR1-3 with the sequences shown in Table 27 by Chothia. 【0340】 [Table 15] 【0341】 [Table 16] 【0342】 [Table 17] 【0343】 Example CD19 CAR F In some embodiments, the CD19 CAR is encoded by a nucleotide sequence from Table 34 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions to the nucleotide sequence from Table 34. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence from Table 34 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions to the nucleic acid sequence from Table 34. In some embodiments, the CD19 CAR includes the polypeptide sequence of Table 34 or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR includes a polypeptide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer amino acid substitutions to the amino acid sequence of Table 34. In some embodiments, the CD19 CAR includes the heavy chain CDR1-3 and light chain CDR1-3 of the sequence of Table 34. In some embodiments, the CD19 CAR includes the heavy chain CDR1-3 and light chain CDR1-3 of the sequence of Table 34 by Kabat. In some embodiments, the CD19 CAR includes the heavy chain CDR1-3 and light chain CDR1-3 of the sequence of Table 34 by Chothia. In some embodiments, the CD19 CAR includes the heavy chain CDR1-3 of the sequence of Table 34. In some embodiments, the CD19 CAR includes heavy chains CDR1-3 of the sequence shown in Table 34 by Kabat. In some embodiments, the CD19 CAR includes heavy chains CDR1-3 of the sequence shown in Table 34 by Chothia. In some embodiments, the CD19 CAR includes light chains CDR1-3 of the sequence shown in Table 34. In some embodiments, the CD19 CAR includes light chains CDR1-3 of the sequence shown in Table 34 by Kabat.In some embodiments, the CD19 CAR comprises light chains CDR1-3 of the sequence shown in Table 34 by Chothia. 【0344】 [Table 18] 【0345】 [Table 19] 【0346】 Exemplary CD19-CD20 CAR G In some embodiments, the CD19 CAR is a bispecific CAR. In certain embodiments, the CD19 bispecific CAR includes a light chain variable domain targeting CD19 and a heavy chain variable domain targeting a different target (e.g., CD20). In some embodiments, the bispecific CAR is an anti-CD19 and anti-CD20 CAR. In some embodiments, the bispecific CAR is encoded by a nucleotide sequence from Table 35 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the bispecific CAR is encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions to the nucleotide sequences from Table 35. In some embodiments, the bispecific CAR includes a polypeptide encoded by a nucleotide sequence from Table 35 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the bispecific CAR comprises a polypeptide encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions relative to the nucleic acid sequence in Table 35. In some embodiments, the bispecific CAR comprises a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with respect to the polypeptide sequence in Table 35. In some embodiments, the bispecific CAR comprises a polypeptide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer amino acid substitutions relative to the amino acid sequence in Table 35. In some embodiments, the bispecific CAR comprises the heavy chain CDR1-3 and light chain CDR1-3 of the sequence in Table 35. In some embodiments, the bispecific CAR comprises the heavy chain CDR1-3 and light chain CDR1-3 of the sequence in Table 35 by Kabat. In some embodiments, the bispecific CAR comprises the heavy chain CDR1-3 and light chain CDR1-3 of the sequence in Table 35 by Chothia. In some embodiments, the bispecific CAR includes heavy chains CDR1-3 of the sequences in Table 35.In some embodiments, the bispecific CAR includes the heavy chain CDR1-3 of the sequence in Table 35 by Chothia. In some embodiments, the bispecific CAR includes the light chain CDR1-3 of the sequence in Table 35. In some embodiments, the bispecific CAR includes the light chain CDR1-3 of the sequence in Table 35 by Kabat. In some embodiments, the bispecific CAR includes the light chain CDR1-3 of the sequence in Table 35 by Chothia. 【0347】 [Table 20] 【0348】 [Table 21] 【0349】 [Table 22] 【0350】 BCMA A non-exclusive exemplary tumor antigen is BCMA. CARs that bind to BCMA are known in the art. For example, those disclosed in International Publication No. 2016 / 014565 or International Publication No. 2019 / 241426 can be used in accordance with this disclosure. In the art, any known BCMA CAR, for example, the BCMA antigen-binding domain of any known BCMA CAR, can be used in accordance with this disclosure. For example, BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, B disclosed in International Publication No. 2016 / 014565 CMA_EBB-C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB- C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2 or C13F12.1. 【0351】 In some embodiments, BCMA CAR is BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB-C It contains one or more CDR, VH, VL, scFV, or full-length sequences of 1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2, or C13F12.1, or a sequence that is substantially (e.g., 95-99%) identical thereto. 【0352】 Exemplary antigen-binding domains that bind to BCMA are described in International Publication Nos. 2012 / 0163805, 2017 / 021450, 2017 / 011804, 2017 / 025038, 2016 / 090327, 2016 / 130598, 2016 / 210293, 2016 / 090320, and 2016 / 014789. Pamphlets No. 2016 / 094304, No. 2016 / 154055, No. 2015 / 166073, No. 2015 / 188119, No. 2015 / 158671, U.S. Patent No. 9,243,058, No. 8,920,776, No. 9,273,141, No. 7,083,785, No. 9,034,324, U.S. Patent Application Publication No. 2007 / 0049735, and the same No. 2 Specification No. 015 / 0284467, Specification No. 2015 / 0051266, Specification No. 2015 / 0344844, Specification No. 2016 / 0131655, Specification No. 2016 / 0297884, Specification No. 2016 / 0297885, Specification No. 2017 / 0051308, Specification No. 2017 / 0051252, Specification No. 2016 / 020332, International Publication Brochure No. 2016 / 087531, Brochure No. 2016 / 079177, and No. 201 These are disclosed in brochure 5 / 172800, brochure 2017 / 008169, U.S. Patent No. 9,340,621, U.S. Patent Publication No. 2013 / 0273055, 2016 / 0176973, 2015 / 0368351, 2017 / 0051068, 2016 / 0368988, and 2015 / 0232557, which are incorporated herein by reference in their entirety. In some embodiments, these are one or more antigen-binding domains of one or more BCMA antigen-binding domains disclosed herein. 【0353】 In some embodiments, the antigen-binding domain comprises a human antibody or human antibody fragment that binds to BCMA. In some embodiments, the antigen-binding domain comprises one or more (e.g., all three) human anti-BCMA binding domains described herein (e.g., Tables 2-14) LC CDR1, LC CDR2, and LC CDR3 and / or one or more (e.g., all three) human anti-BCMA binding domains described herein (e.g., Tables 2-14) HC CDR1, HC CDR2, and HC CDR3. In some embodiments, the human anti-BCMA binding domain comprises human VL and / or human VH as described herein (e.g., Tables 2, 6, and 10). In some embodiments, the antigen-binding domain is an scFv comprising VL and VH of the amino acid sequences in Tables 2, 6, and 10. In some embodiments, the antigen-binding domain (e.g., scFv) includes: VL, which includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences provided in Tables 2, 6, and 10, provided that the modifications are 30, 20, or 10 or fewer, or a sequence having 95-99% identity with the amino acid sequences in Tables 2, 6, and 10; and / or VH, which includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequences provided in Tables 2, 6, and 10, provided that the modifications are 30, 20, or 10 or fewer, or a sequence having 95-99% identity with the amino acid sequences in Tables 2, 6, and 10. 【0354】 In certain embodiments, the antigen-binding domain described herein includes: (1) One, two, or three light chain (LC) CDRs selected from the following: (i) LC CDR1 of SEQ ID NO: 54, LC CDR2 of SEQ ID NO: 55, and LC CDR3 of SEQ ID NO: 56; and / or (2) One, two, or three heavy chain (HC) CDRs derived from any one of the following: (i) HC CDR1 of sequence number 44, HC CDR2 of sequence number 45, and HC CDR3 of sequence number 84; (ii) HC CDR1 of sequence number 44, HC CDR2 of sequence number 45, and HC CDR3 of sequence number 46; (iii) HC CDR1 of sequence number 44, HC CDR2 of sequence number 45, and HC CDR3 of sequence number 68; or (iv) HC CDR1 of sequence number 44, HC CDR2 of sequence number 45, and HC CDR3 of sequence number 76. 【0355】 In certain embodiments, the antigen-binding domain described herein includes: (1) One, two, or three light chain (LC) CDRs derived from any one of the following: (i) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 131, and LC CDR3 of SEQ ID NO: 132; (ii) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 96, and LC CDR3 of SEQ ID NO: 97; (iii) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 114, and LC CDR3 of SEQ ID NO: 115; or (iv) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 114, and LC CDR3 of SEQ ID NO: 97; and / or (2) One, two, or three heavy chain (HC) CDRs derived from any one of the following: (i) HC CDR1 of sequence number 86, HC CDR2 of sequence number 130, and HC CDR3 of sequence number 88; (ii) HC CDR1 of sequence number 86, HC CDR2 of sequence number 87, and HC CDR3 of sequence number 88; or (iii) HC CDR1 of sequence number 86, HC CDR2 of sequence number 109, and HC CDR3 of sequence number 88. 【0356】 In certain embodiments, the antigen-binding domain described herein includes: (1) One, two, or three light chain (LC) CDRs derived from any one of the following: (i) LC CDR1 of SEQ ID NO: 147, LC CDR2 of SEQ ID NO: 182, and LC CDR3 of SEQ ID NO: 183; (ii) LC CDR1 of SEQ ID NO: 147, LC CDR2 of SEQ ID NO: 148, and LC CDR3 of SEQ ID NO: 149; or (iii) LC CDR1 of SEQ ID NO: 147, LC CDR2 of SEQ ID NO: 170, and LC CDR3 of SEQ ID NO: 171; and / or (2) One, two, or three heavy chain (HC) CDRs derived from any one of the following: (i) HC CDR1 of sequence number 179, HC CDR2 of sequence number 180, and HC CDR3 of sequence number 181; (ii) HC CDR1 of sequence number 137, HC CDR2 of sequence number 138, and HC CDR3 of sequence number 139; or (iii) HC CDR1 of sequence number 160, HC CDR2 of sequence number 161, and HC CDR3 of sequence number 162. 【0357】 In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, 84, 54, 55, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, 46, 54, 55, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, 68, 54, 55, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 44, 45, 76, 54, 55, and 56, respectively. 【0358】 In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 47, 48, 84, 57, 58, and 59, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 47, 48, 46, 57, 58, and 59, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 47, 48, 68, 57, 58, and 59, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 47, 48, 76, 57, 58, and 59, respectively. 【0359】 In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 49, 50, 85, 60, 58, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 49, 50, 51, 60, 58, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 49, 50, 69, 60, 58, and 56, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each contain the amino acid sequences of SEQ ID NOs. 49, 50, 77, 60, 58, and 56, respectively. 【0360】 In some embodiments, the BCMA CAR includes sequences, such as the CDR, VH, VL, scFv, or full-length CAR sequences disclosed in Tables 2-14, or sequences having at least 80%, 85%, 90%, 95%, or 99% identity thereto. 【0361】 Table 23 【0362】 Table 24 【0363】 Table 25 【0364】 Table 26 【0365】 Table 27 【0366】 Table 28 【0367】 Table 29 【0368】 Table 30 【0369】 Table 31 【0370】 Table 32 【0371】 Table 33 【0372】 Table 34 【0373】 Table 35 【0374】 Table 36 【0375】 Table 37 【0376】 Table 38 【0377】 Table 39 【0378】 Table 40 【0379】 Table 41 【0380】 Table 42 【0381】 Table 43 【0382】 Table 44 【0383】 Table 45 【0384】 Table 46 【0385】 Table 47 【0386】 Table 48 【0387】 Table 49 【0388】 Table 50 【0389】 Table 51 【0390】 Table 52 【0391】 Table 53 【0392】 Table 54 【0393】 Table 55 【0394】 Table 56 【0395】 Table 57 【0396】 Table 58 【0397】 Table 59 【0398】 Table 60 【0399】 Table 61 【0400】 Table 62 【0401】 Table 63 【0402】 Table 64 【0403】 [Table 65] 【0404】 [Table 66] 【0405】 [Table 67] 【0406】 [Table 68] 【0407】 In some embodiments, BCMA CARs may be generated using VH and VL sequences from International Publication No. 2012 / 0163805 (the contents thereof are incorporated herein by reference in their entirety). In some embodiments, BCMA CARs may be generated using CDR, VH, VL, scFv, or full-length CAR sequences from International Publication No. 2019 / 241426 (the contents thereof are incorporated herein by reference in their entirety). 