Viral Vector Production System

Abstract

The present disclosure provides, at least in part, methods for producing high titer lentiviral vectors and lentiviral particles carrying a transgene of interest under satisfactory safety conditions. The present disclosure also provides, at least in part, methods for purifying such lentiviral particles, for example from cell culture. The present disclosure also provides formulation into lentiviral preparations that maintain the structural integrity of viral vectors during purification, storage and gene transfer events.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 180,423, filed April 27, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Viruses often evade detection by the immune system of an infected host, yet are highly efficient at delivering nucleic acids to specific cell types, characteristics that make certain viruses attractive candidates as gene delivery vehicles for use in gene therapy.

[0003] Lentiviral vectors are viral vectors that can be used for gene therapy. These vectors include reassortment viral vector systems derived from human immunodeficiency virus-1 (HIV-1) and can introduce genes of interest into animal and human primary cells or cell lines. Lentiviral vector-mediated gene expression can be used to achieve continuous and stable protein production because the gene of interest is integrated into the host cell's 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 in which lentiviral vector-mediated long-term expression of a gene of interest can occur include the brain, liver, muscle cells, retina, hematopoietic stem cells, bone marrow mesenchymal stem cells, and macrophages, among others. Summary of the Invention [Problem to be solved by the invention]

[0004] The large-scale production of lentiviral vectors has been hindered by several challenges, such as low viral titer production and low vector stability.In addition, lentiviral vectors are susceptible to inactivation during the purification process, which may contribute to a decrease in the final quality and efficacy of vector preparations, creating another obstacle to the large-scale production of purified lentiviral vectors.Thus, there remains a need for a large-scale production process and purification process for higher-titer lentiviral vectors, as well as a formulation buffer that maintains the stability of the vectors. [Means for solving the problem]

[0005] The present disclosure provides, at least in part, methods for producing high-titer lentiviral vectors carrying a transgene of interest under satisfactory safety conditions. The present disclosure also provides, at least in part, methods for purifying such lentiviral particles from, for example, cell culture. The present 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 embodiments, the present disclosure provides a method of producing a lentiviral vector, the method comprising: a) providing a plurality of mammalian (e.g., human) cells; b) a plurality of mammalian cells; i) FectoVIR®-AAV transfection reagent, and ii) a nucleic acid encoding a therapeutic effector, such as a therapeutic protein (e.g., a CAR) and sufficient LTR sequence for packaging into a viral particle, and optionally a lentiviral packaging protein, a lentiviral envelope protein under conditions that allow the nucleic acid to be introduced into at least a subset of cells; c) culturing the cells under conditions suitable for the production of lentiviral vectors; Includes:

[0007] In certain embodiments, a plurality of mammalian cells in a 50 L culture provides a number of transducing units per ml of culture that is 50%, 60%, 70%, or 80% or more of the number of transducing units per ml of culture in an otherwise similar 100 ml culture.

[0008] In certain embodiments, the method produces at least 1 x 10 cells when used under the conditions described in Example 5. 7 Or 3 x 10 7 or at least 1 × 10 8 resulting in transducing units of

[0009] In certain embodiments, the method results in a ratio of PP (physical particles):IP (infectious particles) of less than or equal to 1188:1, 953:1, and 1800:1.

[0010] In certain embodiments, the mammalian cells are 293 cells, such as Expi293F cells.

[0011] In certain embodiments, FectoVIR®-AAV is used at a concentration of 0.3-0.6 μl of FectoVIR®-AAV / million cells, for example, about 0.4 μl / million cells.

[0012] In certain embodiments, the nucleic acid is used at a concentration of 0.3 to 0.6 μg of nucleic acid per million cells, for example about 0.4 μg per million cells.

[0013] In certain embodiments, the ratio of FectoVIR®-AAV:DNA for transfection is 1:0.5 to 1:2, e.g., 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).

[0014] In certain embodiments, the FectoVIR®-AAV transfection reagent is complexed with the nucleic acid.

[0015] In some embodiments, the method further comprises the step of mixing FectoVIR®-AAV transfection reagent with the nucleic acid prior to step b).

[0016] In a specific embodiment, the amount of complex formed between the transfection reagent and the nucleic acid is about 1% to about 15%, for example, about 1% to about 10% (eg, about 5 to 7.5% or 7.5 to 10%).

[0017] In some embodiments, the amount of complex formed is 3-7%, 4-6%, or about 5%.

[0018] In certain embodiments, the FectoVIR®-AAV transfection reagent and nucleic acid are incubated for a time sufficient to allow complex formation to occur, for example, about 10-90 minutes, for example, 15-60, for example, 15-30, 30-45, or 45-60 minutes.

[0019] In some embodiments, the present disclosure provides a method of producing a lentiviral vector, the method comprising: a) culturing a plurality of mammalian (e.g., human) cells at a pH greater than about 6.9 or between about 6.9 and 7.3, e.g., between about 7.0 and 7.1; b) following step a), adjusting the pH of the culture to about 6.0 to 6.8, such as 6.6 to 6.8, for example about 6.7; c) following step b), contacting the culture with a transfection reagent and DNA. Includes:

[0020] In certain embodiments, the transfection reagent comprises FectoVIR®-AAV transfection reagent.

[0021] In some embodiments, the DNA encodes one or more retroviral packaging proteins, a retroviral envelope protein, and a therapeutic effector, such as a therapeutic protein (e.g., a CAR).

[0022] In some embodiments, a) comprises culturing the cells for about 2-4 days, for example, about 3 days.

[0023] In certain embodiments, the method further comprises an additional step of culturing the cells between steps b) and c).

[0024] In some embodiments, the method further comprises the additional step of culturing the cells after step c).

[0025] In some embodiments, step b) comprises decreasing the pH by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.

[0026] In certain embodiments, prior to step a), the plurality of mammalian cells is 0.1 x 10 6 cells / mL~0.3×10 6 cells / mL (e.g., approximately 0.15 x 10 6 cells / mL or approximately 0.2 x 10 6 The cells are inoculated at 1000 x 1000 cells / mL into a final volume of culture medium (e.g., FreeStyle™ medium).

[0027] In some embodiments, the plurality of mammalian cells is inoculated 50 to 80 hours (e.g., about 55 hours, about 60 hours, about 65 hours, about 70 hours, about 72 hours, about 75 hours, or about 80 hours) prior to step a).

[0028] In certain embodiments, the plurality of mammalian cells is grown at a suitable cell density for transfection (e.g., about 1.0×10 6 cells / mL ~ approx. 3.0×10 6 cells / mL (e.g., 1.5 x 10 6cells / mL~2.5×10 6 The cells are cultured under suitable conditions to allow cell growth and amplification to a total volume of 1000 cells / mL.

[0029] In some embodiments, the present disclosure provides a method of producing a lentiviral vector, the method comprising: a) providing a composition comprising a lentiviral vector and at least one impurity (e.g., where the composition comprises a clarified cell harvest or filtrate); b) contacting the composition with arginine or a salt thereof; Includes:

[0030] In certain embodiments, one or more of the following: i) arginine is at a concentration of about 25 to 50 mM (about 50 mM), 50 to 100 mM (e.g., about 75 mM), 100 to 200 mM (e.g., about 150 mM), or 200 to 400 (e.g., about 300) mM arginine; or ii) arginine is at a concentration sufficient to increase the level of transducing units of the lentiviral vector by about 10% to 300%, 20% to 180%, 30% to 160%, 50% to 150%, 75% to 125%, or about 100%, compared to an otherwise similar composition, e.g., in an assay according to Example 7; iii) after step b), the composition exhibits a total particle concentration of less than 400,000, 300,000, 200,000 or 100,000 particles per ml as measured by microflow imaging, e.g., in the assay described in Example 10, and optionally the particles comprise aggregated lentivirus; iv) after step b), the composition exhibits a concentration of less than about 5,000, 4,500, 4,000, 3,500, 3,000, or 2,500 particles >= 10 μm per ml as measured by microflow imaging, e.g., in the assay described in Example 10, and optionally the particles comprise aggregated lentivirus; v) after step b), the composition exhibits a concentration of less than about 500, 400, 300, or 200 particles >= 25 μm per ml as measured by microflow imaging, e.g., in the assay described in Example 10, and optionally the particles comprise aggregated lentivirus; vi) after step b), the composition exhibits reduced aggregation of lentiviral vectors compared to an otherwise similar filtrate without the addition of arginine or a salt thereof; vii) The recovery of transducing units of the lentiviral vector is, for example, at least about 10%, 20%, 50%, 100% or 200% higher than a similar control except that no arginine is added, as measured, for example, in an assay according to Example 7.

[0031] In some embodiments, b) comprises contacting the composition with a solution comprising arginine and a buffer, optionally wherein the buffer is PIPES, and optionally wherein the PIPES is at a concentration of about 10 mM to about 50 mM, e.g., about 20 mM, in the solution. In certain embodiments, the solution has a pH of about 6.0 to about 7.0, e.g., about 6.5.

[0032] In some embodiments, the solution further comprises a salt, optionally the salt is selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride, for example sodium chloride.

[0033] In some embodiments, the salt is present in the solution at a concentration of about 25-150 mM, such as 50-100 mM, for example about 75 mM.

[0034] In certain embodiments, the concentration of salt in solution has a pH of about 6.5.

[0035] In some embodiments, the solution further comprises a carbohydrate, eg, a non-reducing carbohydrate, such as sucrose or trehalose.

[0036] In certain embodiments, the carbohydrate is present in the solution at a concentration of about 1 to about 10% w / v of the solution, such as about 2 to about 5% w / v of the solution, such as about 2.5% w / v of the solution.

[0037] In certain embodiments, the carbohydrate is present in the solution at a concentration of about 30 to 150 mM (about 73 mM) or 150 to 300 (eg, about 220) mM.