【0408】 Exemplary BCMA CAR D In some embodiments, the BCMA CAR comprises a mouse extracellular single-strand variable fragment (scFv) specific to the recognition of B cell maturation antigen (BCMA), followed by a tandem transmembrane domain fused to the human CD8α hinge and the CD137(4-1BB) and CD3ζ chain T cell cytoplasmic signaling domains. When BCMA CAR D binds to BCMA-expressing target cells, signaling initiated by the CD3ζ and 4-1BB domains occurs, followed by activation of CAR-positive T cells. Antigen-specific activation of BCMA CAR D leads to proliferation of CAR-positive T cells, cytokine secretion, and subsequent cytolytic death of BCMA-expressing cells. In some embodiments, the BCMA CAR is encoded by the nucleotide sequences in Table 28 or nucleotide sequences having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the BCMA CAR is encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions relative to the nucleotide sequence in Table 28. In some embodiments, the BCMA CAR includes a polypeptide encoded by a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the BCMA CAR includes a polypeptide encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions relative to the nucleic acid sequence in Table 28. In some embodiments, the BCMA CAR includes a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the BCMA CAR includes a polypeptide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer amino acid substitutions relative to the amino acid sequence in Table 28. In some embodiments, the BCMA CAR includes heavy chain CDR1-3 and light chain CDR1-3 in the sequence shown in Table 28.In some embodiments, the BCMA CAR includes heavy chain CDR1-3 and light chain CDR1-3 of the sequence shown in Table 28 by Chothia. In some embodiments, the BCMA CAR includes heavy chain CDR1-3 of the sequence shown in Table 28 by Kabat. In some embodiments, the BCMA CAR includes heavy chain CDR1-3 of the sequence shown in Table 28 by Chothia. In some embodiments, the BCMA CAR includes light chain CDR1-3 of the sequence shown in Table 28 by Chothia. In some embodiments, the BCMA CAR includes light chain CDR1-3 of the sequence shown in Table 28 by Kabat. In some embodiments, the BCMA CAR includes light chain CDR1-3 of the sequence shown in Table 28 by Chothia. 【0409】 [Table 69] 【0410】 [Table 70] 【0411】 Exemplary BCMA CAR E In some embodiments, the BCMA CAR comprises two single-domain antibodies linked to a 4-1BB costimulatory domain and a CD3-ζ signaling domain. In some embodiments, the chimeric antigen receptor described herein comprises (a) an extracellular antigen-binding domain comprising a first anti-BCMA single-domain antibody (sdAb) and a polypeptide comprising a second anti-BCMA sdAb. In some embodiments, each of the first and second anti-BCMA antibodies is independently a VhH domain. In certain embodiments, the first anti-BCMA sdAb comprises CDR1, CDR2, and CDR3 represented in a VhH domain comprising the amino acid sequence of SEQ ID NO: 377 or a peptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the first anti-BCMA sdAb comprises CDR1, CDR2, and CDR3 represented in a VhH domain containing an amino acid sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions relative to the amino acid sequence of SEQ ID NO: 377. In certain embodiments, the second anti-BCMA sdAb comprises CDR1, CDR2, and CDR3 represented in a VhH domain containing a peptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 381. In some embodiments, the second anti-BCMA sdAb comprises CDR1, CDR2, and CDR3 represented in a VhH domain containing an amino acid sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions relative to the amino acid sequence of SEQ ID NO: 381. In certain embodiments, the BCMA CAR is any BCMA CAR described in U.S. Patent No. 11,186,647, the contents of which are incorporated herein by reference. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence from Table 29 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions with respect to the nucleotide sequences from Table 29.In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence from Table 29 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer substitutions to the nucleic acid sequence from Table 29. In some embodiments, the CD19 CAR comprises a polypeptide sequence from Table 29 or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide sequence having 20, 15, 10, 8, 5, 4, 3, 2, or 1 or fewer amino acid substitutions to the amino acid sequence from Table 29. In some embodiments, the BCMA CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence from Table 29. In some embodiments, the BCMA CAR includes heavy chain CDR1-3 and light chain CDR1-3 in the sequence of Table 29 by Kabat. In some embodiments, the BCMA CAR includes heavy chain CDR1-3 and light chain CDR1-3 in the sequence of Table 29 by Chothia. In some embodiments, the BCMA CAR includes heavy chain CDR1-3 in the sequence of Table 29. In some embodiments, the BCMA CAR includes heavy chain CDR1-3 in the sequence of Table 29 by Kabat. In some embodiments, the BCMA CAR includes heavy chain CDR1-3 in the sequence of Table 29 by Chothia. In some embodiments, the BCMA CAR includes light chain CDR1-3 in the sequence of Table 29. In some embodiments, the BCMA CAR includes light chain CDR1-3 in the sequence of Table 29 by Kabat. In some embodiments, the BCMA CAR includes light chain CDR1-3 in the sequence of Table 29 by Chothia. 【0412】 [Table 71] 【0413】 [Table 72] 【0414】 [Table 73] 【0415】 Other exemplary targets Further non-exclusive exemplary tumor antigens include CD20, CD22, EGFR, CD123, and CLL-1. 【0416】 CD20-binding CARs are known in the art. For example, those disclosed in International Publication No. 2018 / 067992 or International Publication No. 2016 / 164731, which are incorporated herein by reference, may be used in accordance with this disclosure. In the art, any known CD20 CAR, for example, the CD20 antigen-binding domain of any known CD20 CAR, may be used in accordance with this disclosure. Exemplary CD20-binding sequences or CD20 CAR sequences are disclosed, for example, in Tables 1 to 5 of International Publication No. 2018 / 067992, which are incorporated herein by reference. In some embodiments, the CD20 CAR includes the CDR, variable region, scFv, or full-length sequence of a CD20 CAR disclosed in International Publication No. 2018 / 067992 or International Publication No. 2016 / 164731 (both incorporated herein by reference). In some embodiments, CD20CAR includes sequences disclosed in Table 23 below, such as CDR, VH, VL, scFv, or all CAR sequences, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to them. 【0417】 Exemplary antigen-binding domains that bind to CD20 are described in International Publication Brochures 2016 / 164731 and 2018 / 067992, which are incorporated herein by reference. In some embodiments, one or more of the CD20 antigen-binding domains disclosed therein are used. 【0418】 Exemplary antigen-binding domains that bind to CD22 are described in International Publication Nos. 2016 / 164731 and 2018 / 067992, which are incorporated herein by reference. 【0419】 In some embodiments, the antigen-binding domain comprises HC CDR1, HC CDR2, and HC CDR3 of any heavy chain-binding domain amino acid sequences listed in Table 15. In some embodiments, the antigen-binding domain further comprises LC CDR1, LC CDR2, and LC CDR3. In some embodiments, the antigen-binding domain comprises LC CDR1, LC CDR2, and LC CDR3 amino acid sequences listed in Table 16. 【0420】 In some embodiments, the antigen-binding domain includes one, two, or all of the LC CDR1, LC CDR2, and LC CDR3 of any light chain-binding domain amino acid sequences listed in Table 16, and one, two, or all of the HC CDR1, HC CDR2, and HC CDR3 of any heavy chain-binding domain amino acid sequences listed in Table 15. 【0421】 Exemplary antigen-binding domains that bind to EGFRvIII are described in International Publication No. 2014 / 130657. 【0422】 Exemplary antigen-binding domains that bind to CD123 are described in International Publication Nos. 2014 / 130635 and 2016 / 028896, which are incorporated herein by reference. 【0423】 In some embodiments, the antigen-binding domain includes sequences from Tables 1-2 of International Publication No. 2014 / 130635, which are incorporated herein by reference. 【0424】 In some embodiments, the antigen-binding domain includes sequences from Tables 2, 6, and 9 of International Publication No. 2016 / 028896, which is incorporated herein by reference. 【0425】 Exemplary antigen-binding domains that bind to CLL-1 are disclosed in International Publication No. 2016 / 014535, which is incorporated herein by reference. 【0426】 In some embodiments, the antigen-binding domain is one, two, or three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC derived from the antibodies described herein (e.g., the antibodies described in International Publication No. 2015 / 142675, U.S. Patent Application Publication No. 2015-0283178-A1, No. 2016-0046724-A1, No. 2014 / 0322212A1, No. 2016 / 0068601A1, No. 2016 / 0051651A1, No. 2016 / 0096892A1, No. 2014 / 0322275A1, or International Publication No. 2015 / 090230, which are incorporated herein by reference) Includes one, two, or three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3 derived from CDR3 and / or the antibodies described herein (e.g., the antibodies described in International Publication No. 2015 / 142675, U.S. Patent Application Publication No. 2015-0283178-A1, No. 2016-0046724-A1, No. 2014 / 0322212A1, No. 2016 / 0068601A1, No. 2016 / 0051651A1, No. 2016 / 0096892A1, No. 2014 / 0322275A1, or International Publication No. 2015 / 090230, which are incorporated herein by reference). In some embodiments, the antigen-binding domain includes the heavy chain variable region and / or light chain variable region of the antibody described above. 【0427】 In several embodiments, the antigen-binding domain is an antigen-binding domain described in International Publication No. 2015 / 142675, U.S. Patent Application Publication No. 2015-0283178-A1, U.S. Patent Application Publication No. 2016-0046724-A1, U.S. Patent Application Publication No. 2014 / 0322212A1, U.S. Patent Application Publication No. 2016 / 0068601A1, U.S. Patent Application Publication No. 2016 / 0051651A1, U.S. Patent Application Publication No. 2016 / 0096892A1, U.S. Patent Application Publication No. 2014 / 0322275A1, or International Publication No. 2015 / 090230, which are incorporated herein by reference. 【0428】 Exemplary target antigens that can be targeted using CAR-expressing cells include, but are not limited to, CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4, particularly those described in International Publications No. 2014 / 153270, 2014 / 130635, 2016 / 028896, 2014 / 130657, 2016 / 014576, 2015 / 090230, 2016 / 014565, 2016 / 014535, and 2016 / 025880, each of which is incorporated herein by reference in whole. 【0429】 In some embodiments, the antigen-binding domain of any of the CARs described herein (e.g., CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4) comprises one, two, or three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3 derived from the antibodies listed above, and / or one, two, or three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3 derived from the antigen-binding domains listed above. In some embodiments, the antigen-binding domain comprises the heavy chain variable region and / or variable light chain region of the antibodies listed or described above. 【0430】 In some embodiments, the antigen-binding domain includes one, two, or three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3 of the antibodies listed above, and / or one, two, or three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3 derived from the antibodies listed above. In some embodiments, the antigen-binding domain includes the heavy chain variable region and / or the variable light chain region of the antibodies listed or described above. 【0431】 [Table 74] 【0432】 [Table 75] 【0433】 [Table 76] 【0434】 [Table 77] 【0435】 [Table 78] 【0436】 [Table 79] 【0437】 [Table 80] 【0438】 [Table 81] 【0439】 Table 82 【0440】 Table 83 【0441】 Table 84 【0442】 Table 85 【0443】 Table 86 【0444】 Table 87 【0445】 Table 88 【0446】 Table 89 【0447】 Table 90 【0448】 Table 91 【0449】 Table 92 【0450】 Table 93 【0451】 Table 94 【0452】 Table 95 【0453】 Table 96 【0454】 Table 97 【0455】 Table 98 【0456】 Table 99 【0457】 Table 100 【0458】 Table 101 【0459】 Table 102 【0460】 [Table 103] 【0461】 [Table 104] 【0462】 [Table 105] 【0463】 CD22-binding CARs are known in the art. For example, those disclosed in International Publication No. 2018 / 067992 or International Publication No. 2016 / 164731 may be used in accordance with this disclosure. In the art, any known CD22 CAR, for example, the CD22 antigen-binding domain of any known CD22 CAR, may be used in accordance with this disclosure. 【0464】 Exemplary CD22 binding sequences or CD22 CAR sequences are disclosed, for example, in Tables 6A, 6B, 7A, 7B, 7C, 8A, 8B, 9A, 9B, 10A, and 10B of International Publication No. 2016164731 and in Tables 6-10 of International Publication No. 2018067992. In some embodiments, the CD22 CAR sequence includes the CDR, variable region, scFv, or full-length sequence of the CD22 CAR disclosed in International Publication No. 2018067992 or International Publication No. 2016164731. 【0465】 In some embodiments, the CAR includes an antigen-binding domain (CD22 CAR) that binds to CD22. In some embodiments, the antigen-binding domain targets human CD22. In some embodiments, the antigen-binding domain includes a single-chain Fv sequence as described herein. 【0466】 The sequence of human CD22 CAR is provided below. In some embodiments, the human CD22 CAR is CAR22-65. Human CD22 CAR scFv sequence [ka] Human CD22 CAR heavy chain variable region [ka] Human CD22 CAR light chain variable region QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGTGTQLTVL (Sequence ID 755) 【0467】 In some embodiments, CD22CAR includes sequences disclosed in Tables 15-16 and Table 24 below, such as CDR, VH, VL, scFv, or all CAR sequences, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to them. 【0468】 [Table 106] 【0469】 [Table 107] 【0470】 [Table 108] 【0471】 [Table 109] 【0472】 Table 110 【0473】 Table 111 【0474】 Table 112 【0475】 Table 113 【0476】 Table 114 【0477】 Table 115 【0478】 Table 116 【0479】 Table 117 【0480】 Table 118 【0481】 Table 119 【0482】 Table 120 【0483】 [Table 121] 【0484】 [Table 122] 【0485】 [Table 123] 【0486】 EGFR-binding CARs are known in the art. For example, those disclosed in International Publication No. 2014 / 130657, incorporated herein by reference, may be used in accordance with this disclosure. In the art, any known EGFR CAR, for example, the EGFR antigen-binding domain of any known EGFR CAR, may be used in accordance with this disclosure. Exemplary EGFRvIII CARs may include CDRs, variable regions, scFvs, or full-length CAR sequences, as disclosed in Table 2 of International Publication No. 2014 / 130657, incorporated herein by reference. 