[0038] 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% w / v of the solution).

[0039] In some embodiments, the solution comprises 20 mM PIPES, 75 mM sodium chloride, and 2.5% sucrose by weight / volume of the solution, 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% sucrose by weight / volume of the solution, and the solution has a pH of about 6.5.

[0040] In certain embodiments, the solution comprises 20 mM PIPES, 75 mM sodium chloride, and 73 mM 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 about 73 mM sucrose, and the solution has a pH of about 6.5.

[0041] 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. In some embodiments, the solution comprises about 20 mM PIPES, about 75 mM sodium chloride, and about 220 mM sucrose, and the solution has a pH of about 6.5.

[0042] In some embodiments, the solution further comprises 20 mM PIPES, 75 mM arginine, e.g., arginine-HCl, and the solution has a pH of about 6.5. In some embodiments, the solution further comprises about 20 mM PIPES, about 75 mM arginine, e.g., arginine-HCl, and the solution has a pH of about 6.5.

[0043] In some embodiments, the osmolality of the solution is about 270 mOsm / kg to about 330 mOsm / kg, for example, about 275 mOsm / kg to about 300 mOsm / kg, for example, about 285 mOsm / kg.

[0044] In certain embodiments, the method further comprises c) performing a purification step, e.g., a filtration step, on the composition of b), thereby producing a semi-purified composition comprising the lentiviral vector.

[0045] In certain embodiments, the method further comprises, after step c), contacting the semi-purified composition with arginine or a salt thereof.

[0046] In some embodiments, arginine encapsulates the lentiviral vector.

[0047] In certain embodiments, arginine stabilizes the lentiviral vector.

[0048] 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.

[0049] In certain embodiments, the cell density at transfection is about 1.0 x 10 6 cells / mL ~ approx. 3.0×10 6 cells / mL (e.g., 1.5 x 10 6 cells / mL~2.5×10 6 cells / mL).

[0050] In certain embodiments, cell viability is or is assessed to be at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) at the time of transfection.

[0051] In certain embodiments, cell viability is measured at or around the time of transfection (eg, within 30 minutes before transfection).

[0052] In some embodiments, the methods are used in processes using two or more nucleic acids (eg, two or more plasmids, such as two plasmids, three plasmids, four plasmids, or five plasmids).

[0053] In some embodiments, the present disclosure provides a method of producing a lentiviral vector, the method comprising: a) providing a population of human cells (e.g., 293 cells); b) introducing into the cell a nucleic acid encoding a retroviral packaging protein, a retroviral envelope protein, and a therapeutic effector, such as a therapeutic protein (e.g., a CAR); c) contacting the cells with benzonase at about 6 to 40 to 10 to 40 to 10 to 30 or about 20 hours after step b); d) culturing the cells under conditions suitable for the production of lentiviral vectors; Includes.

[0054] In some embodiments, the present disclosure provides a method of producing a lentiviral vector, the method comprising: a) providing a population of human cells (e.g., 293 cells); b) introducing into the cell a nucleic acid encoding a retroviral packaging protein, a retroviral envelope protein, and a therapeutic effector, such as a therapeutic protein (e.g., a CAR); c) contacting the cells with benzonase; d) culturing the cells under conditions suitable for the production of lentiviral vectors; e) harvesting the lentiviral vector from the cells 6 to 10 hours, 10 to 20 hours, 20 to 30 hours, 30 to 40 hours, or 40 to 50 hours after step c). Includes.

[0055] In some embodiments, the present disclosure provides a method of producing a lentiviral vector, the method comprising: a) providing a plurality of mammalian (e.g., human) cells, wherein the plurality of cells are fibroblasts (e.g., fetal kidney fibroblasts, e.g., Expi293F cells), and the cells comprise nucleic acid (e.g., DNA) encoding one or more retroviral packaging proteins, retroviral envelope proteins, and a therapeutic effector, e.g., a therapeutic protein (e.g., CAR); b) culturing the cells under conditions suitable for the production of the lentiviral vector Includes.

[0056] In some aspects, the present disclosure provides an aqueous composition comprising a lentiviral vector, arginine, 1,4-piperazinediethanesulfonic acid (PIPES) buffer, and a salt.

[0057] In certain embodiments, the arginine in the aqueous composition is at a concentration 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 (e.g., about 300) mM arginine), and optionally, the PIPES aqueous composition is at a concentration of about 10 mM to about 50 mM, e.g., about, e.g., 20 mM.

[0058] In some embodiments, the aqueous composition has a pH of about 6.0 to about 7.0, for example, about 6.5.

[0059] In certain embodiments, the aqueous composition further comprises a salt, optionally selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride.

[0060] In some embodiments, the salt is sodium chloride (NaCl).

[0061] In certain embodiments, the salt in the aqueous composition is from about 25 mM to about 150 mM, for example, from about 50 mM to about 75 mM.

[0062] 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.

[0063] In certain embodiments, the aqueous composition further comprises a carbohydrate, eg, a non-reducing carbohydrate, such as sucrose or trehalose.

[0064] In some embodiments, the carbohydrate is present in the aqueous composition at a concentration of about 1 to about 10% w / v of the solution, e.g., about 2 to about 5% w / v of the aqueous composition, e.g., about 2.5% w / v of the aqueous composition.

[0065] In one embodiment, the carbohydrate is present in the aqueous composition at a concentration of about 30 to 150 mM (about 73 mM) or 150 to 300 (eg, about 220) mM.

[0066] 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% weight / volume of the aqueous composition).

[0067] In one embodiment, the aqueous composition comprises 20 mM PIPES, 75 mM sodium chloride, and 2.5% sucrose weight / volume of the aqueous composition, wherein the aqueous composition has a pH of about 6.5.

[0068] In a particular embodiment, 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.

[0069] In one embodiment, the aqueous composition comprises 20 mM PIPES, 75 mM sodium chloride, and 220 mM sucrose, wherein the aqueous composition has a pH of about 6.5.

[0070] In certain embodiments, the osmolality of the aqueous composition is from about 270 mOsm / kg to about 330 mOsm / kg, for example, from about 275 mOsm / kg to about 300 mOsm / k, for example, about 285 mOsm / kg.

[0071] In one embodiment, the lentiviral vector of any preceding claim comprises about 3 x 10 8 TU / mL ~ approx. 5 x 10 8 It is present at a concentration of TU / mL.

[0072] In certain embodiments, the aqueous composition does not comprise 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.

[0073] In one embodiment, the lentiviral vector comprises a transgene, e.g., a transgene encoding a protein, e.g., a protein comprising a chimeric antigen receptor (CAR).

[0074] In certain embodiments, the CAR comprises, from N- to C-terminal, an antigen-binding domain, a transmembrane domain, and one or more signaling domains.

[0075] In some embodiments, the signaling domain comprises one or more primary signaling domains and / or one or more costimulatory signaling domains.

[0076] In a specific embodiment, one of the one or more primary signaling domains comprises a CD3-zeta stimulatory domain.

[0077] In some embodiments, one or more of the costimulatory signaling domains are selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), CDS, 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, C The protein comprises an intracellular domain selected from a costimulatory protein selected from the group consisting of D18, 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 a ligand that specifically binds to CD83, such as a 4-1BB (CD137) costimulatory domain or a CD28 costimulatory domain.

[0078] In some embodiments, one or more of the costimulatory signaling domains are selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, 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, The protein comprises an intracellular domain selected from a costimulatory protein selected from the group consisting of CD11b, 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.

[0079] In certain embodiments, the antigen-binding domain is an scFv.

[0080] In some embodiments, the antigen binding domain is selected from the group consisting of CD19; CD123; CD22; CD30; CD171; CS-1; C-type lectin-like molecule-1, CD33; epidermal growth factor receptor variant III (EGFRvIIIl); 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 embryonic antigen-4 (SSEA-4); CD20; folate receptor α; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein α (FAP) ); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropein) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral 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 beta; 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 differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 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 / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation mutant 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 cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutants; prostein; survivin; telomerase; prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells; rat sarcoma (Ras) mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (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 avian myelocytomatosis 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 recognized by T cells (SART3); 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-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mutated) hsp70-2); 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 lambda-like polypeptide 1 (IGLL1), which binds to an antigen selected from the group consisting of, for example, CD19, CD22, mesothelin, or CD123;

[0081] In a specific embodiment, the CAR comprises an anti-CD19 antibody or fragment thereof, a 4-1BB (CD137) transmembrane domain, and a CD3-zeta signaling domain.

[0082] In some embodiments, the lentiviral vector comprises a second transgene, e.g., a second transgene encoding a second protein, e.g., a second protein comprising a second chimeric antigen receptor (CAR).

[0083] In one aspect, the present disclosure provides a method of producing a lentiviral vector, the method comprising: a) providing a population of human cells (e.g., 293 cells); b) introducing into the cell a nucleic acid encoding a retroviral packaging protein, a retroviral envelope protein, and a therapeutic effector, such as a therapeutic protein (e.g., a CAR); c) contacting the cells with benzonase about 2-6 (e.g., about 3), 4-10 (e.g., 6), 6-40, 10-40, 10-30 (e.g., about 24), or about 20 hours after step b); d) culturing the cells under conditions suitable for the production of lentiviral vectors; Includes.

[0084] In certain embodiments, benzonase is added 6 to 10 hours, 10 to 20 hours, 20 to 30 hours, 30 to 40 hours, or 40 to 50 hours before harvesting the lentiviral vector from the cells.

[0085] In one aspect, the present disclosure provides a method of producing a lentiviral vector, the method comprising: a) providing a population of human cells (e.g., 293 cells); b) introducing into the cell a nucleic acid encoding a retroviral packaging protein, a retroviral envelope protein, and a therapeutic effector, such as a therapeutic protein (e.g., a CAR); c) contacting the cells with benzonase (e.g., 3-24 hours after step b); d) culturing the cells under conditions suitable for the production of lentiviral vectors; e) harvesting the lentiviral vector from the cells 6 to 10 hours, 10 to 20 hours, 20 to 30 hours, 30 to 40 hours, or 40 to 50 hours after step c). Includes.