【0487】 CARs that bind to CD123 are known in the art. For example, those disclosed in International Publication No. 2014 / 130635 or International Publication No. 2016 / 028896 may be used in accordance with this disclosure. In the art, any known CD123 CAR, for example, the CD123 antigen-binding domain of any known CD123 CAR may be used in accordance with this disclosure. For example, CAR1 to CAR8 disclosed in International Publication No. 2014 / 130635; or CAR123-1 to CAR123-4 and hzCAR123-1 to hzCAR123-32 disclosed in International Publication No. 2016 / 028896. The amino acid and nucleotide sequences encoding the CD123 CAR molecule and antigen-binding domain (e.g., including one, two, or three VH CDRs and one, two, or three VL CDRs by Kabat or Chothia) are designated in International Publication No. 2014 / 130635 and International Publication No. 2016 / 028896. 【0488】 CARs that bind to CLL-1 are known in the art, for example, those disclosed in U.S. Patent Application Publication No. 2016 / 0051651A1, which is incorporated herein by reference. In the art, any known CLL-1 CAR, for example, the CLL-1 antigen-binding domain of any known CLL-1 CAR, may be used in accordance with this disclosure. 【0489】 In some embodiments, the CAR comprises a CLL-1 CAR or antigen-binding domain as shown in Table 2 of International Publication No. 2016 / 014535, which is incorporated herein by reference. The amino acid and nucleotide sequences encoding the CLL-1 CAR molecule and antigen-binding domain (including, for example, one, two, or three VH CDRs and one, two, or three VL CDRs by Kabat or Chothia) are specified in International Publication No. 2016 / 014535. 【0490】 CD33-binding CARs are known in the art. For example, those disclosed in U.S. Patent Application Publication 2016 / 0096892A1 and International Publication Brochure 2016 / 014576, which are incorporated herein by reference, may be used in accordance with this disclosure. Any known CD33 CAR in the art, for example, the CD33 antigen-binding domain of any known CD33 CAR, may be used in accordance with this disclosure. For example, CAR33-1 to CAR33-9 disclosed in International Publication Brochure 2016 / 014576 may be used in accordance with this disclosure. 【0491】 In some embodiments, the CAR comprises a CD33 CAR or antigen-binding domain as shown in Table 2 or 9 of International Publication No. 2016 / 014576, which is incorporated herein by reference. The amino acid and nucleotide sequences encoding the CD33 CAR molecule and antigen-binding domain (including, for example, one, two, or three VH CDRs and one, two, or three VL CDRs by Kabat or Chothia) are specified in International Publication No. 2016 / 014576. 【0492】 In one embodiment, the antigen-binding domain for CD33 is, for example, Bross et al., Clin Cancer Res 7(6):1490-1496(2001) (Gemtuzumab Ozogamicin, hP67.6), Caron et al., Cancer Res 52(24):6761-6767(1992) (Lintuzumab, HuM195), Lapusan et al., Invest New Drugs 30(3):1121-1131(2012) (AVE9633), Aigner et al., Leukemia 27(5):1107-1115(2013) (AMG330, CD33 BiTE), Dutour et al., Adv hematol The antigen-binding portion of an antibody, such as a CDR, is described in 2012:683065 (2012) and Pizzitola et al., Leukemia doi:10.1038 / Lue.2014.62 (2014). In one embodiment, the antigen-binding domain for CD33 is the antigen-binding portion of an antibody, antigen-binding fragment, or CAR, such as a CDR, as described in International Publication No. 2016 / 014576. 【0493】 In one embodiment, the antigen-binding domain for GD2 is, for example, the antigen-binding portion of an antibody described in Mujoo et al., Cancer Res. 47(4):1098-1104 (1987); Cheung et al., Cancer Res 45(6):2642-2649 (1985); Cheung et al., J Clin Oncol 5(9):1430-1440 (1987); Cheung et al., J Clin Oncol 16(9):3053-3060 (1998); Handgretinger et al., Cancer Immunol Immunother 35(3):199-204 (1992), such as CDR. In some embodiments, the antigen-binding domain for GD2 is the antigen-binding portion of an antibody selected from mAb14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9. See, for example, International Publication Nos. 2012033885, 2013040371, 2013192294, 2013061273, 2013123061, 2013074916, and 201385552. In some embodiments, the antigen-binding domain for GD2 is the antigen-binding portion of the antibody described in U.S. Patent Application Publication No. 20100150910 or International Publication Brochure No. 2011160119. 【0494】 In one embodiment, the antigen-binding domain for the Tn antigen is, for example, the antigen-binding portion of an antibody described in U.S. Patent No. 8,440,798, Brooks et al., PNAS 107(22):10056-10061 (2010), and Stone et al., OncoImmunology 1(6):863-873 (2012), such as a CDR. 【0495】 In one embodiment, the antigen-binding domain for PSMA is, for example, the antigen-binding portion of antibodies and single-chain antibody fragments (scFv A5 and D7) described in Parker et al., Protein Expr Purif 89(2):136-145 (2013), U.S. Patent No. 20110268656 (J591 ScFv); Frigerio et al., European J Cancer 49(9):2223-2232 (2013) (scFvD2B); and International Publication No. 2006125481 (mAb 3 / A12, 3 / E7 and 3 / F11), such as CDR. 【0496】 In one embodiment, the antigen-binding domain for ROR1 is, for example, the antigen-binding portion of an antibody described in Hudecek et al, Clin Cancer Res 19(12):3153-3164(2013); International Publication No. 2011159847; and U.S. Patent No. 20130101607, such as CDR. 【0497】 In one embodiment, the antigen-binding domain for FLT3 is, for example, the antigen-binding portion of antibodies described in International Publication No. 2011076922, U.S. Patent No. 5777084, European Patent No. 0754230, U.S. Patent No. 20090297529 and several commercially available catalog antibodies (R&D, ebiosciences, Abcam), such as CDR. 【0498】 In one embodiment, the antigen-binding domain for TAG72 is, for example, the antibody described in Hombach et al, Gastroenterology 113(4):1163-1170 (1997); and the antigen-binding portion of Abcam ab691, for example, CDR. 【0499】 In one embodiment, the antigen-binding domain for FAP is, for example, the antigen-binding portion of an antibody described in Ostermann et al, Clinical Cancer Research 14:4584-4592 (2008) (FAP5), U.S. Patent Application Publication No. 2009 / 0304718; cibrotuzumab (see, for example, Hofheinz et al., Oncology Research and Treatment 26(1), 2003); and Tran et al., J Exp Med 210(6):1125-1135 (2013), e.g., CDR. 【0500】 In one embodiment, the antigen-binding domain for CD38 is the antigen-binding portion of an antibody described in U.S. Patent No. 8,263,746, e.g., CDR, such as daratumumab (see, e.g., Groen et al, Blood 116(21):1261-1262 (2010)); MOR202 (see, e.g., U.S. Patent No. 8,263,746); or U.S. Patent No. 8,362,211. 【0501】 In one embodiment, the antigen-binding domain for CD44v6 is, for example, the antigen-binding portion of an antibody described in Casucci et al, Blood 122(20):3461-3472 (2013), e.g., CDR. 【0502】 In one embodiment, the antigen-binding domain for CEA is, for example, the antigen-binding portion of an antibody described in Chmielewski et al, Gastoenterology 143(4):1095-1107 (2012), such as a CDR. 【0503】 In one embodiment, the antigen-binding domain for EPCAM is an antibody selected from MT110, an EpCAM-CD3 bispecificity Ab (see, for example, clinicaltrials.gov / ct2 / show / NCT00635596); edrecolomab; 3622W94; ING-1; and the antigen-binding moiety of adecatumumab (MT201), e.g., CDR. 【0504】 In one embodiment, the antigen-binding domain for PRSS21 is the antigen-binding portion of an antibody described in U.S. Patent No. 8,080,650, for example, a CDR. 【0505】 In one embodiment, the antigen-binding domain for B7H3 is the antigen-binding portion of the antibody MGA271 (Macrogenics), for example, the CDR. 【0506】 In one embodiment, the antigen-binding domain for KIT is, for example, the antigen-binding portion of antibodies described in U.S. Patent No. 7,915,391, U.S. Patent Application Publication No. 20120288506, and several commercially available catalog antibodies, such as CDRs. 【0507】 In one embodiment, the antigen-binding domain for IL-13Ra2 is, for example, the antigen-binding portion of the antibodies described in International Publication No. 2008 / 146911, International Publication No. 2004087758, several commercially available catalog antibodies, and the CDR of the antibodies described in International Publication No. 2004087758. 【0508】 In one embodiment, the antigen-binding domain for CD30 is, for example, the antigen-binding portion of an antibody described in U.S. Patent No. 7090843B1 and European Patent No. 0805871, such as CDR. 【0509】 In one embodiment, the antigen-binding domain for GD3 is, for example, the antigen-binding portion of an antibody described in U.S. Patent No. 7,253,263; U.S. Patent No. 8,207,308; U.S. Patent Publication No. 20120276046; European Patent No. 1013761; International Publication No. 2005035577; and U.S. Patent No. 6437098, such as a CDR. 【0510】 In one embodiment, the antigen-binding domain for CD171 is, for example, the antigen-binding portion of an antibody described in Hong et al., J Immunother 37(2):93-104 (2014), such as CDR. 【0511】 In one embodiment, the antigen-binding domain for IL-11Ra is the antigen-binding portion of an antibody available from Abcam (cat#ab55262) ​​or Novus Biologicals (cat#EPR5446), e.g., a CDR. In another embodiment, the antigen-binding domain for IL-11Ra is a peptide; see, for example, Huang et al., Cancer Res 72(1):271-281 (2012). 【0512】 In one embodiment, the antigen-binding domain for PSCA is, for example, the antigen-binding portion of an antibody described in Morgenroth et al., Prostate 67(10):1121-1131(2007)(scFv 7F5); Nejatollahi et al., J of Oncology 2013(2013), paper ID 839831(scFv C5-II); and U.S. Patent Application Publication No. 20090311181, such as CDR. 【0513】 In one embodiment, the antigen-binding domain for VEGFR2 is, for example, the antigen-binding portion of an antibody described in Chinnasamy et al., J Clin Invest 120(11):3953-3968(2010), such as CDR. 【0514】 In one embodiment, the antigen-binding domain for Lewis Y is, for example, the antigen-binding portion of an antibody described in Kelly et al., Cancer Biother Radiopharm 23(4):411-423(2008)(hu3S193 Ab(scFvs)); Dolezal et al., Protein Engineering 16(1):47-56(2003)(NC10 scFv), such as CDR. 【0515】 In one embodiment, the antigen-binding domain for CD24 is, for example, the antigen-binding portion of an antibody described in Malial et al., Gastroenterology 143(5):1375-1384 (2012), such as CDR. 【0516】 In one embodiment, the antigen-binding domain for PDGFR-β is the antigen-binding portion of the antibody Abcam ab32570, for example, the CDR. 【0517】 In one embodiment, the antigen-binding domain for SSEA-4 is the antigen-binding portion of the antibody MC813 (Cell Signaling) or another commercially available antibody, such as CDR. 【0518】 In one embodiment, the antigen-binding domain for CD20 is an antibody such as rituximab, ofatumumab, ocrelizumab, vertuzumab, or GA101; or the antigen-binding portion of an antibody described in International Publication No. 2016 / 164731, for example, CDR. 【0519】 In one embodiment, the antigen-binding domain for folate receptor α is the antigen-binding portion of antibody IMGN853 or the antibody described in U.S. Patent Application Publication No. 20120009181; U.S. Patent No. 4851332, LK26: U.S. Patent No. 5952484, for example, the CDR. 【0520】 In one embodiment, the antigen-binding domain (Her2 / neu) for ERBB2 is the antigen-binding portion of the antibody: trastuzumab or pertuzumab, for example, the CDR. 【0521】 In one embodiment, the antigen-binding domain for MUC1 is the antigen-binding portion of the antibody SAR566658, for example, the CDR. 【0522】 In one embodiment, the antigen-binding domain for EGFR is the antigen-binding portion of an antibody: cetuximab, panitumumab, zaltumumab, nimotuzumab, or matuzumab, for example, the CDR. 【0523】 In one embodiment, the antigen-binding domain for NCAM is the antigen-binding portion of antibody clone 2-2B:MAB5324 (EMD Millipore), for example, the CDR. 【0524】 In one embodiment, the antigen-binding domain for ephrin B2 is, for example, the antigen-binding portion of an antibody described in Abengozar et al., Blood 119(19):4565-4576 (2012), such as CDR. 【0525】 In one embodiment, the antigen-binding domain for the IGF-I receptor is, for example, the antigen-binding portion of an antibody described in U.S. Patent No. 8344112B2; European Patent Application Publication No. 2322550A1; International Publication No. 2006 / 138315 or PCT / US2006 / 022995, e.g., a CDR. 【0526】 In one embodiment, the antigen-binding domain for CAIX is the antigen-binding portion of antibody clone 303123 (R&D Systems), for example, CDR. 【0527】 In one embodiment, the antigen-binding domain for LMP2 is, for example, the antigen-binding portion of an antibody described in U.S. Patent No. 7,410,640 or U.S. Patent Application Publication No. 20050129701, such as a CDR. 【0528】 In one embodiment, the antigen-binding domain for gp100 is an antigen-binding portion of an antibody described in International Publication No. 2013165940 or U.S. Patent Application Publication No. 20130295007, such as a CDR. 【0529】 In one embodiment, the antigen-binding domain for tyrosinase is, for example, the antigen-binding portion of an antibody described in U.S. Patent No. 5,843,674 or U.S. Patent No. 1,995,050,4048, such as a CDR. 