[0086] In some embodiments, the benzonase is at a concentration of about 10-40 U / mL, such as 20-30 U / mL, for example about 25 U / mL.

[0087] In some embodiments, the benzonase is at a concentration of about 3-60 U / mL, 3-10 U / mL, 3-7 U / mL, 4-6 U / mL, or about 5 U / mL.

[0088] In one embodiment, the benzonase is at a concentration of 5 to 50, 5 to 15, 15 to 25, or 25 to 50 U / mL.

[0089] In certain embodiments, the method further comprises contacting the benzonase with, for example, about 1-5 mM, 1-3 mM, or about 2 mM MgCl 2 prior to step c).

[0090] In one aspect, the present disclosure provides a method of producing a lentiviral vector, the method comprising: a) providing a plurality of mammalian (e.g., human) cells, wherein the plurality of mammalian cells do not comprise SV40 large T antigen (e.g., where the cells are fibroblasts, e.g., fetal kidney fibroblasts, e.g., Expi293F cells), and the plurality of mammalian cells comprise one or more retroviral packaging proteins, retroviral envelope proteins, and nucleic acid (e.g., DNA) encoding a therapeutic effector, e.g., a therapeutic protein (e.g., CAR); b) culturing the cells under conditions suitable for the production of the lentiviral vector Includes.

[0091] In certain embodiments, a) comprises introducing the nucleic acid into a plurality of mammalian cells.

[0092] In some embodiments, the method further comprises at least partially isolating the lentiviral vector from the plurality of mammalian cells.

[0093] In one embodiment, the one or more retroviral packaging proteins comprise lentiviral gag, lentiviral pol, or lentiviral rev, or any combination thereof.

[0094] In certain embodiments, the retroviral envelope protein comprises VSV-G.

[0095] In some embodiments, the present disclosure provides a preparation of a lentiviral vector, the preparation comprising: a) a lentiviral genome encoding a therapeutic effector, such as a therapeutic protein (e.g., a CAR); b) an envelope that encapsulates the lentiviral genome (wherein, optionally, the envelope contains VSV-G); a plurality of lentiviral vectors comprising Preparations should contain at least 5 x 10 7 , 1×10 8 , 1×10 9 or 1 x 10 10 transducing units of The preparation contains less than 90% SV40 large T antigen or less than 10 μg / ml, less than 1 μg / ml of nucleic acid (eg, DNA) encoding SV40 large T antigen.

[0096] In some embodiments, the plurality of lentiviral vectors is at least 1 x 10 9 , 2 × 10 9 , 5×10 9 or 1 x 10 10 , 2 × 10 10 , 5×10 10 , 1×10 11 , 2 × 10 11 , 5×10 1 or 1 x 10 12 Contains cells of.

[0097] In certain embodiments, the plurality of mammalian cells is in a culture volume of at least 5, 10, 20, 50, 100, 200, or 500 L.

[0098] In one embodiment, the method includes culturing a plurality of mammalian cells in serum-free medium.

[0099] In certain embodiments, the plurality of mammalian cells is grown in suspension.

[0100] In some embodiments, the CAR comprises a CD19 CAR (e.g., a humanized CD19 CAR, e.g., as described in WO2014153270A1).

[0101] In certain embodiments, the CAR comprises a dual CAR (e.g., a humanized CD19-CD22 CAR, e.g., as described in WO2016164731A2).

[0102] In some embodiments, the nucleic acid encoding the CAR further encodes an shRNA, for example, as described in WO2017049166A.

[0103] In one embodiment, the lentiviral vector is produced in cells cultured in the absence of serum.

[0104] In certain embodiments, the lentiviral vector is characterized by a hydrodynamic radius of 100±25 nm as measured by dynamic light scattering (DLS).

[0105] In certain embodiments, the lentiviral vector maintains the 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).

[0106] In some embodiments, the lentiviral vector is characterized by a polydispersity of 10% to 25% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%).

[0107] In one embodiment, the lentiviral vector maintains the 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).

[0108] 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, each of the freeze / thaw cycles comprising freezing the aqueous composition and subsequently thawing the aqueous composition at room temperature.

[0109] In some embodiments, the lentiviral vector maintains the 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.

[0110] 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.

[0111] In some embodiments, the salt is selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride.

[0112] In one embodiment, the aqueous composition further comprises a non-reducing carbohydrate selected from the group consisting of sucrose and trehalose.

[0113] In some aspects, the present disclosure provides a scalable process for producing large quantities of viral vectors (e.g., lentiviral vectors), e.g., for prophylactic, diagnostic, immunotherapeutic, or therapeutic applications. The process can be performed using suspension cells (e.g., HEK293 cells, e.g., Expi293F cells). In some embodiments, substantially all of the suspension cells do not express large T antigen, e.g., SV40 T antigen. In some embodiments, the method can be performed using a bioreactor.

[0114] In some embodiments, the present disclosure provides a highly reproducible, efficient, and scalable process for producing large quantities of viral vectors (e.g., lentiviral vectors) with one or both of high viral titers or high viral yields.

[0115] In some embodiments, the present disclosure provides a highly reproducible, efficient, and scalable process for purifying large quantities of viral (e.g., lentiviral) vectors with one or both of high viral titers or high viral yields.

[0116] In another aspect, the present disclosure provides compositions and methods for stabilizing viral vectors, such as lentiviral vectors, during the purification process. [Brief explanation of the drawings]

[0117] [Figure 1A] Expi293F cells showed an LV productivity of approximately 1.5E7TU / mL and HEK293T / 17 cells showed an LV productivity of approximately 3.9E7TU / mL, with the PP / IP ratio obtained with Expi293F cells being approximately 1900 compared to a PP / IP ratio of approximately 1000 achieved with HEK293T / 17 cells. [Figure 1B]It shows that the cell densities observed at each passage were comparable between both cell lines (approximately 3 x 106 cells / mL). [Figure 1C] Both Expi293F and HEK293T / 17 cells exhibit high viability (>90%) in culture. [Figure 2A] Figure 1 shows that the transfection reagent FectoVIR®-AAV significantly increases LV productivity in Expi293F cells by 1.9- to 2.8-fold depending on the gene of interest. [Figure 2B] Figure 1 shows a consistent and robust increase in LV productivity of Expi293F cells when FectoVIR®-AAV was used as the transfection reagent in different culture volumes. [Figure 3] The amount of lentivirus obtained using different amounts of DNA for transfection is shown. In this experiment, the highest virus production and lowest PP / IP were obtained using 0.4 μg of DNA / 1E6 cells. [Figure 4] The amount of lentivirus obtained using two different lentiviral vectors was shown, and a roughly three-fold increase in productivity was induced by changing the pH to 6.7 prior to transfection. A 2.5 L scale bioreactor was used. [Figure 5] A comparison of lentiviral productivity using different CAR constructs is shown in two production systems: (i) Expi293F cells using FectoVIR®-AAV as the transfection reagent, and (ii) HEK293T cells using PEIpro® as the transfection reagent. [Figure 6] Compared to the control run where the sample was subjected to ultrafiltration, the filtration process time was reduced from 244 minutes to 145 minutes in the presence of arginine. [Figure 7] We show that adding arginine before TFF improved vector recovery in the subsequent process from approximately 40% to over 80%. [Figure 8]Addition of arginine before both filtration steps further increased vector recovery. [Figure 9] It is shown that the addition of arginine reduces the particle number and size in a concentration-dependent manner. [Figure 10] 1 is a bar graph showing the productivity of infectious LVV (TU / mL-TU assay) and the ratio PP / IP (physical particles / infectious particles) when harvested at different concentrations of benzonase and different addition times. [Figure 11] 1 is a bar graph showing the amount of DNA (ng / 1E+7 TU) recovered at different concentrations of benzonase and different addition times. [Figure 12] 1 is a bar graph showing the amount of DNA (ng / mL) recovered at different concentrations of benzonase and different addition times. [Figure 13] 1 is a bar graph showing the productivity of infectious LVV (TU / mL-TU assay) and the ratio PP / IP (physical particles / infectious particles) when harvested at different incubation times and amounts of complex formation. [Figure 14] 1 is a bar graph showing the productivity of infectious LVV (TU / mL-TU assay) and the ratio PP / IP (physical particles / infectious particles) upon harvesting at different incubation times. [Figure 15] 1 is a bar graph showing cell density and viability at the 50 L scale (n=4) during LVV treatment. [Figure 16] 1 is a bar graph showing the productivity of infectious LVV (TU / mL-TU assay) at different scales using two different products (C1 and I1). DETAILED DESCRIPTION OF THE INVENTION

[0118] The present disclosure is based, at least in part, on methods for producing high-titer lentiviral vectors carrying a transgene of interest under satisfactory safety conditions. The present disclosure also provides, at least in part, methods for purifying such lentiviral particles from, for example, cell culture. The present disclosure also provides for formulation into lentiviral preparations that maintain the structural integrity of viral vectors during purification, storage, and ex vivo gene transfer events.

[0119] definition Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings:

[0120] As used herein, the singular forms "a" or "an" include plural referents unless otherwise stated.

[0121] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0122] "About" and "approximately" are generally intended to mean an acceptable degree of error for the quantity measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0123] The term "amino acid" refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar 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-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0124] As used herein, the term "buffer" 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, "1,4-piperazinediethanesulfonic acid buffer" refers to a mixture containing 1,4-piperazinediethanesulfonic acid and the 1,4-piperazinediethanesulfonic acid anion (e.g., sodium 1,4-piperazinediethanesulfonate). Similarly, as used herein, "sodium citrate buffer" refers to a mixture containing sodium citrate and its conjugate acid, citric acid. Because a chemical equilibrium is established between a weak acid and its conjugate base, a buffered solution resists sudden changes in pH when small amounts of acid or base are added to the solution.