【0530】 In one embodiment, the antigen-binding domain for EphA2 is, for example, the antigen-binding portion of an antibody described in Yu et al., Mol Ther 22(1):102-111(2014), such as a CDR. 【0531】 In one embodiment, the antigen-binding domain for GD3 is, for example, the antigen-binding portion of an antibody described in U.S. Patent No. 7,253,263; U.S. Patent No. 8,207,308; U.S. Patent Application Publication No. 20120276046; European Patent Application Publication No. 1013761 A3; 20120276046; International Publication No. 2005035577; or U.S. Patent No. 6,437,098, such as a CDR. 【0532】 In one embodiment, the antigen-binding domain for fucosyl GM1 is, for example, the antigen-binding portion of an antibody described in U.S. Patent Application Publication No. 20100297138; or International Publication Brochure No. 2007 / 067992, such as a CDR. 【0533】 In one embodiment, the antigen-binding domain for sLe is the antigen-binding portion of antibody G193 (in the case of Lewis Y), for example, CDR (see Scott AM et al., Cancer Res 60:3254-61 (2000), and also as described in Neeson et al., J Immunol May 2013 190 (Meeting Abstract Supplement) 177.10). 【0534】 In one embodiment, the antigen-binding domain for GM3 is the antigen-binding portion of the antibody CA 2523449 (mAb 14F7), for example, CDR. 【0535】 In one embodiment, the antigen-binding domain for HMWMAA is, for example, the antigen-binding portion of an antibody described in Kmiecik et al., Oncoimmunology 3(1):e27185(2014)(PMID:24575382)(mAb9.2.27); U.S. Patent No. 6,528,481; International Publication No. 2010033866; or U.S. Patent Application Publication No. 20140004124, such as a CDR. 【0536】 In one embodiment, the antigen-binding domain for o-acetyl-GD2 is the antigen-binding portion of antibody 8B6, for example, CDR. 【0537】 In one embodiment, the antigen-binding domain for TEM1 / CD248 is, for example, the antigen-binding portion of an antibody described in Marty et al., Cancer Lett 235(2):298-308(2006); Zhao et al., J Immunol Methods 363(2):221-232(2011), such as CDR. 【0538】 In one embodiment, the antigen-binding domain for CLDN6 is the antigen-binding portion of the antibody IMAB027 (Ganymed Pharmaceuticals), for example, CDR (see, for example, clinicaltrial.gov / show / NCT02054351). 【0539】 In one embodiment, the antigen-binding domain for TSHR is, for example, the antigen-binding portion of an antibody described in U.S. Patent No. 8,603,466; No. 8,501,415; or No. 8,309,693, such as a CDR. 【0540】 In one embodiment, the antigen-binding domain for GPRC5D is the antigen-binding portion of an antibody: FAB6300A (R&D Systems); or LS-A4180 (Lifespan Biosciences), for example, the CDR. 【0541】 In one embodiment, the antigen-binding domain for CD97 is, for example, the antibody described in U.S. Patent No. 6,846,911; de Groot et al., J Immunol 183(6):4127-4134 (2009); or the antigen-binding portion of the antibody from R&D:MAB3734, e.g., CDR. 【0542】 In one embodiment, the antigen-binding domain for ALK is, for example, the antigen-binding portion of an antibody described in Mino-Kenudson et al., Clin Cancer Res 16(5):1561-1571 (2010), such as CDR. 【0543】 In one embodiment, the antigen-binding domain for polysialic acid is, for example, the antigen-binding portion of an antibody described in Nagae et al., J Biol Chem 288(47):33784-33796(2013), such as a CDR. 【0544】 In one embodiment, the antigen-binding domain for PLAC1 is, for example, the antigen-binding portion of an antibody described in Ghods et al., Biotechnol Appl Biochem 2013 doi:10.1002 / bab.1177, such as a CDR. 【0545】 In one embodiment, the antigen-binding domain for GloboH is antibody VK9; or the antigen-binding portion of an antibody described, for example, Kudryashov V et al., Glycoconj J.15(3):243-9(1998), Lou et al., Proc Natl Acad Sci USA 111(7):2482-2487(2014); MBr1: Bremer EG et al. J Biol Chem 259:14773-14777(1984). 【0546】 In one embodiment, the antigen-binding domain for NY-BR-1 is the antigen-binding moiety, for example, the CDR of an antibody described in Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007). 【0547】 In one embodiment, the antigen-binding domain for WT-1 is, for example, the antigen-binding portion of an antibody described in Dao et al., Sci Transl Med 5(176):176ra33(2013); or International Publication No. 2012 / 135854, such as the CDR. 【0548】 In one embodiment, the antigen-binding domain for MAGE-A1 is, for example, the antigen-binding portion of an antibody described in Willemsen et al., J Immunol 174(12):7853-7858(2005) (TCR-like scFv), such as a CDR. 【0549】 In one embodiment, the antigen-binding domain for sperm protein 17 is, for example, the antigen-binding portion of an antibody described in Song et al., Target Oncol 2013 Aug 14 (PMID:23943313); Song et al., Med Oncol 29(4):2923-2931 (2012), such as a CDR. 【0550】 In one embodiment, the antigen-binding domain for Tie 2 is the antigen-binding portion of antibody AB33 (Cell Signaling Technology), for example, CDR. 【0551】 In one embodiment, the antigen-binding domain for MAD-CT-2 is, for example, the antigen-binding portion of an antibody described in PMID:2450952; U.S. Patent No. 7635753, e.g., CDR. 【0552】 In one embodiment, the antigen-binding domain for Fos-related antigen 1 is the antigen-binding portion of antibody 12F9 (Novus Biologicals), for example, the CDR. 【0553】 In one embodiment, the antigen-binding domain for MelanA / MART1 is the antigen-binding portion of an antibody described in European Patent Publication No. 2514766A2; or U.S. Patent No. 7,749,719, for example, a CDR. 【0554】 In one embodiment, the antigen-binding domain for the sarcoma translocation breakpoint is, for example, the antigen-binding portion of an antibody described in Luo et al., EMBO Mol. Med. 4(6):453-461 (2012), such as a CDR. 【0555】 In one embodiment, the antigen-binding domain for TRP-2 is, for example, the antigen-binding portion of an antibody described in Wang et al., J Exp Med. 184(6):2207-16 (1996), such as a CDR. 【0556】 In one embodiment, the antigen-binding domain for CYP1B1 is, for example, the antigen-binding portion of an antibody described in Maecker et al., Blood 102(9):3287-3294 (2003), such as a CDR. 【0557】 In one embodiment, the antigen-binding domain for RAGE-1 is the antigen-binding portion of the antibody MAB5328 (EMD Millipore), for example, the CDR. 【0558】 In one embodiment, the antigen-binding domain for human telomerase reverse transcriptase is the antigen-binding portion of the antibody (cat no: LS-B95-100, Lifespan Biosciences), for example, the CDR. 【0559】 In one embodiment, the antigen-binding domain for intestinal carboxylesterase is the antigen-binding portion of the antibody 4F12 (cat no. LS-B6190-50, Lifespan Biosciences), for example, the CDR. 【0560】 In one embodiment, the antigen-binding domain for mut hsp70-2 is the antigen-binding portion of the antibody:Lifespan Biosciences:monoclonal:cat no:LS-C133261-100(Lifespan Biosciences), for example, the CDR. 【0561】 In one embodiment, the antigen-binding domain for CD79a is the antigen-binding portion, e.g., CDR, of the following antibodies: an antibody available from Abcam, anti-CD79a antibody [HM47 / A9](ab3121); an antibody available from Cell Signalling Technology, CD79A antibody #3351; or an antibody available from Sigma Aldrich, HPA017748 (an anti-CD79A antibody produced in rabbits). 【0562】 In one embodiment, the antigen-binding domain for CD79b is polatuzumab vedotin, anti-CD79b, or "4507 Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b / CD3 As a Potential Therapy for B Cell Malignancies" Abstracts of 56th ASH Annual Meeting and Exposition, San Francisco, CA December 6-9 This refers to the antigen-binding portion of the bispecific antibody Anti-CD79b / CD3 described in 2014, for example, the CDR. 【0563】 In one embodiment, the antigen-binding domain for CD72 is the antigen-binding portion of the following antibody: J3-109 described in Myers, and Uckun, “An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia.” Leuk Lymphoma. 1995 Jun;18(1-2):119-22, or the antigen-binding portion of anti-CD72 (10D6.8.1, mIgG1) described in Polson et al., “Antibody-Drug Conjugates for the Treatment of Non-Hodgkin's Lymphoma: Target and Linker-Drug Selection” Cancer Res March 15, 2009 69;2358, for example, the CDR. 【0564】 In one embodiment, the antigen-binding domain for LAIR1 is the following antibody: ANT-301 LAIR1 antibody available from ProSpec; or the antigen-binding portion of an anti-human CD305 (LAIR1) antibody available from BioLegend, e.g., CDR. 【0565】 In one embodiment, the antigen-binding domain for FCAR is the antigen-binding portion of an antibody available from Sino Biological Inc.: CD89 / FCARAntibody (catalog #10414-H08H), such as the CDR. 【0566】 In one embodiment, the antigen-binding domain for LILRA2 is the antigen-binding portion, e.g., CDR, of the following antibodies: LILRA2 monoclonal antibody (M17), clone 3C7, available from Abnova, or mouse anti-LILRA2 antibody, monoclonal (2D7), available from Lifespan Biosciences. 【0567】 In one embodiment, the antigen-binding domain for CD300LF is the antigen-binding portion of the following antibodies: mouse anti-CMRF35-like molecule 1 antibody, monoclonal [UP-D2] available from BioLegend or rat anti-CMRF35-like molecule 1 antibody, monoclonal [234903] available from R&D Systems, e.g., CDR. 【0568】 In one embodiment, the antigen-binding domain for CLEC12A is the following antibody: bispecific T cell derivative (BiTE) scFv antibody and the ADC described in Noordhuis et al., “Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1xCD3 BiTE Antibody” 53rd ASH Annual Meeting and Exposition, December 10-13, 2011, as well as the antigen-binding portion of MCLA-117(Merus), for example, the CDR. 【0569】 In one embodiment, the antigen-binding domain for BST2 (also called CD317) is the antigen-binding portion of the following antibody: mouse anti-CD317 antibody, monoclonal [3H4] available from Antibodies-Online or mouse anti-CD317 antibody, monoclonal [696739] available from R&D Systems, e.g., CDR. 【0570】 In one embodiment, the antigen-binding domain for EMR2 (also called CD312) is the antigen-binding portion, e.g., CDR, of the following antibodies: mouse anti-CD312 antibody, monoclonal [LS-B8033] available from Lifespan Biosciences or mouse anti-CD312 antibody, monoclonal [494025] available from R&D Systems. 【0571】 In one embodiment, the antigen-binding domain for LY75 is the antigen-binding portion, e.g., CDR, of the following antibodies: mouse anti-lymphocyte antigen 75 antibody, monoclonal [HD30] available from EMD Millipore or mouse anti-lymphocyte antigen 75 antibody, monoclonal [A15797] available from Life Technologies. 【0572】 In one embodiment, the antigen-binding domain for GPC3 is the antigen-binding portion, for example, the CDR, of the following antibody: hGC33 or MDX-1414, HN3 or YP7, as described in Nakano K, Ishiguro T, Konishi H, et al. Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization. Anticancer Drugs. 2010 Nov;21(10):907-916 (all three of these are described in Feng et al., “Glypican-3 antibodies: a new therapeutic target for liver cancer.” FEBS Lett. 2014 Jan 21;588(2):377-82). 【0573】 In one embodiment, the antigen-binding domain for FCRL5 is the antigen-binding portion of an anti-FcRL5 antibody described in Elkins et al., “FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma,” Mol Cancer Ther. 2012 Oct;11(10):2222-32, for example, a CDR. In one embodiment, the antigen-binding domain for FCRL5 is, for example, the antigen-binding portion of an anti-FcRL5 antibody described in International Publication Nos. 2001 / 038490, 2005 / 117986, 2006 / 039238, 2006 / 076691, 2010 / 114940, 2010 / 120561, or 2014 / 210064, for example, a CDR. 【0574】 In one embodiment, the antigen-binding domain for IGLL1 is the antigen-binding portion of the mouse anti-immunoglobulin λ-like polypeptide 1 antibody, monoclonal [AT1G4] available from Lifespan Biosciences, or the mouse anti-immunoglobulin λ-like polypeptide 1 antibody, monoclonal [HSL11] available from BioLegend, for example, the CDR. 【0575】 In one embodiment, the antigen-binding domain includes one, two, or three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3 derived from the antibodies listed above, and / or one, two, or three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3 derived from the antibodies listed above. In one embodiment, the antigen-binding domain includes the heavy chain variable region and / or the variable light chain region of the antibodies listed above. 【0576】 In another embodiment, the antigen-binding domain comprises a humanized antibody or antibody fragment. In some embodiments, a non-human antibody is humanized, and a specific sequence or region of the antibody is modified to increase its similarity to an antibody or fragment naturally produced in humans. In one embodiment, the antigen-binding domain is humanized. 【0577】 CARs that bind to mesothelin are known in the art. For example, those that bind to human mesothelin are disclosed in International Publication No. 2015090230 and International Publication No. 2017112741, which are incorporated herein by reference, for example in Tables 2, 3, 4, and 5 of International Publication No. 2017112741. In the art, any known mesothelin CAR, for example, the mesothelin antigen-binding domain of any known mesothelin CAR, may be used in accordance with this disclosure. 【0578】 CARs that bind to GFR ALPHA-4 are known in the art. For example, those disclosed in International Publication No. 2016 / 025880 may be used in accordance with this disclosure. In the art, any known GFR ALPHA-4 CAR, for example, the GFR ALPHA-4 antigen-binding domain of any known GFR ALPHA-4 CAR may be used in accordance with this disclosure. The amino acid and nucleotide sequences encoding the GFR ALPHA-4 CAR molecules and antigen-binding domains (e.g., including one, two, or three VH CDRs and one, two, or three VL CDRs by Kabat or Chothia) are specified in International Publication No. 2016 / 025880. 【0579】 antigen-binding domain structure In some embodiments, the antigen-binding domain of the encoded CAR molecule includes an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single-domain antibody (SDAB), VH or VL domain, camelid VHH domain, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). 【0580】 In some cases, scFv can be produced by methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking the VH and VL regions together using a mobile polypeptide linker. The scFv molecule contains a linker (e.g., a Ser-Gly linker) having an optimal length and / or amino acid composition. The linker length can significantly influence how the variable regions of the scFv fold and interact. In fact, when using short polypeptide linkers (e.g., 5-10 amino acids), intrachain folding is inhibited. Intrachain folding is also necessary for the two variable regions to unite and form a functional epitope binding site. For examples of linker orientations and sizes, see, for example, Hollinger et al. 1993 Proc Natl Acad.Sci.USA90:6444-6448, U.S. Patent Application Publication No. 2005 / 0100543, U.S. Patent Application Publication No. 2005 / 0175606, U.S. Patent Application Publication No. 2007 / 0014794, and International Publication Brochures No. 2006 / 020258 and International Publication Brochures No. 2007 / 024715, which are incorporated herein by reference. 【0581】 scFv may contain a linker between its VL region and VH region of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more amino acid residues. The linker sequence may contain any naturally occurring amino acid. In some embodiments, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains a series of glycine and serine repeats such as (Gly4Ser)n (where n is one or more positive integers) (SEQ ID NO: 22). In some embodiments, the linker may be (Gly4Ser)4 (SEQ ID NO: 29) or (Gly4Ser)3 (SEQ ID NO: 30). Variations in linker length can maintain or enhance activity, potentially leading to superior efficacy in activity tests. 【0582】 In another embodiment, the antigen-binding domain is a T cell receptor ("TCR") or a fragment thereof, such as a single-chain TCR (scTCR). Methods for constructing such TCRs are known in the art. See, for example, Willemsen RA et al, Gene Therapy 7:1369-1377 (2000); Zhang T et al, Cancer Gene Ther 11:487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) (the entire reference is incorporated herein). For example, scTCRs containing Vα and Vβ genes derived from T cell clones linked by a linker (e.g., a flexible peptide) can be manipulated. This approach is very useful for cancer-related targets that are themselves intracellular, but fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC. 【0583】 In certain embodiments, the encoded antigen-binding domain is 10 -4 M~10 -8 It has binding affinity KD for M. 【0584】 In some embodiments, the encoded CAR molecule is 10 for the target antigen. -4 M~10 -8 M, for example 10 -5 M~10 -7 M, for example 10 -6 M or 10 -7 Binding affinity K of M D It comprises an antigen-binding domain having a binding affinity at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, or at least 1000 times lower than a reference antibody, e.g., an antibody described herein. In some embodiments, the encoded antigen-binding domain has a binding affinity at least 5 times lower than a reference antibody (e.g., an antibody from which the antigen-binding domain is derived). In some embodiments, such antibody fragments are functional in that they provide a biological response that includes, but is not limited to, activation of an immune response and inhibition of signal initiation from its target antigen, as understood by those skilled in the art. 【0585】 In some embodiments, the antigen-binding domain of the CAR is a humanized scFv antibody fragment compared to the mouse sequence of the scFv from which it is derived. 【0586】 In some embodiments, the antigen-binding domain (e.g., scFv) of the CAR described herein is encoded by a nucleic acid molecule whose sequence is codon-optimized for expression in mammalian cells. In some embodiments, the entire CAR construct is encoded by a nucleic acid molecule whose entire sequence is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons encoding the same amino acid) in coding DNA is biased across different species. Such codon degeneracy allows the same polypeptide to be encoded by various nucleotide sequences. Various codon optimization methods are known in the art and include, for example, the methods disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148. 【0587】 As will be understood by those skilled in the art, percentage (%) amino acid sequence identity for nucleic acids (e.g., DNA or RNA), peptides, and polypeptides can be calculated as the percentage of nucleotide or amino acid residues in a candidate sequence that are identical to a nucleotide or amino acid residue in a particular nucleotide, peptide, or polypeptide sequence, after aligning the sequences and introducing gaps as necessary, without considering any conservative substitutions as part of sequence identity, and achieving maximum percentage sequence identity. Alignment aimed at determining the percentage of nucleic acid / amino acid sequence identity can be achieved in various ways within the scope of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. Unless otherwise indicated herein, percentage identity is calculated using BLAST. 【0588】 Certain antigen-antibody pairs are known in the art. Non-limiting exemplary embodiments of antigen-antibody pairs and their components are provided in the section above titled “Targets” and below. 【0589】 Bispecific CAR In certain embodiments, the antigen-binding domain is a bispecific or multispecific molecule (e.g., a multispecific antibody molecule). In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody is specific to two or fewer antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In some embodiments, the first and second epitopes are the same antigen, e.g., the same protein (or a subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, the bispecific antibody molecule includes a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a second epitope. In some embodiments, the bispecific antibody molecule includes a semi-antibody having binding specificity to a first epitope and a semi-antibody having binding specificity to a second epitope. In some embodiments, the bispecific antibody molecule includes a semi-antibody or a fragment thereof having binding specificity to a first epitope and a semi-antibody or a fragment thereof having binding specificity to a second epitope. In some embodiments, the bispecific antibody molecule includes an scFv or a fragment thereof having binding specificity to a first epitope and an scFv or a fragment thereof having binding specificity to a second epitope. 【0590】 In some embodiments, the antibody molecule is a multispecific (e.g., bispecific or triplicate) antibody molecule. Such molecules include, for example, bispecific fusion proteins, expression constructs comprising, for example, two scFvs, a hydrophilic helic peptide linker between them, and a fully constant region, as described in, for example, U.S. Patent No. 5,637,481; minibody constructs having linked VL and VH chains, further linked by peptide spacers to the antibody hinge region and CH3 region, which can dimerize to form a bispecific / multivalent molecule, as described in, for example, U.S. Patent No. 5,837,821; and crosslinkable groups linked at the C-terminus by peptide bonds, further linked to the VL domain. This includes strings of VH domains (or VL domains of family members) that associate with ions to form a series of FVs (or scFvs), e.g., as described in U.S. Patent No. 5,864,019; and single-chain linked polypeptides having both VH and VL domains linked by peptide linkers that combine into multivalent structures by non-covalent bonding or chemical crosslinking, e.g., homobivalent, heterobivalent, trivalent, and tetravalent structures, using both scFV or bispecific antibody type formats, e.g., as described in U.S. Patent No. 5,869,620. The contents of the above-referenced applications are incorporated herein by reference in their entirety. 【0591】 Within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, VH can be upstream or downstream of VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is positioned with its VH (VH1) upstream of its VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) is positioned with its VH (VH2) upstream of its VL (VL2), resulting in the overall bispecific antibody molecule having the configuration VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is positioned with its VL (VL1) upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is positioned with its VL (VL2) upstream of its VH (VH2), resulting in the overall bispecific antibody molecule having the configuration VL1-VH1-VH2-VL2. Optionally, a linker is positioned between two antibodies or antibody fragments (e.g., scFv), for example, between VL1 and VL2 when the construct is sequenced as VH1-VL1-VL2-VH2, or between VH1 and VH2 when the construct is arranged as VL1-VH1-VH2-VL2. The linker may be one of the linkers described herein, for example, a (Gly4-Ser)n linker (where n is 1, 2, 3, 4, 5, or 6, preferably 4) (SEQ ID NO: 691). In general, the linker between two scFvs must be long enough to avoid mispairing between the domains of the two scFvs. Optionally, the linker is positioned between VL and VH of the first scFv. Optionally, the linker is positioned between VL and VH of the second scFv. In constructs having multiple linkers, any two or more linkers may be identical or different. Accordingly, in some embodiments, the dual-specific CAR comprises a VL, a VH, and optionally one or more linkers in the configuration described herein. 【0592】 transmembrane domain With respect to the transmembrane domain, in various embodiments, the chimeric molecules (e.g., CARs) described herein can be designed to include a transmembrane domain that binds to the extracellular domain of the chimeric molecule. The transmembrane domain may include one or more further amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular domain of the protein from which the transmembrane originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 from the extracellular domain) and / or one or more further amino acids associated with the intracellular domain of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 from the intracellular domain). In some embodiments, the transmembrane domain is associated with one of the other domains of the chimeric protein (e.g., CAR), and in some embodiments, for example, the transmembrane domain may originate from the same protein from which the signaling domain, co-stimulatory domain, or hinge domain originates. In other embodiments, the transmembrane domain does not originate from the same protein from which any other domain of the chimeric protein (e.g., CAR) originates. In some cases, transmembrane domains can be selectively modified or modified by amino acid substitution to minimize interactions with other members of the receptor complex, for example, to avoid binding to the transmembrane domains of the same or different surface membrane proteins. In some embodiments, transmembrane domains can homodimerize with another CAR on the cell surface of a CAR-expressing cell. In other embodiments, the amino acid sequence of a transmembrane domain can be modified or substituted to minimize interactions with the binding domain of a native binding partner present in the same CAR-expressing cell. 【0593】 The transmembrane domain may be of natural origin or recombinant origin. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain can always signal to the intracellular domain when the CAR binds to a target. In some embodiments, the transmembrane domain may include, for example, the alpha, beta, or zeta chain of a T cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154 transmembrane regions. In some embodiments, the transmembrane domains are, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGA It may include at least the transmembrane region of M, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C. 【0594】 In some embodiments, the transmembrane domain can bind to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. For example, in some embodiments, the hinge may be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge or an IgD hinge), a GS linker (e.g., the GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge. In some embodiments, the hinge or spacer comprises (e.g., derived from) the amino acid sequence of SEQ ID NO: 4. In some embodiments, the transmembrane domain comprises (e.g., derived from) the transmembrane domain of SEQ ID NO: 12. 【0595】 In some embodiments, the encoded transmembrane domain includes an amino acid sequence of a CD8 transmembrane domain having at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 12, but with no more than 20, 10, or 5 modifications, or a sequence that is 95-99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the encoded transmembrane domain includes the sequence of SEQ ID NO: 12. 