[0125] As used herein, the term "binding domain" or "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In some embodiments, an antibody molecule is a multispecific antibody molecule, e.g., comprises multiple immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence of the multiple sequences has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the multiple sequences has binding specificity for a second epitope. In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for only two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0126] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, which usually determines the class to which the antibody belongs.

[0127] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) light chains and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0128] The term "antigen-binding fragment," as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of binding fragments include, but are not limited to, single-chain Fvs (scFv), camelid antibodies, disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single antibody arm; dAb fragments consisting of the VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs) or other epitope-binding fragments of antibodies.

[0129] The portion of the described CAR comprising an antibody or antibody fragment thereof can exist in various forms in which the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, 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 comprises an antibody fragment. In another embodiment, the CAR comprises an antibody fragment comprising an scFv. The precise amino acid sequence boundaries of a given CDR can be determined using any 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), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme).

[0130] Furthermore, although the two domains of an Fv fragment, i.e., VL and VH, are encoded by separate genes, recombinant methods can be used to join them with a synthetic linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a "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 encompassed by the term "antigen-binding fragment." These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0131] The term "chimeric antigen receptor" or alternatively "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 an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule, as defined below. In some embodiments, the domains of a CAR polypeptide construct are on the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the domains of a CAR polypeptide construct are not contiguous with one another, e.g., are on different polypeptide chains, e.g., as provided in an RCAR.

[0132] In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In some embodiments, the costimulatory molecule is selected from 41BB (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 costimulatory 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 costimulatory 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, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and cell membrane localization of the CAR.

[0133] A CAR comprising an antigen-binding domain (e.g., an scFv, single-domain antibody, or TCR (e.g., a TCR alpha-binding domain or a TCR beta-binding domain)) that targets a specific tumor marker X (where X can be a tumor marker as described herein) is also referred to as an XCAR. For example, a CAR comprising an antigen-binding domain that targets BCMA is referred to as a BCMA CAR. CARs can be expressed in any cell, such as an immune effector cell (e.g., a T cell or an NK cell) described herein.

[0134] The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information intracellularly to regulate cellular activity through a defined signaling pathway by generating a second messenger or functioning as an effector in response to such a messenger. The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides a cytoplasmic signaling sequence that stimulatorily regulates immune cell activation for at least some aspects of an immune cell signaling pathway. In some embodiments, the signal is a primary signal, e.g., initiated by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that function in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing cytoplasmic signaling sequences that are particularly useful in the present invention 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 of the invention comprises an intracellular signaling sequence, e.g., the primary signaling sequence of CD3ζ. In certain CARs of the invention, the primary signaling sequence of CD3ζ is a human sequence or equivalent residues from a non-human species, such as mouse, rodent, monkey, ape, etc.

[0135] "Intracellular signaling domain," as that term is used herein, refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of the CAR-containing cell, e.g., a CAR T cell. Examples of immune effector function (e.g., in CAR T cells) include cytolytic activity and helper activity, including cytokine secretion.

[0136] In some embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of a CAR T, the primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0137] The primary intracellular signaling domain can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 beta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0138] The term "zeta" or alternatively "zeta chain," "CD3zeta," or "TCRzeta" refers to CD247. Swiss-Prot Accession No. P20963 provides an exemplary human CD3zeta amino acid sequence. "Zeta stimulatory domain" or alternatively "CD3zeta stimulatory domain" or "TCRzeta stimulatory domain" refers to the stimulatory domain of CD3zeta or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion). In some embodiments, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Accession No. BAG36664.1 or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion). Alternatively or additionally, the terms "ζ" or alternatively "ζ chain," "CD3-ζ" (or "CD3ζ, CD3ζ, or CD3z"), or "TCR-ζ" are defined as the protein provided under GenBank Accession No. BAG36664.1 or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "ζ stimulatory domain" or alternatively a "CD3-ζ stimulatory domain" or a "TCR-ζ stimulatory domain" is defined as amino acid residues from the cytoplasmic domain of the ζ chain sufficient to functionally transmit an initial signal necessary for T cell activation, or a functional derivative thereof. In some embodiments, the cytoplasmic domain of ζ comprises residues 52-164 of GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species that is a functional ortholog thereof, e.g., mouse, rodent, monkey, ape, etc.

[0139] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, and include CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12, which contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, 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, 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 ligands that specifically bind to CD83.

[0140] The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83.

[0141] The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived, or the entire naturally occurring intracellular signaling domain, or a functional fragment or derivative thereof.

[0142] "Complementarity-determining domain" or "complementarity-determining region (CDR)" refers interchangeably to the hypervariable regions of VL and VH. CDRs are the target protein-binding sites of antibody chains, which have specificity for such target proteins. Each human VL or VH has three CDRs (CDRs 1 to 3, numbered sequentially from the N-terminus), which constitute approximately 15 to 20% of the variable domain. CDRs are structurally complementary to the epitope of the target protein and are therefore directly responsible for binding specificity. The remaining sections of VL or VH, the so-called framework regions, exhibit less amino acid sequence variability (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

[0143] The positions of CDRs and framework regions can be determined using various definitions known in the art, such as Kabat, Chothia, the international ImMunoGeneTics database (IMGT) (World Wide Web: www.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 references: 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).

[0144] As used herein, the term "contaminating polynucleotide" refers to a polynucleotide that is not derived from a lentiviral vector. Contaminating polynucleotides can include, for example, non-lentiviral polynucleotides derived from the cell in which the lentiviral vector was produced, such as chromosomal mammalian DNA (e.g., human DNA) not contained in the transgene or other components of the lentiviral vector.

[0145] "Derived from," as the term is 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 a limitation on the manner or source by which the first molecule is derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include a limitation to a particular method of making the intracellular signaling domain, e.g., it does not mean that one must start with the CD3ζ sequence and delete unnecessary sequence or make mutations to arrive at the intracellular signaling domain in order to provide the intracellular signaling domain.

[0146] As used herein, the term "freeze / thaw cycling" refers to 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 with a temperature of about -80°C to about -20°C. The mixture can be kept frozen for, 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 with a temperature of about 2°C to about 8°C, or by storing the mixture at room temperature (e.g., ambient laboratory temperature or about 25°C). Alternatively, thawing can be carried out using a water bath (e.g., 37°C).

[0147] As used herein, the term "hydrodynamic radius" refers to the apparent radius (Rh in nm) of a particle in solution, as estimated from the particle's diffusion properties. The hydrodynamic radius of a virus particle is one factor that determines the virus particle's diffusion rate in aqueous solution and the particle's ability to move through a polymeric gel. The hydrodynamic radius of a virus particle is determined in part by the mass and molecular structure of each of the particle's components 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.

[0148] 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 (Cu). Exemplary non-reducing carbohydrates include, but are not limited to, disaccharides such as sucrose, trehalose, and palatinitol, trisaccharides such as raffinose and melezitose, and tetrasaccharides such as stachyose. Non-reducing carbohydrates further include monosaccharide derivatives such as sorbitol, mannitol, erythritol, and xylitol, disaccharide derivatives such as lacitol and maltitol, aldonic acid and its lactones such as gluconic acid and gluconic acid gamma lactone, aldaric acid and its lactones such as rivaranic acid, arabinaric acid, and galactaric acid, uronic acids such as glucuronic acid, galacuronic acid, and ithianuronic acid, ester derivatives such as trehalose octaacetate, sucrose octaacetate, and cellobiose octaacetate, and ether derivatives in which the hydroxyl group has been O-alkylated. Non-reducing carbohydrates include those with D or L stereochemical orientation.

[0149] As used herein, the term "osmolality" refers to a measure of the osmotic pressure of solute particles in an aqueous solution. Solute particles include both ionic and non-ionized molecules. Osmolality is expressed as the concentration (i.e., osmoles) of osmotically active particles dissolved in 1 kg of solvent (i.e., water). Osmolality is expressed herein in units of milliosmoles per kg of water (mOsm / kg).

[0150] As used herein, the term "weight / volume percent" or "w / v%" refers to the percentage by weight (in grams) of a single component relative to the total volume of the 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%.

[0151] As used herein, the term "polydispersity" refers to the degree of size homogeneity of particles, such as lentiviral particles, within a sample. Higher polydispersity indicates lower homogeneity, while lower polydispersity indicates higher homogeneity. For example, when the level of homogeneity is high, the lentiviral particles can be considered to be similar in size and therefore monodisperse. As will be understood by those skilled in the art, as the polydispersity decreases, the level of homogeneity increases. Therefore, lower polydispersity indicates a higher level of homogeneity. For example, a preparation with a polydispersity of 15% is less homogeneous than a preparation with a polydispersity of 10%. When the level of homogeneity is low, the particle population can be considered polydisperse because it contains significantly different sizes.

[0152] As used herein, the terms "prevent," "preventing," or "prevention" of any disease or disorder refers to prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.

[0153] As used herein, the term "recognize" refers to an antibody or antigen-binding fragment thereof that finds and interacts with (e.g., binds to) the epitope, regardless of whether the epitope is linear or conformational. The term "epitope" refers to the site on an antigen to which an antibody or antigen-binding fragment of the present disclosure specifically binds. Epitopes can be formed both from contiguous amino acids juxtaposed by tertiary folding of a protein or from non-contiguous amino acids. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include techniques within the art, such as x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0154] As used herein, the term "retroviral packaging protein" refers to a protein derived from a retrovirus or variant thereof, which assists in packaging nucleic acid (e.g., a viral genome) into an envelope. Exemplary 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 thereto. In some embodiments, one or more retroviral packaging proteins are provided as a polyprotein.