【0596】 In other embodiments, the nucleic acid encoding the CAR molecule includes, for example, a nucleotide sequence of the CD8 transmembrane domain containing the sequence of SEQ ID NO: 13 or a sequence having 95-99% identity thereto. 【0597】 In some embodiments, the encoded antigen-binding domain is linked to the transmembrane domain by a hinge region. In some embodiments, the encoded hinge region includes the amino acid sequence of the CD8 hinge, e.g., SEQ ID NO: 4; or the amino acid sequence of the IgG4 hinge, e.g., SEQ ID NO: 6 or a sequence having 95-99% identity with SEQ ID NO: 4 or 6. In other embodiments, the nucleic acid sequence encoding the hinge region includes the sequence of SEQ ID NO: 5 or SEQ ID NO: 7, or a sequence having 95-99% identity with SEQ ID NO: 5 or 7, corresponding to the CD8 hinge or the IgG4 hinge, respectively. 【0598】 In some embodiments, the hinge or spacer includes an IgG4 hinge. For example, in some embodiments, the hinge or spacer is an amino acid sequence [ka] Includes a hinge. In some embodiments, the hinge or spacer is [ka] It contains a hinge encoded by the nucleotide sequence. 【0599】 In some embodiments, the hinge or spacer includes an IgD hinge. For example, in some embodiments, the hinge or spacer is an amino acid sequence [ka] Includes a hinge. In some embodiments, the hinge or spacer is [ka] It contains a hinge encoded by the nucleotide sequence. 【0600】 In some embodiments, the transmembrane domain may be recombinant, in which case it predominantly contains hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain. 【0601】 Optionally, short oligo or polypeptide linkers of 2 to 10 amino acids in length may form a linkage between the transmembrane domain and the cytoplasmic region of the CAR. Glycine-serine doublets provide particularly suitable linkers. For example, in some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 10). In some embodiments, the linker is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 11). In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 877). In some embodiments, the linker is encoded by the nucleotide sequence of SEQ ID NO: 876. 【0602】 In some embodiments, the hinge or spacer includes a KIR2DS2 hinge. 【0603】 Signal transduction domains In some embodiments of the CAR described herein, which have an intracellular signaling domain, such domain may include, for example, one or more primary signaling domains and / or co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain includes a sequence encoding a primary signaling domain. In some embodiments, the intracellular signaling domain includes a co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain includes a primary signaling domain and a co-stimulatory signaling domain. 【0604】 The intracellular signaling sequences within the cytoplasmic portion of CARs can be linked to each other randomly or in a specific order. Optionally, short oligo or polypeptide linkers of, for example, 2 to 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) can form links between the intracellular signaling sequences. In some embodiments, a glycine-serine doublet can be used as a suitable linker. In some embodiments, a single amino acid, such as alanine or glycine, can be used as a suitable linker. 【0605】 In some embodiments, the intracellular signaling domain is designed to include two or more, for example, two, three, four, five, or more, co-stimulatory signaling domains. In some embodiments, two or more, for example, two, three, four, five, or more, co-stimulatory signaling domains are separated by a linker molecule, for example, the linker molecule described herein. In some embodiments, the intracellular signaling domain includes two co-stimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue. 【0606】 Primary signal transduction domains The primary signaling domain controls the primary activation of the TCR complex in either a stimulative or inhibitory manner. Primary intracellular signaling domains that act in a stimulative manner may contain an immunoreceptor tyrosine-based activation motif or a signaling motif known as ITAM. In CARs, such domains are used for the same purpose. 【0607】 Examples of primary intracellular signaling domains containing ITAM include those of CD3ζ, common FcRγ (FCER1G), FcgammaRIIa, FcRbeta (FcepsilonR1b), CD3gamma, CD3delta, CD3epsilon, CD79a, CD79b, DAP10, and DAP12. In some embodiments, the CAR includes an intracellular signaling domain, such as the primary signaling domain of CD3ζ. 【0608】 In some embodiments, the encoded primary signaling domain includes a functional signaling domain of CD3ζ. The encoded CD3ζ primary signaling domain may include an amino acid sequence having at least one, two, or three modifications to SEQ ID NO: 18 or SEQ ID NO: 20, but with no more than 20, 10, or 5 modifications, or a sequence that is 95-99% identical to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, the encoded primary signaling domain includes the sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In other embodiments, the nucleic acid sequence encoding the primary signaling domain includes the sequence of SEQ ID NO: 19 or SEQ ID NO: 21, or a sequence that is 95-99% identical thereto. 【0609】 Co-stimulatory signaling domain In some embodiments, the encoded intracellular signaling domain includes a co-stimulatory signaling domain. For example, the intracellular signaling domain may include a primary signaling domain and a co-stimulatory signaling domain. In some embodiments, the encoded co-stimulatory signaling domain is ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD1 It contains a functional signaling domain of a protein selected from one or more of the following: 1b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D. 【0610】 In some embodiments, the encoded co-stimulatory signaling domain includes an amino acid sequence having at least one, two, or three modifications to SEQ ID NO: 14 or SEQ ID NO: 16, but with no more than 20, 10, or 5 modifications, or a sequence having 95-99% identity with SEQ ID NO: 14 or SEQ ID NO: 16. In some embodiments, the encoded co-stimulatory signaling domain includes the sequence of SEQ ID NO: 14 or SEQ ID NO: 16. In other embodiments, the nucleic acid sequence encoding the co-stimulatory signaling domain includes the sequence of SEQ ID NO: 15 or SEQ ID NO: 17, or a sequence having 95-99% identity thereto. 【0611】 In other embodiments, the encoded intracellular domain includes the sequence of SEQ ID NO: 14 or SEQ ID NO: 16 and the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, and the sequences containing these intracellular signaling domains are expressed in the same frame and as a single polypeptide chain. 【0612】 In some embodiments, the nucleic acid sequence encoding the intracellular signaling domain includes the sequence of SEQ ID NO: 15 or SEQ ID NO: 17 or a sequence having 95-99% identity thereto, and the sequence of SEQ ID NO: 19 or SEQ ID NO: 21 or a sequence having 95-99% identity thereto. 【0613】 In some embodiments, the nucleic acid molecule further encodes a leader sequence. In some embodiments, the leader sequence includes the sequence of SEQ ID NO: 2. 【0614】 In some embodiments, the intracellular signaling domain is designed to include a CD3ζ signaling domain and a CD28 signaling domain. In some embodiments, the intracellular signaling domain is designed to include a CD3ζ signaling domain and a 4-1BB signaling domain. In some embodiments, the 4-1BB signaling domain is the signaling domain of SEQ ID NO: 14. In some embodiments, the CD3ζ signaling domain is the signaling domain of SEQ ID NO: 18. 【0615】 In some embodiments, the intracellular signaling domain is designed to include a CD3ζ signaling domain and a CD27 signaling domain. In some embodiments, the CD27 signaling domain includes the amino acid sequence QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 16). In some embodiments, the CD27 signaling domain is [ka] It is encoded by the nucleic acid sequence. 【0616】 Inhibitory domain In some embodiments, the vector comprises a nucleic acid sequence encoding an inhibitor molecule, including a CAR, for example, a nucleic acid sequence encoding a CAR as described herein and an inhibitor KIR (inhKIR) cytoplasmic domain; a transmembrane domain, for example, a KIR transmembrane domain; and an inhibitor cytoplasmic domain, for example, an ITIM domain, for example, an inhKIR ITIM domain. In some embodiments, the inhibitor molecule is a naturally occurring inhKIR, or a sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homology with a naturally occurring inhKIR, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues. 【0617】 In some embodiments, the nucleic acid sequence encoding the inhibitory molecule includes a SLAM family cytoplasmic domain; a transmembrane domain, e.g., a SLAM family transmembrane domain; and an inhibitory cytoplasmic domain, e.g., a SLAM family domain, e.g., a SLAM family ITIM domain. In some embodiments, the inhibitory molecule is a naturally occurring SLAM family member, or a sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homology with a naturally occurring SLAM family member, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues. 【0618】 In some embodiments, the vector is an in vitro transcription vector, for example, a vector for transcribing RNA of a nucleic acid molecule described herein. In some embodiments, the nucleic acid sequence of the vector further comprises a poly(A) tail, for example, a poly-A tail. In some embodiments, the nucleic acid sequence of the vector further comprises a 3'UTR, for example, a 3'UTR comprising at least one repeat of a 3'UTR derived from human β-globulin, as described herein. In some embodiments, the nucleic acid sequence of the vector further comprises a promoter, for example, a T2A promoter. 【0619】 promoter In some embodiments, the vector further comprises a promoter. In some embodiments, the promoter is selected from the EF-1 promoter, the CMV IE gene promoter, the EF-1α promoter, the ubiquitin C promoter, or the phosphoglycerate kinase (PGK) promoter. In some embodiments, the promoter is the EF-1 promoter. In some embodiments, the EF-1 promoter comprises the sequence of SEQ ID NO: 1. 【0620】 In some embodiments, immunoeffector cells, such as T cells, can be obtained from units of blood collected from a subject using any technique known to those skilled in the art, such as Ficoll® isolation. In some embodiments, cells are obtained from the circulating blood of an individual by apheresis. Apheresis products typically include lymphocytes, erythrocytes, and platelets, including T cells, monocytes, granulocytes, B cells, and other nucleated leukocytes. In some embodiments, cells obtained by apheresis are washed to remove the plasma fraction, and optionally the cells can be suspended in a buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or many, if not all, divalent cations. 【0621】 [Table 124] 【0622】 [Table 125] 【0623】 [Table 126] 【0624】 [Table 127] 【0625】 [Table 128] 【0626】 [Table 129] 【0627】 Manufacturing method The lentiviral vectors described herein (for example, those prepared using the methods described herein) can be used, for example, in the in vitro production of CAR-T cells. 【0628】 The CARTs disclosed herein may be manufactured ex vivo by any method known in the art. For example, the methods described in International Publication No. 2012 / 079000 or International Publication No. 2020 / 047452 (both incorporated herein by reference) may be used. The CARTs disclosed herein may also be manufactured in vivo by any method known in the art. For example, the method described in International Publication No. 2020 / 176397 (incorporated herein by reference) is used. In vivo CART production is also described in International Publication No. 2022 / 040586 (in its entirety incorporated herein by reference), for example. 【0629】 Immune effector cells (e.g., T cells or NK cells) may express one or more CARs. 【0630】 In some embodiments, the methods disclosed herein can produce immunoeffector cells engineered to express one or more CARs in less than 24 hours. While not wishing to be bound by theory, the methods provided herein preserve the undifferentiated phenotype of T cells, such as naive T cells, during the manufacturing process. These CAR-expressing cells with the undifferentiated phenotype may persist longer and / or proliferate better in vivo after injection. In some embodiments, CART cells produced by the manufacturing methods provided herein contain a higher percentage of stem cell memory T cells compared to CART cells produced by conventional manufacturing processes, as measured, for example, using scRNA-seq. 【0631】 In some embodiments, CART cells produced by the manufacturing methods provided herein contain a higher percentage of effector T cells compared to CART cells produced by conventional manufacturing processes, as measured, for example, using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein retain better stem cell properties of T cells compared to CART cells produced by conventional manufacturing processes, as measured, for example, using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein exhibit lower levels of hypoxia compared to CART cells produced by conventional manufacturing processes, as measured, for example, using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein exhibit lower levels of autophagy compared to CART cells produced by conventional manufacturing processes, as measured, for example, using scRNA-seq. In some embodiments, immune effector cells are engineered to contain one or more CAR-encoding nucleic acid molecules disclosed herein. 【0632】 In some embodiments, the methods disclosed herein do not involve the use of beads such as Dynabeads® (e.g., CD3 / CD28 Dynabeads®) and therefore do not involve a bead removal step. In some embodiments, CART cells produced by the methods disclosed herein can be administered to a subject with minimal ex vivo proliferation, e.g., less than 1 day, less than 12 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or without ex vivo proliferation. Accordingly, the methods disclosed herein provide a rapid production process for producing improved CAR-expressing cell products for use in the treatment of diseases in subjects. 【0633】 In some embodiments, the present disclosure provides a method for producing a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), the method comprising the steps of: (i) contacting a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukocyte apheresis products) with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates a co-stimulatory molecule on the cell surface; (ii) contacting a population of cells (e.g., T cells) with a nucleic acid molecule encoding a CAR (e.g., a DNA or RNA molecule), thereby providing a population of cells (e.g., T cells) containing the nucleic acid molecule; and (iii) collecting a population of cells (e.g., T cells) for storage (e.g., reconstitution of the cell population in cryopreservation medium) or administration, wherein (a) step (ii) is performed together with step (i) or within 20 hours after the start of step (i), for example, within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example (b) Step (ii) shall be carried out within 18 hours of the commencement of Step (i), and Step (iii) shall be carried out within 26 hours of the commencement of Step (i), for example within 22, 23 or 24 hours of the commencement of Step (i), for example within 24 hours of the commencement of Step (i); (b) Step (ii) shall be carried out together with Step (i) or within 20 hours of the commencement of Step (i), for example within 12, 13, 14, 15, 16, 17 or 18 hours of the commencement of Step (i), for example within 18 hours of the commencement of Step (i) (c) Step (iii) is performed within 30 hours of the start of step (ii), for example, within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours of the start of step (ii), or (c) the population of cells from step (iii) has not proliferated, or, as evaluated by the number of viable cells, has proliferated by 5, 10, 15, 20, 25, 30, 35, or 40% or less compared to the population of cells at the start of step (i), for example, about 10%. In some embodiments, the nucleic acid molecule in step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is an RNA molecule.In some embodiments, the nucleic acid molecule of step (ii) is located on a viral vector, such as a lentiviral vector, adenovirus vector, or retroviral vector. In some embodiments, the nucleic acid molecule of step (ii) is located on a non-viral vector. In some embodiments, the nucleic acid molecule of step (ii) is located on a plasmid. In some embodiments, the nucleic acid molecule of step (ii) is not located on any vector. In some embodiments, step (ii) includes transducing a population of cells (e.g., T cells) with a viral vector containing a nucleic acid molecule encoding a CAR. 【0634】 In some embodiments, a population of cells (e.g., T cells) is collected from an apheresis sample (e.g., a leukocyte apheresis sample) from a subject. 【0635】 In some embodiments, apheresis samples (e.g., leukocyte apheresis samples) are collected from subjects and transported to a cell manufacturing facility as frozen samples (e.g., cryopreserved samples). The frozen apheresis samples are then thawed, and T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis samples using, for example, a cell sorting machine (e.g., a CliniMACS® Prodigy® instrument). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then seeded for CART production using the activation process described herein. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are subjected to one or more rounds of freeze-thaw cycles before seeding for CART production. 【0636】 In some embodiments, an apheresis sample (e.g., a leukocyte apheresis sample) is collected from the subject and transported to a cell manufacturing facility as a fresh product (e.g., an unfrozen product). For example, T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample using a cell sorter (e.g., a CliniMACS® Prodigy® instrument). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then seeded for CART production using the activation process described herein. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are subjected to one or more rounds of freeze-thaw cycles before seeding for CART production. 【0637】 In some embodiments, an apheresis sample (e.g., a leukocyte apheresis sample) is collected from the subject. For example, T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample using a cell sorting machine (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then transported to a cell manufacturing facility as a frozen sample (e.g., a cryopreserved sample). Subsequently, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are thawed and seeded for CART production using the activation process described herein. 【0638】 In some embodiments, cells (e.g., T cells) are exposed to anti-CD3 and anti-CD28 antibodies for, for example, 12 hours, followed by transduction (e.g., one or more CARs) using a vector encoding a CAR (e.g., a lentiviral vector) (e.g., one or more vectors). Twenty-four hours after the start of culture, the cells are washed and formulated for storage or administration. 【0639】 While we do not wish to be bound by theory, short-term CD3 and CD28 stimulation may promote efficient transduction of self-replicating T cells. Compared to conventional CART manufacturing approaches, the activation process provided herein does not involve prolonged ex vivo proliferation. Similar to cytokine processes, the activation process provided herein also preserves undifferentiated T cells during CART manufacturing. 【0640】 In some embodiments, a population of cells comes into contact with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates a co-stimulatory molecule on the cell surface. 【0641】 In some embodiments, the agent stimulating the CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent stimulating the co-stimulator is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent stimulating the co-stimulator is an agent that stimulates CD28. In some embodiments, the agent stimulating the CD3 / TCR complex is selected from antibodies (e.g., single-domain antibodies (e.g., heavy-chain variable-domain antibodies), peptide bodies, Fab fragments, or scFv), small molecules, or ligands (e.g., native ligands, recombinant ligands, or chimeric ligands). In some embodiments, the agent stimulating the CD3 / TCR complex is an antibody. In some embodiments, the agent stimulating the CD3 / TCR complex is an anti-CD3 antibody. In some embodiments, the agent stimulating the co-stimulatory molecule is selected from antibodies (e.g., single-domain antibodies (e.g., heavy-chain variable-domain antibodies), peptide bodies, Fab fragments, or scFv), small molecules, or ligands (e.g., native ligands, recombinant ligands, or chimeric ligands). In some embodiments, the agent stimulating the co-stimulatory molecule is an antibody. In some embodiments, the agent stimulating the co-stimulatory molecule is an anti-CD28 antibody. In some embodiments, the agent stimulating the CD3 / TCR complex or the agent stimulating the co-stimulatory molecule does not contain beads. In some embodiments, the agent stimulating the CD3 / TCR complex includes an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix. In some embodiments, the agent stimulating the co-stimulatory molecule includes an anti-CD28 antibody covalently bound to a colloidal polymer nanomatrix. In some embodiments, the agent stimulating the CD3 / TCR complex and the agent stimulating the co-stimulatory molecule include T Cell TransAct®. 【0642】 In some embodiments, the matrix includes or is composed of polymers that are, for example, biodegradable or biocompatible inert materials, such that these materials are nontoxic to cells. In some embodiments, the matrix is ​​composed of hydrophilic polymer chains that obtain maximum mobility in aqueous solution by hydration of the chains. In some embodiments, the mobile matrix may consist of collagen, purified protein, purified peptide, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. Examples of polysaccharides include cellulose ether, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan gum, guar gum, or alginates. Other polymers include polyester, polyether, polyacrylate, polyacrylamide, polyamine, polyethyleneimine, polyquaternium polymer, polyphosphazene, polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, block copolymer, or polyurethane. In some embodiments, the mobile matrix is ​​a polymer of dextran. 【0643】 In some embodiments, a population of cells is brought into contact with nucleic acid molecules (e.g., one or more nucleic acid molecules) encoding CARs (e.g., one or more CARs). In some embodiments, a population of cells is transduced with DNA molecules (e.g., one or more DNA molecules) encoding CARs (e.g., one or more CARs). 【0644】 In some embodiments, in the case of cotransduction of two nucleic acid molecules (e.g., lentiviral vectors) each encoding a CAR as disclosed herein, each of the vectors containing the CAR-encoding nucleic acid molecules can be added to the reaction mixture (e.g., including a population of cells) at different modalities (MOIs). 【0645】 While we do not wish to be bound by theory, in some embodiments, it is conceivable that using different MOIs for vectors containing nucleic acid molecules encoding different CAR molecules could affect the final composition of the cell population. For example, in the case of co-transduction of a lentiviral vector encoding one CAR and another lentiviral vector encoding a different CAR targeting a different target, using different MOIs could maximize the percentage of desirable mono-CART and dual-CART cells while simultaneously reducing undesirable mono-CART and non-transduced cells. 【0646】 The exact MOI used for each vector can be adjusted or determined based on many factors, including but not limited to the characteristics of the viral vector batch, the characteristics of the cells being transduced, and the transduction efficiency. In some embodiments, the step of contacting the CAR-encoding nucleic acid molecule with a population of cells is performed concurrently with the step of contacting the population of cells with a drug that stimulates the CD3 / TCR complex and / or a drug that stimulates a co-stimulatory molecule on the cell surface, as described above. In some embodiments, the contact between the CAR-encoding nucleic acid molecule and the population of cells is performed within 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 hours after the start of contact between the population of cells and the drug that stimulates the CD3 / TCR complex and / or a co-stimulatory molecule on the cell surface, as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 20 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the cell surface, as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 19 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the cell surface, as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 18 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the cell surface, as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 17 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the cell surface, as described above. 【0647】 In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 16 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules on the cell surface as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 15 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules on the cell surface as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 14 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules on the cell surface as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 14 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules on the cell surface as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 13 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules on the cell surface, as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 12 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules on the cell surface, as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 11 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules on the cell surface, as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 10 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules on the cell surface, as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 9 hours of the initiation of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the cell surface, as described above.In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 8 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the cell surface as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 7 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the cell surface as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 6 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the cell surface as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell population is performed within 5 hours of the start of contact between the cell population and the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the cell surface as described above. In some embodiments, contact between the CAR-encoding nucleic acid molecule and the cell pop...