[0155] As used herein, the term "retroviral envelope protein" refers to a protein or mutant thereof derived from a retrovirus that can assemble to form an envelope around nucleic acid (e.g., a viral genome). An exemplary retroviral envelope protein is env, e.g., wild-type or a mutant thereof. In some embodiments, the retroviral envelope protein is a lentiviral envelope protein, e.g., wild-type or a mutant thereof. In some embodiments, the retroviral envelope protein is VSV-G, e.g., wild-type or a mutant thereof. In some embodiments, the retroviral envelope protein is pseudotyped. In some embodiments, the retroviral envelope protein is derived from a different virus than the retroviral packaging proteins or one or more of the LTRs of the nucleic acid to be packaged.

[0156] As used herein, the phrases "specifically bind" and "binding" refer to a binding reaction that determines the presence of a specific protein, e.g., recognized by a specific ligand, in a heterogeneous population of proteins and other biomolecules. A ligand that specifically binds to a protein (e.g., a protein, proteoglycan, glycosaminoglycan, etc.) binds to the protein with a KD of less than 500 nM. For example, a ligand that specifically binds to a protein binds to the protein with a KD of up to 500 nM (e.g., 1 pM to 500 nM). A ligand that does not specifically bind to a protein or its domains exhibits a KD of 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) for that particular protein or its domain. Various assay formats can be used to determine the affinity of a ligand for a particular protein. For example, solid-phase ELISA assays are routinely 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, e.g., 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).

[0157] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" and "subject" are used interchangeably herein.

[0158] The term "therapeutic effector," as used herein, refers to a molecule (eg, RNA or polypeptide) that, at effective levels, can have a therapeutic effect on a subject.

[0159] The terms "therapeutically acceptable amount" or "therapeutically effective dose" refer interchangeably to an amount sufficient to produce a desired result (i.e., reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal, or parasitic infection). In some embodiments, a therapeutically acceptable amount does not induce or elicit undesirable side effects. In some embodiments, a therapeutically acceptable amount induces or elicits side effects, but only as tolerated by a healthcare provider given the patient's condition. A therapeutically acceptable amount can be determined by administering a low dose initially and then gradually increasing the dose until the desired effect is achieved. A "prophylactically effective dose" and a "therapeutically effective dose" can, in some embodiments, prevent the onset of or result in a reduction in the severity of disease symptoms, such as symptoms associated with cancer, respectively.

[0160] As used herein, the term "transfection" refers to the introduction of DNA into eukaryotic cells. Transfection can be achieved by a variety of means, including but not limited to, calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0161] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refer, in one embodiment, to ameliorating the disease or disorder (i.e., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including one that may not be discernible by the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both.

[0162] 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 the particle to a target cell. Viral titer can be measured by a functional assay, such as that described in Xiao et al., Exp. Neurobiol 144:1 13-124, 1997, or Fisher et al., J. Virol. 70:520-532, 1996, the disclosures of both of which are incorporated by reference in their entirety. Alternatively, viral titer can be measured by determining the amount of viral DNA integrated into the host cell genome, for example, using polymerase chain reaction (PCR) techniques known in the art.

[0163] As used herein, the term "viral vector" refers to a viral particle capable of introducing a nucleic acid molecule into a host. Viral vectors carrying exogenous genes are typically packaged into infectious viral particles via viral packaging by using a specific cell line to package the plasmid. The infectious viral particles infect cells to achieve expression of the exogenous gene. A "recombinant" viral vector refers to a viral vector constructed by genetic recombination technology. Recombinant viral vectors can be constructed using any suitable method, for example, by transducing or transfecting a nucleic acid encoding the viral genome into a packaging cell line and then isolating the newly packaged viral particles. It is understood that recombinant technology can be performed at a step upstream of the production of the viral vector itself. For example, recombinant technology can be used to produce a plasmid, which can then be produced on a large scale, and finally the plasmid can be introduced into a cell line for packaging to produce the viral vector.

[0164] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, such as a self-inactivating lentiviral vector such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in medical settings include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology and LENTIMAX™ vector systems from Lentigen. Non-clinical forms of lentiviral vectors are also available and would be known to those skilled in the art.

[0165] Lentivirus production method The present disclosure provides, among other things, improved methods 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 large T antigen, are described in more detail in the section entitled "Host Cells" herein. As described in Example 1 herein, host cells lacking large T antigen can provide manufacturing advantages compared to host cells containing large T antigen.

[0166] In some embodiments, to produce large quantities of cells, host cells are cultured in successively larger vessels (eg, bioreactors) until a sufficiently large number of cells are produced.

[0167] Once a sufficient number of host cells are obtained, the desired nucleic acid can be introduced into the host cells. The nucleic acid can be introduced by transfection, for example, using FectoVIR®-AAV transfection reagent, as described in the section entitled "Transfection" herein. The advantages of FectoVIR®-AAV transfection reagent are described in Examples 2 and 3 herein. The transfected nucleic acid can include the viral genome to be packaged, which contains a therapeutic gene of interest and sufficient long terminal repeat (LTR) sequences for packaging into viral particles. Additional nucleic acids that can 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 can be altered downward before transfection, for example, from about 7.1 to about 6.7, as described in the section entitled "Culture and Transfection Conditions" herein and in Example 4 herein. The cells then begin to produce lentivirus.

[0168] After transfection, a nuclease such as benzonase can be added to the culture medium, for example, as described in the section entitled "Culture Medium" herein and in Example 5. Without wishing 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 from lysed host cells. Thus, adding benzonase to the cell culture medium can degrade contaminating nucleic acids, allowing for improved purification of the lentivirus.

[0169] The lentivirus can then be harvested from the host cell culture to begin lentivirus purification. In some embodiments, harvesting the lentivirus involves separating the supernatant or cell culture medium from the cells. In some embodiments, the cells are not lysed prior to clarification. In some embodiments, the cells may be lysed and the lysate may be clarified.

[0170] Purification of lentivirus from cell culture medium or cell lysate typically involves several consecutive purification steps. The purification steps may include filtration (e.g., ultrafiltration) and chromatography steps. In some embodiments, arginine can 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 the section entitled "Purification" herein and in Examples 8-12. Without wishing to be bound by theory, in some embodiments, arginine stabilizes lentiviral vectors and / or reduces their aggregation.

[0171] Purified lentivirus can be used for various purposes.For example, lentivirus can be used to deliver genes to cells ex vivo, for example, to generate CART cells from immune effector cells derived from apheresis samples.As another example, lentivirus can be administered to a subject to deliver genes to cells of the subject in situ.For example, lentivirus can be used for in vivo CART.In some embodiments, lentivirus is suitable for administration to a human subject, and for example, a lentivirus encoding a CAR can be administered to a subject to allow the introduction of CAR-encoding nucleic acid into immune effector cells in the subject's body.

[0172] Naturally occurring lentiviruses are a genus of viruses in the Retroviridae family, characterized by long latency periods. Lentiviruses are generally capable of delivering large amounts of genetic information into the DNA of host cells. Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV types 1 and 2), the etiological agent of acquired immunodeficiency syndrome (AIDS) in humans; visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep; caprine 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 infection in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in great apes. The diseases caused by these viruses are characterized by a long incubation period and a protracted course. Usually, the viruses latently infect monocytes and macrophages, from where they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T cells).

[0173] Transgene In some embodiments, the lentivirus or lentiviral vector disclosed herein can comprise a nucleic acid, e.g., a transgene, e.g., a transgene encoding a protein. The nucleic acid can comprise, for example, a transgene as described herein in the section entitled "Transgene." The transgene can be operably linked to a promoter sequence. The nucleic acid can also comprise one or more (e.g., two) LTR sequences. Without wishing to be bound by theory, the LTR can facilitate insertion of the transgene and promoter into the host cell genome. The LTR sequence can comprise a wild-type lentiviral LTR sequence or a mutant thereof. For example, the 3' LTR can contain a deletion that self-inactivates the virus after integration. Additionally, the 5' LTR can be a chimeric LTR. In some embodiments, the transgene can be integrated into the chromosomal DNA of the target cell.

[0174] Exemplary transgenes include those encoding chimeric antigen receptors (CARs). CARs can include several domains, such as an antigen-binding domain, a transmembrane domain, and one or more signaling domains. In such cases, the signaling domains can include one or more primary signaling domains (such as a CD3-zeta stimulatory domain) and / or one or more costimulatory signaling domains (such as CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, or a ligand that specifically binds to CD83.

[0175] In some embodiments, a transgene, e.g., a transgene comprising a CAR, may encode an antigen binding domain (such as an scFv) that binds to a specific target protein or carbohydrate. Exemplary antigens include CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), 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 (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 embryonic antigen-4 (SSEA-4), CD20, folate receptor α, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), mucin 1, cell surface-associated (MUC1), epidermal growth factor receptor (EGFR), and neurotrophic factor receptor (N2). The oncogene fusion protein consisting of cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrin B2, fibroblast activation protein α (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetran) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100), breakpoint cluster region (BCR), and Abelson murine leukemia viral 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 moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor β3 (ADRB3), pannexin 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 / testis antigen 1 (NY-ESO-1), cancer / testis 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 cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutants, prostein, survivin, telomerase, prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells, rat sarcoma (Ras) mutants, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane receptor agonist) Protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis 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 recognized by T cells (SART3), 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-breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 mutant (mutated)hsp70-2), 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 lambda-like polypeptide 1 (IGLL1).

[0176] In some embodiments, the lentiviral vectors described herein comprise two or more transgenes, e.g., a first transgene encoding a first CAR, e.g., a CD19 CAR, and a second transgene encoding a second CAR, e.g., a CD22 CAR.

[0177] In some embodiments, the dual CAR lentiviral vectors described herein encode 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 and are 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., that binds CD22), optionally a first hinge domain, a first transmembrane domain, a first costimulatory 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., that binds CD19), optionally a second hinge domain, a second transmembrane domain, a second costimulatory 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 costimulatory domains have the same sequence. In some embodiments, the first and second primary signaling domains have the same sequence.

[0178] Additional CARs that can be encoded by the transgenes described herein are provided, for example, in the section entitled "CAR Targets" herein.

[0179] In some embodiments, the lentiviral vectors described herein encode siRNA or shRNA that targets a nucleic acid in an immune effector cell. For example, the siRNA or shRNA can target a nucleic acid encoding a TCR and / or HLA and / or an inhibitory molecule (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 a T cell. siRNA and shRNA expression systems and exemplary shRNAs are described, for example, in paragraphs 649 and 650 of International Publication No. WO 2015 / 142675, filed March 13, 2015, which is incorporated by reference in its entirety. These nucleic acids can also be targeted using, for example, the CRISPER system, zinc finger nucleases, or TALENs. Immune effector cells can be autologous or allogeneic to the subject to be treated.

[0180] In some embodiments, the lentiviral vectors described herein comprise or encode one or more inhibitors of methylcytosine dioxygenase genes (e.g., Tet1, Tet2, or Tet3). Use of such compositions and methods to increase the functional activity of engineered cells (e.g., genetically modified antigen-specific T cells, such as CAR-T cells) is also contemplated. Without being bound by theory, disruption of a single allele of the Tet gene (Tet1, Tet2, or Tet3) reduces the total level of 5-hydroxymethylcytosine, accompanied by enhanced proliferation, modulation of effector cytokine production, and degranulation, thereby enhancing CAR T cell proliferation and / or function. In some embodiments, Tet2 expression and / or function in the cells is reduced or eliminated.

[0181] In some embodiments, the inhibitor of Tet1, Tet2, and / or Tet3 is an siRNA or shRNA specific to Tet1, Tet2, Tet3, or a nucleic acid encoding the siRNA or shRNA. In some embodiments, the siRNA or shRNA comprises a sequence complementary to the sequence of Tet2 mRNA, e.g., a target sequence of an shRNA listed in Table 4 of WO 2017 / 049166, which is incorporated herein by reference in its entirety, including Table 4. In some embodiments, the inhibitor of Tet1, Tet2, and / or Tet3 is (1) a gene editing system that targets one or more sites in a gene encoding Tet1, Tet2, and / or Tet3 or a regulatory element thereof, e.g., Tet2 or a regulatory element thereof; (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 a CRISPR / Cas9 system, a zinc finger nuclease system, a TALEN system, and a meganuclease system.

[0182] In some embodiments, the lentiviral vectors described herein comprise a transgene, such as a transgene encoding a chimeric antigen receptor (CAR), and further comprise an siRNA or shRNA that targets a nucleic acid in an immune effector cell.

[0183] Characteristics of lentiviral vectors In some embodiments, the lentiviral vector is characterized by a hydrodynamic radius of 100±25 nm as measured by dynamic light scattering (DLS). For example, the lentiviral vector can maintain a hydrodynamic radius of 100±25 nm within a temperature range of 25°C to 55°C.

[0184] In some embodiments, the lentiviral vector is characterized by a polydispersity of 10% to 25%. For example, the lentiviral vector can maintain a polydispersity of 10% to 25% within a temperature range of 25°C to 55°C.

[0185] In some embodiments, the lentiviral vector maintains a concentration of about 70% to about 100% relative to the concentration of the lentiviral vector in the aqueous composition before the freeze / thaw cycles 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.

[0186] In some embodiments, a lentivirus prepared, purified, or stored using any of the methods or formulations disclosed herein may have a lower vector copy number (VCN), e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 60% lower, when tested at an MOI of 1, compared to a lentivirus that is not produced, purified, or stored by a method described herein or in a formulation described herein.

[0187] Lentiviral Vector Packaging System A packaging system can be used to package nucleic acids, such as RNA encoding a transgene, into a lentiviral vector. Thus, the systems and methods described herein can include, for example, a lentiviral packaging system comprising at least one plasmid suitable for producing a lentiviral vector, e.g., a lentiviral vector optionally comprising a transgene. Various lentiviral components useful for producing 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. Various functions suitable for lentiviral vector production can be imparted to a host cell 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, e.g., Gag and Pol proteins (packaging plasmid or Gag-Pol plasmid), and one plasmid encodes a lentiviral Rev protein (Rev plasmid) and one or more plasmids comprising at least one transgene of interest (TOI) expression cassette. In some embodiments, the lentiviral packaging system further comprises, or the methods described herein include the use of, at least one, two, three, or four plasmids. In some embodiments, the lentiviral packaging system further comprises, or the methods described herein include the use of, a fifth plasmid. In certain embodiments, the methods described herein include transfecting five plasmids into a host cell, where the fifth plasmid does not encode a protein of the lentiviral vector packaging system.In some embodiments, the lentiviral packaging system comprises one or more nucleic acids (e.g., plasmids), for example, five plasmids, where 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), e.g., as described in Rout-Pitt et al., J. Biol. Methods 5(2):1-9, 2018). In some embodiments, the plasmids can comprise a dual gene expression cassette, e.g., a bicistronic cassette, e.g., a bicistronic construct encoding two transgenes of interest. In some embodiments, a first transgene of interest encodes a first CAR, e.g., a CD19 CAR, and a second transgene of interest encodes a second CAR, e.g., a CD22 CAR. In some embodiments, the retroviral packaging proteins are derived from a lentivirus, e.g., lentiviral packaging proteins, e.g., lentiviral gag and pol proteins. In some embodiments, the lentiviral gag protein is a wild-type lentiviral gag protein, which in other embodiments 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, which in other embodiments has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the rev protein is a wild-type rev protein, which in other embodiments has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral vector can be a pseudotype vector containing a modified envelope protein, e.g., an envelope protein from a different virus or a chimeric envelope protein; for example, the Env plasmid can encode a VSV-G Env protein, e.g., a wild-type VSV-G protein or a modified mutant.

[0188] In some embodiments, lentiviral vectors are produced using a packaging system including pMDLgpRRE, pRSV-Rev, and pMD.G plasmids (Dull et al., supra), but instead of the ampicillin gene, a kanamycin resistance marker is used, e.g., neomycin phosphotransferase II, a marker that confers resistance to both kanamycin and neomycin.

[0189] In some embodiments, the systems described herein include a transfer vector that includes a kanamycin resistance marker, e.g., a marker that confers resistance to both kanamycin and neomycin, neomycin phosphotransferase II, e.g., in place of the ampicillin gene. In some embodiments, the transfer vector includes sequences from a pELPS construct, e.g., as disclosed in WO2017087861A or Milone et al., Mol. Ther. 17(8):1453-1464, 2009 (each incorporated by reference in its entirety). In some embodiments, the therapeutic protein is encoded by a self-inactivating transfer vector that includes one or more, e.g., all, of a lentiviral 5' LTR (e.g., a truncated lentiviral 5' LTR), a lentiviral 3' LTR, a cPPT, and a WPRE. In some embodiments, the transcription vector is deleted of one or more, e.g., all, of a promoter active in bacteria (e.g., lacking all of the T7 promoter, T3 promoter, and lac promoter), an M13 primer binding site (e.g., lacking both the M13 forward primer binding site and the M13 reverse primer binding site), a phage origin (e.g., f1 ori), and a gene encoding a fluorescent protein (e.g., GFP, e.g., EGFP). In some embodiments, the transfer vector is deleted of both the CAP binding site and the lac operator. In some embodiments, the transfer vector comprises a pELPS construct as disclosed in WO2017087861, except that the transfer vector is deleted of the T7 promoter, the M13 forward primer binding site, the f1 ori, the CAP binding site, the IPTG-inducible promoter, the lac operator, the M13 reverse primer binding site, the T3 promoter, and EGFP, 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) is more stable than an otherwise similar control transfer vector, (b) produces less cytotoxicity than an otherwise similar control transfer vector, or (c) results in a lower vector copy number (VCN) when integrated into a target cell, e.g., 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.

[0190] 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 the gene expression cassette are described in this disclosure.

[0191] Transfection In some embodiments, various functions for lentiviral vector production are imparted to a plurality of host cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells (e.g., a plurality of Expi293F cells grown in suspension under serum-free conditions), by transfection, e.g., transient or stable transfection, with 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%, or at least 95% of the host cells, e.g., HEK293 cells, e.g., Expi293F cells, are transfected. Methods of transfection or infection are well known to those of skill in the art. In some embodiments, 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, at least 0.9 μg, or at least 1.0 μg of lentiviral packaging system per million cells is provided for transfection. 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 available from commercial suppliers. Examples of transfection reagents include, but are not limited to, Lipofectamine™ (Invitrogen), Polifectamine, LentiTran (Origene), PEIpro® (Polyplus), FectoVIR®-AAV (Polyplus), and ProFection® (Promega). In some embodiments, the transfection reagent, e.g., FectoVIR®-AAV, is used at a level 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, e.g., FectoVIR®-AAV, are used for transfection at a ratio of about 1:0.5, 1:0.75, 1:1, 1:1.5, or 1:2, or any range therebetween.

[0192] In some embodiments, the transfection reagent comprises 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 chemical-based, animal-origin-free transfection reagent.

[0193] In some embodiments, at the time of transfection, cells (e.g., Expi293F cells) are transfected at a density of about 0.5×10 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 In some embodiments, at the time of transfection, the cell population has a viability of at least about 80%, 90%, or 95%.

[0194] In some embodiments, the PP / IP (physical particles / infectious particles) ratio is less than 500, 700, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 after transfection.