Claims

[Claim 1] 1) Lentiviral vector and, 2) An aqueous composition, a) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), b) One or both of free positively charged amino acids and free nonpolar amino acids an aqueous composition containing and A composition containing the following: [Claim 2] The aqueous composition according to claim 1, wherein the aqueous composition is substantially free of inorganic salts. [Claim 3] The aqueous composition, NaCl and MgCl ２ The composition according to claim 1, which substantially does not contain one or both of the above. [Claim 4] The aqueous composition according to claim 1, wherein the aqueous composition contains less than 20 mM of inorganic salts in total. [Claim 5] The composition according to any one of claims 1 to 4, wherein HEPES is concentrated in a concentration of 10-200, 10-150, 10-100, 10-50, 10-40, 10-30, 15-25, or 20 mM. [Claim 6] The composition according to any one of claims 1 to 5, wherein the HEPES has a concentration of about 20 mM. [Claim 7] The composition according to any one of claims 1 to 6, comprising the positively charged amino acid, wherein the positively charged amino acid is arginine, lysine, or histidine. [Claim 8] The composition according to claim 7, wherein the positively charged amino acid is arginine. [Claim 9] The composition according to claim 8, wherein the arginine is L-arginine. [Claim 10] The composition according to any one of claims 7 to 9, wherein the arginine is at a concentration of at least 50, at least 75, at least 100, or at least 150 mM. [Claim 11] The composition according to any one of claims 7 to 10, wherein the arginine is at a concentration of 25-50, 50-75, 75-100, 75-125, 100-200, 125-175, or 150 mM. [Claim 12] The composition according to any one of claims 7 to 10, wherein the arginine is concentrated at a concentration of about 150 mM. [Claim 13] The composition according to any one of claims 1 to 12, comprising the nonpolar amino acid, wherein the nonpolar amino acid is proline, methionine, or tryptophan. [Claim 14] The composition according to claim 13, wherein the nonpolar amino acid is proline. [Claim 15] The composition according to claim 14, wherein the proline is L-proline. [Claim 16] The composition according to any one of claims 13 to 15, wherein the proline is present in a concentration of 25 to 200, 50 to 200, 100 to 200, 125 to 175, or 150 mM. [Claim 17] The composition according to any one of claims 13 to 15, wherein the proline is at a concentration of about 150 mM. [Claim 18] The composition according to any one of claims 1 to 17, further comprising a freeze-protecting agent. [Claim 19] The composition according to claim 18, wherein the cryoprotectant is a carbohydrate. [Claim 20] The composition according to claim 19, wherein the carbohydrate is a non-reducing carbohydrate. [Claim 21] The composition according to claim 19, wherein the carbohydrate is sucrose. [Claim 22] The composition according to any one of claims 1 to 17, further comprising sucrose. [Claim 23] The composition according to claim 22, wherein the sucrose is concentrated at a concentration of 25-200, 50-200, 100-200, 125-175, or 150 mM. [Claim 24] The composition according to claim 22, wherein the sucrose is concentrated to a concentration of about 150 mM. [Claim 25] The aqueous composition according to any one of claims 1 to 24, further comprising a stabilizer. [Claim 26] The aqueous composition is the composition according to any one of claims 1 to 24, comprising HSA. [Claim 27] The composition according to claim 26, wherein the HSA comprises recombinant HSA (rHSA) or human-derived HSA. [Claim 28] The composition according to claim 26 or 27, wherein the HSA is present in an amount of 0.5-3%, 0.5-2%, 0.5-1%, 1-2%, 1.5-2.5%, or 2% w / v. [Claim 29] The composition according to claim 28, wherein the HSA is present at approximately 2% w / v. [Claim 30] The aqueous composition is substantially free of HSA, as described in any one of claims 1 to 24. [Claim 31] A composition according to any one of claims 1 to 30, having a pH of 6.0 to 7.5, 6.0 to 7.0, 6.0 to 6.5, 6.5 to 7.0, 6.2 to 6.8, 6.4 to 6.6, or 6.

5. [Claim 32] A composition according to any one of claims 1 to 31, having a pH of approximately 6.

5. [Claim 33] The aqueous composition according to any one of claims 1 to 32, wherein the aqueous composition substantially does not contain one, two or three of PEG lipids, F108, and cholesterol. [Claim 34] The aqueous composition contains the positively charged amino acid, and the positively charged amino acid contains L-arginine. The aqueous composition comprises the nonpolar amino acid, the nonpolar amino acid comprising L-proline, The aqueous composition further comprises sucrose, and The aqueous composition is substantially free of inorganic salts, as described in any one of claims 1 to 33. [Claim 35] The L-arginine is at a concentration of 100 to 200 mM. The L-proline is at a concentration of 25 to 200 mM, and The composition according to claim 34, wherein the sucrose is concentrated to a concentration of 25 to 200 mM. [Claim 36] The composition according to any one of claims 1 to 35, wherein the osmotic pressure of the aqueous composition is about 400 mOsm / kg to about 700 mOsm / kg. [Claim 37] The composition according to claim 36, wherein the osmotic pressure of the aqueous composition is about 415 mOsm / kg to about 689 mOsm / kg. [Claim 38] At least 1 × 10 ６ 、 at least 1 × 10 ７ 、 at least 5 × 10 ７ 、 at least 1 × 10 ８ 、 at least 1 × 10 ９ 、 at least 2 × 10 ９ 、 at least 3 × 10 ９ 、 at least 4 × 10 ９ 、 at least 5 × 10 ９ or at least 6 × 10 ９ The composition according to any one of claims 1 to 37, comprising the lentiviral vector of transduction units / milliliter (TU / mL). [Claim 39] The lentiviral vector comprises a transgene, as described in any one of claims 1 to 38. [Claim 40] The composition according to claim 39, wherein the introduced gene encodes a chimeric antigen receptor (CAR). [Claim 41] CAR is TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PD GFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrinB2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, Tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, Regmine, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostain, survivor and telomerase, PCTA-1 / galectin 8, Melan A / MART1, Ras variant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe composition according to claim 39, comprising an antigen-binding domain that binds to a tumor antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1, and any combination thereof. [Claim 42] The composition according to claim 40 or 41, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, one or more primary signaling domains and / or one or more costimulatory signaling domains. [Claim 43] The composition according to claim 42, wherein one or more primary signaling domains include a CD3-ζ stimulating domain. [Claim 44] The one or more co-stimulus signaling domains described above are: (a) OX40, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4 , CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C D49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 , CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SL AMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL 1. A co-stimulatory protein selected from the group consisting of CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and CD19a, and (b) A ligand that specifically binds to CD83, selected from the 4-1BB (CD137) costimulatory domain and the CD28 costimulatory domain. The composition according to claim 42 or 43, comprising an intracellular domain selected from. [Claim 45] (a) The transmembrane domain includes the transmembrane domain of a protein selected from the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, (b) The transmembrane domain includes the transmembrane domain of CD8, (c) The transmembrane domain contains the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 90% sequence identity thereto, or (d) The composition according to any one of claims 42 to 44, wherein the transmembrane domain is encoded by the nucleic acid sequence of Sequence ID No. 17 or a nucleic acid sequence having at least about 90% sequence identity therewith. [Claim 46] The composition according to any one of claims 1 to 45, exhibiting a level of subvisible particles of 25 μm or larger, of approximately 50 to 100 particles / mL or less. [Claim 47] The composition according to any one of claims 1 to 46, exhibiting a level of subvisible particles of 10 μm or larger, of approximately 1100 to 2000 particles / mL or less. [Claim 48] The composition according to any one of claims 1 to 47, wherein the lentiviral vector in the composition has a hydrodynamic diameter of about 85 to 200, 90 to 200, or 85 to 130 nm. [Claim 49] A kit comprising a lentiviral vector and an aqueous composition according to any one of claims 1 to 48. [Claim 50] A method for producing CAR-expressing cells, A step of providing the composition according to any one of claims 1 to 48, The steps include: bringing the composition into contact with immune effector cells under conditions that enable transduction of one or more of the immune effector cells; A method for producing CAR-expressing cells, comprising the above. [Claim 51] The method according to claim 50, which is an in vitro or ex vivo method. [Claim 52] The method according to claim 50 or 51, wherein the population of immune effector cells includes one or both T cells and NK cells. [Claim 53] The method according to any one of claims 50 to 52, wherein the composition comprises HSA. [Claim 54] The method according to claim 53, wherein the HSA has a concentration of about 0.5% to 3% w / v. [Claim 55] The method according to claim 53 or 54, wherein the HSA includes recombinant HSA (rHSA) or human-derived HSA. [Claim 56] A method for delivering a transgene to a target, comprising the step of administering a composition according to any one of claims 1 to 48 to the target, wherein the lentiviral vector of the composition contains the transgene. [Claim 57] The method according to claim 56, wherein the introduced gene includes CAR. [Claim 58] The method according to claim 56 or 57, further comprising particles in the composition. [Claim 59] The method according to claim 58, wherein the particles are silica particles. [Claim 60] The method according to claim 59, wherein the silica particles are mesoporous particles (MSPs). [Claim 61] The method according to claim 60, wherein the mesoporous silica particles are mesoporous silica rods. [Claim 62] (i) The retroviral vector associates with the mesoporous silica particles noncovalently or covalently, and / or (ii) The method according to claim 60 or 61, wherein the cell activator associates with the mesoporous silica particles non-covalently or covalently. [Claim 63] The method according to any one of claims 56 to 61, further comprising a cell activator. [Claim 64] The aforementioned cell activator is (a) A drug that stimulates the CD3 / TCR complex and / or a co-stimulatory molecule and / or a drug that stimulates growth factor receptors, (b) A multispecific binding molecule comprising (i) an anti-CD3 binding domain and (ii) a costimulatory molecule binding domain, and / or (c) The method according to claim 63, wherein the particles are conjugated or adsorbed thereon. [Claim 65] The method according to claim 64, wherein the costimulatory molecule binding domain is an anti-CD2 binding domain or an anti-CD28 binding domain. [Claim 66] The method according to claim 64 or 65, wherein the particles are mesoporous silica particles.

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