[0195] host cell The methods disclosed herein can be carried out using any host cell suitable for expressing a viral vector, e.g., a lentiviral vector, e.g., a lentiviral vector disclosed herein. In some embodiments, a suitable host cell is a eukaryotic cell, e.g., a mammalian cell. In some embodiments, the mammalian cell can be a genetically modified mammalian cell for expressing a virus, e.g., a lentivirus, e.g., a lentiviral vector, or a lentivirus of interest. Some mammalian cell lines are suitable host cells for recombinant expression of viruses. Mammalian host cell lines include, for example, 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, murine myelomas (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 culture of primary tissues and primary explants. In some embodiments, the host cells are HEK293 cells, including cells derived from HEK293 cells, e.g., 293F cells, e.g., Expi293F cells. In some embodiments, at least 80%, at least 85%, at least 90%, at least 90%, or at least 95% of the host cells in the culture express a large T antigen, e.g., a polyomavirus large T antigen, e.g., an SV40 large T antigen, e.g., a mutant SV40 large T antigen. In some embodiments, at least 99%, at least 98%, at least 97%, at least 96%, or at least 95% of the host cells in the culture do not express a large T cell antigen. In some embodiments, the host cells are suitable for growth in suspension.

[0196] Cultivation Process The cell lines described herein can be cultured under conditions that allow for the production of high-titer lentiviral vector particles. Eukaryotic cells, such as mammalian cells, such as HEK293 cells, e.g., Expi293F cells, can be cultured as non-anchorage-dependent cells that grow freely in suspension throughout the majority of the culture; or as anchorage-dependent cells that require attachment to a solid substrate for growth (e.g., as a monolayer).

[0197] In some embodiments, a microcarrier system may be used to accommodate cell growth. In some embodiments, the microcarrier system may comprise a suspension culture, e.g., a 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, nutrients are not added and waste products are not removed throughout the culture period. In some embodiments, waste products, including cells, products, by-products, and toxic metabolites, are not removed, but nutrients are continuously supplied to the system to extend the growth cycle. In some embodiments, the culture system may be an open, continuous system, e.g., a perfusion system or a chemostat system. In some embodiments, the system may include one or more cell retention devices. Cell retention devices may include, for example, microcarriers, fine mesh spin filters, hollow fibers, flat-plate membrane filters, sedimentation tubes, ultrasonic cell retention devices, etc. In some embodiments, the concentration of cells in the bioreactor is greater than the concentration of cells present in the supernatant harvested from the bioreactor. In some embodiments, the concentration of cells in the bioreactor is substantially the same as the supernatant harvested from the bioreactor.

[0198] In continuous fermentation processes, a defined medium is often continuously added to a bioreactor while an equal amount of medium is simultaneously removed for product harvest. Continuous cultures generally maintain cells at a constant cell density during the logarithmic phase of growth. Continuous or semi-continuous culture methods allow for the modulation of one factor or any number of factors that affect cell growth or end-product concentration. For example, one approach may limit the carbon source and allow all other parameters to moderate metabolism. In some systems, many factors affecting growth may be continuously varied, while the cell concentration, as measured by medium turbidity, is held constant. Continuous systems often maintain steady-state growth, so cell growth rate is often balanced against cell loss due to medium being removed from the culture. Methods for regulating nutrients and growth factors for continuous culture processes are known, and various methods are known in the art.

[0199] In some embodiments, a suspension cell culture comprises only cells that are in suspension. In some embodiments, a suspension cell culture may comprise, for example, a small number (e.g., less than 1%) of cells that transiently adhere to a surface.

[0200] "Cell culture" can refer to any in vitro culture of cells. The term includes continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and other cell populations maintained in vitro.

[0201] In some embodiments, the systems or methods described herein use 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 contain one or more expression cassettes capable of expressing viral proteins (such as gag, pol, and env), but the expression cassettes do not contain a packaging signal. The packaging cells can be cells cultured in vitro. The packaging cell lines can be used to generate producer cell lines for lentiviral particle production, for example, by providing 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) encoding a therapeutic effector and containing sufficient LTR sequences to enable packaging of an LTR-containing RNA into a viral vector. In some embodiments, the producer cell line stably expresses an expression cassette encoding a therapeutic effector and containing sufficient LTR sequences to enable packaging of an LTR-containing RNA into a viral vector.

[0202] Culture medium The methods of the present disclosure can 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., that supports cell growth 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, and refer to a nutrient solution used to grow, 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 (e.g., sodium, potassium, magnesium, calcium, etc.) that contribute to the osmolality of the medium; (2) an energy source, usually in the form of a carbohydrate such as glucose; (3) all essential amino acids and usually a basis set of 20 amino acids; (4) vitamins and / or other organic compounds required at low concentrations; and (5) trace elements (trace elements are defined as inorganic compounds that are usually required at very low concentrations, usually in the micromolar range). The compositions of such media are known in the art (see, e.g., 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, such as insulin, transferrin, and epidermal growth factor; and (c) plant, yeast, and / or tissue hydrolysates (including protein hydrolysates thereof).

[0203] In some embodiments, the culture medium may contain serum, e.g., fetal bovine serum (FBS). In some embodiments, the culture medium is serum-free. In some embodiments, the medium is chemically defined, e.g., animal-derived component-free. As used herein, an "animal-derived" component is any component produced in an intact animal (e.g., a protein isolated and purified from serum) or any component produced using a component produced in an intact animal (e.g., amino acids produced using enzymes isolated and purified from an animal to hydrolyze plant source material). In contrast, a protein produced in vitro in cell culture (e.g., in recombinant yeast or bacterial cells or in an established continuous eukaryotic cell line, whether recombinant or non-recombinant) using a medium that contains a component produced in an intact animal or that is isolated and purified from an intact animal is not an "animal-derived" component.

[0204] A chemically defined medium is a medium in which all components have a known chemical structure. Chemically defined media 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 aspects, media containing concentrated serum, e.g., higher concentrations of nutrients than those typically required and typically 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.

[0205] In some embodiments, an enzyme, e.g., a nuclease, e.g., an endonuclease, e.g., a recombinant endonuclease, e.g., 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 about 5-40, 10-40, 10-30, 20-30, or about 20 or about 24 hours after transfection of the host cells, e.g., Expi293F cells. In some embodiments, Benzonase is added 20-30 hours (e.g., about 24 hours) after transfection of the host cells, at a concentration of 3-7 U / mL (e.g., about 5 U / mL). In some embodiments, Benzonase is added 1-5 hours (e.g., about 3 hours) after transfection of the host cells, at a concentration of 3-7 U / mL (e.g., about 5 U / mL). In some embodiments, Benzonase is added 4-8 hours (e.g., about 6 hours) after transfection of the host cells, at a concentration of 3-7 U / mL (e.g., about 5 U / mL). 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 transfection of 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 transfection of 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 about 3 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 5 U / mL about 6 hours after transfecting the host cells. In some embodiments, benzonase is added at a concentration of 15 U / mL about 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 aspects, 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, e.g., MgCl2, is added to Benzonase®, e.g., at a concentration of about 1-5 mM, 1-3 mM, or about 2 mM. In some embodiments, the methods disclosed herein can include the addition of Benzonase® during the production and / or purification process.

[0206] In some embodiments, a compound can be added to the medium to affect culture growth, e.g., inhibiting proliferation, inducing differentiation, and inducing or suppressing gene expression. In some embodiments, the compound is sodium butyrate. In some embodiments, the cell culture media described herein include sodium butyrate.

[0207] Culture and transfection conditions Culture conditions can include any culture conditions suitable for maintaining cells (e.g., quiescent or proliferative). For example, culture conditions can include several parameters, including, but not limited to, temperature, oxygen content, nutrient content (e.g., glucose content), pH (e.g., increasing or decreasing pH), agitation level (e.g., revolutions per minute), gas flow rate (e.g., air, oxygen, nitrogen gas), redox potential, cell density (e.g., optical density), cell viability, etc. A change in culture conditions can 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., by adding or removing acid, base, or carbon dioxide), increasing or decreasing oxygen content (e.g., introducing air, oxygen, carbon dioxide, nitrogen), increasing or decreasing air pressure (e.g., by introducing air, oxygen, carbon dioxide, nitrogen), increasing or decreasing agitation, and / or adding or removing nutrients (e.g., one or more sugars or sugar sources, biomass, vitamins, etc.), increasing or decreasing the culture-to-flask volume ratio, or a combination thereof. In some embodiments, a change in culture conditions, e.g., an increase or decrease in pH, is introduced at a specific time during culture, e.g., before transfection. In some embodiments, the pH is corrected, e.g., adjusted to about 6.0-6.8, e.g., 6.2-6.8, e.g., 6.4-6.8, e.g., 6.7-6.75, prior to transfection with a lentiviral packaging system.

[0208] Culture volume and culture unit The disclosed methods can be carried out in small cell cultures, e.g., laboratory scale, or large-scale cultures, e.g., industrial scale. These methods can be carried out in suitable culture units, e.g., culture flasks or bioreactors. Bioreactors can be of any size, so long as they are useful for culturing cells, e.g., mammalian cells. In some embodiments, the disclosed methods are highly scalable, e.g., a plurality of mammalian cells are 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 number of transducing units per ml of culture that is 30%, 40%, 50%, 60%, 70%, or 80% or more of the number of transducing units per ml of culture in an otherwise similar small-scale culture, e.g., 100 ml, 200 ml, e.g., 300 ml, 400 ml, 500 ml). In some embodiments, scale cultures (i.e., having a culture volume greater than 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 the culture conditions. The 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 liters and can be about 100, 200, 300, 500, 800, 1000, 2500, 5000, 8000, 10,000, 12,0000 L or more, or any intermediate volume. A suitable culture unit or production culture unit can be made of (i.e., composed of) any material suitable for maintaining a cell culture suspended in a medium under the culture conditions contemplated herein and that promotes mammalian cell, e.g., HEK293 cell, e.g., Expi293F cell growth and viability. Examples of suitable materials include, but are not limited to, glass, plastic, and / or metal. In some embodiments, the material does not interfere or does not significantly or substantially interfere with the expression and / or stability of a desired product, e.g., a lentiviral vector.

[0209] In some embodiments, the cell culture process is operated 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, the process includes transferring the expanded seed culture from one or more seed culture units to a larger production unit. In some embodiments, cell growth through the production culture unit and production stage can be accomplished in a single physical culture unit, e.g., after expanding the cells to final production scale, the process can be switched to production conditions. Spent medium is collected at the end of the culture period for downstream processing of the lentivirus or lentiviral vector. In some embodiments, harvests can be collected at 24 hours, 48 ​​hours, 72 hours, 96 hours, or 120 hours post-transfection.

[0210] In some embodiments, downstream processing includes purification, formulation, and / or long-term storage of the lentivirus. In some embodiments, the viral harvest collected at the end of the culture period is, for example, about 5×10 6 Transducing units / milliliter (TU / mL) ~ approximately 7 x 10 7 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 or 7 x 10 7 In some embodiments, the viral harvest collected at the end of the culture period is at least 5 x 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 7 TU / mL or 7 x 10 7 In some embodiments, the virus harvest collected at the end of the culture period contains lentivirus at a concentration of 5 x 10 TU / mL. 6 TU / mL ~ 6 x 10 6 TU / mL, 6 × 10 6 TU / mL ~ 7 x 10 6 TU / mL, 7 × 10 6 TU / mL ~ 8 x 10 6 TU / mL, 8 × 10 6 TU / mL ~ 9 x 10 6 TU / m, 9 × 10 6 TU / mL ~ 1 x 10 7TU / mL, 1 × 10 7 TU / mL ~ 2 x 10 7 TU / mL, 2 × 10 7 TU / mL ~ 3 x 10 7 TU / mL, 3 × 10 7 TU / mL ~ 4 x 10 7 TU / mL, 4 × 10 7 TU / mL ~ 5 x 10 7 TU / mL, 5 × 10 7 TU / mL ~ 6 x 10 7 TU / mL, 6 × 10 7 TU / mL ~ 7 x 10 7 Contains lentivirus at a concentration of TU / mL.

[0211] Purification methods including filtration and chromatography In some aspects, the present disclosure provides a process for purifying lentiviral vectors with improved efficiency, e.g., resulting in the recovery of larger amounts of lentiviral vector. In some embodiments, at least one step in the purification process includes improving the purification process by adding an agent, such as an amino acid or a salt thereof, e.g., arginine or a salt thereof, e.g., arginine-HCl, to a purification intermediate composition (an intermediate composition including a buffer before the completion of purification) prior to further purification, e.g., 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, and ion exchange chromatography.

[0212] In some embodiments, lentiviral vectors produced according to the methods described herein have one or more of the following properties: comply with GMP guidelines, are sterile, are substantially free of contaminants, are suitable for pharmaceutical use, are suitable for administration to human subjects or are suitable for ex vivo treatment of human cells.

[0213] In some embodiments of the methods described herein, the solution or suspension is subjected to a semipermeable membrane (filtration), which allows solvents and small solute molecules to pass through but retains larger particles, such as viral particles. In some embodiments, the methods described herein use filters to remove and exchange salts, sugars, and non-aqueous solvents, separate free from bound species, remove low molecular weight materials, and / or cause rapid changes 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 lentiviral particles in the harvest. In some embodiments, the methods described herein utilize a process, technique, or combination of techniques that includes sequential or simultaneous filtration steps (e.g., one or more of microfiltration, ultrafiltration, nanofiltration, and diafiltration). In some embodiments, the filtration is performed using flat-sheet membranes or hollow fibers. In some embodiments, the filtration is performed using an average transmembrane pressure 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, the filtration is between 4 and 100 L / m 2 , for example, about 4-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, or 80-90 loads. In certain embodiments of the present disclosure, a filtration step is used, optionally in combination with chromatography or other purification steps, to exchange various buffers used in connection with the present disclosure and optionally remove impurities from the virus harvest.

[0214] Filtration techniques such as those described above and known in the art can be used to produce lentiviral preparations that are substantially free of the microorganisms and cells (e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells) from which the lentiviral vector is prepared. Additionally or alternatively, the lentiviral vector preparations of the present disclosure can be treated with nucleases to produce preparations that are substantially free of contaminating polynucleotides (e.g., non-lentiviral polynucleotides derived from the cells in which the lentiviral vector was produced, such as chromosomal mammalian DNA, human DNA, RNA, or other polynucleotides not contained in the lentiviral transgene).

[0215] For example, buffers used for purification Aqueous compositions containing various buffers, including buffers used in viral vector purification, are known in the art, including, but not limited to, sulfonic acid-based buffers, such as 1,4-piperazinediethanesulfonic acid (PIPES)-based buffers (PIPES buffers), polyol-based buffers, Tris buffers, phosphate buffers, acetate buffers, and citrate buffers. In some embodiments, the buffer used in connection with the purification processes disclosed herein is a sulfonic acid-based buffer, such as a PIPES buffer. In some embodiments, the PIPES buffer may contain a buffering agent, such as PIPES, at a concentration of about 10 mM to about 50 mM, about 15 mM to about 40 mM, about 20 mM to about 30 mM, e.g., about 20 mM.

[0216] In some embodiments, the buffer may further comprise a salt, such as sodium chloride (NaCl), magnesium chloride (MgCl), or calcium chloride (CaCl), or any combination thereof. The salt may be present, for example, in an aqueous lentivirus preparation at a concentration 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, 250 mM, 350 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 1000 mM, 1250 ...350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, The soluble ... 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 can be 50 mM or 75 mM, as appropriate.

[0217] In some embodiments, the buffer solution may further contain a carbohydrate, e.g., a non-reducing carbohydrate, such as sucrose or trehalose. In some embodiments, the carbohydrate, e.g., sucrose, is present at a concentration 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 150 mM, or about 150 mM to 300 mM. In some embodiments, the buffer solution, e.g., a PIPES buffer solution, e.g., a filtration buffer or exchange buffer solution, contains sucrose at a concentration of about 50 mM to about 80 mM, e.g., about 73 mM. In some embodiments, the buffer solution, e.g., a PIPES buffer solution, e.g., a formulation buffer or storage buffer solution, contains sucrose at a concentration of about 200 mM to 250 mM, e.g., about 220 mM.

[0218] In some embodiments, carbohydrates may be present at a concentration of, for example, about 1% to about 10%, about 2.5% to about 10%, or about 2.5% to about 5% weight / volume (w / v). For example, carbohydrates, such as the non-reducing carbohydrates described herein, may be present in an aqueous lentiviral preparation at a concentration of 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% w / v of the aqueous lentiviral preparation. In some embodiments, carbohydrates, such as non-reducing carbohydrates described herein, may be present in the lentivirus aqueous preparation at a concentration 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 non-reducing carbohydrates described herein, may be present in the lentiviral aqueous preparation at a concentration of 1% to 2% w / v, 2% to 3% w / v, 3% to 4% w / v, 4% to 5% w / v, 5% to 6% w / v, 6% to 7% w / v, 7% to 8% w / v, 8% to 9% w / v, or 9% to 10% w / v.

[0219] In some embodiments, the buffer further comprises, for example, arginine or a salt thereof, such as arginine-HCl. In some embodiments, the agent, such as arginine or a salt thereof, such as arginine monohydrochloride (arginine-HCl), is added at a concentration 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, such as a PIPES buffer used in virus purification (e.g., lentivirus purification using the processes disclosed herein), contains arginine, such as arginine-HCl.

[0220] In some embodiments, the pH of the buffer used in the purification processes disclosed herein is about 5.0 to about 8.0, e.g., 6.0 to about 7.0 (e.g., 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0), e.g., about 6.5.

[0221] In some embodiments, a PIPES buffer may be used as one or more of the exchange buffer, filtration buffer, formulation buffer, and / or storage buffer. In some embodiments, the ratio of the concentrations of PIPES, NaCl, and sucrose is different in the PIPES filtration buffer, PIPES exchange buffer, PIPES formulation buffer, and PIPES storage buffer. In some embodiments, the ratio of the concentrations of PIPES, NaCl, and sucrose is the same in the PIPES filtration buffer, PIPES exchange buffer, PIPES formulation buffer, and PIPES storage buffer. In some embodiments, the ratio of the concentrations of PIPES, NaCl, and sucrose is the same in the PIPES exchange buffer and the PIPES filtration buffer. In some embodiments, the ratio of the concentrations of PIPES, NaCl, sucrose, and optionally arginine, e.g., arginine-HCl, is the same in the PIPES formulation buffer and the PIPES storage buffer.

[0222] Arginine supplement In some embodiments, arginine, e.g., arginine-HCl, is added to a cell culture harvest during purification. In some embodiments, arginine, e.g., arginine-HCl, is added to a buffer during purification, e.g., PIPES buffer or a purification intermediate composition comprising PIPES buffer. In some embodiments, arginine, e.g., arginine-HCl, is added to a PIPES buffer that does not contain arginine. In some embodiments, arginine, e.g., arginine-HCl, is added to a PIPES buffer that contains arginine. In some embodiments, an agent, e.g., arginine or a salt thereof, e.g., arginine monosalt (arginine-HCl), is added at a concentration 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.

[0223] In some embodiments, vector recovery, e.g., the amount of lentiviral transducing units, is increased by about 10% to 300%, 20% to 180%, 30% to 160%, 50% to 150%, 75% to 125%, or about 100% in a purification process that includes a purification step that includes adding arginine to a purified intermediate composition, compared to a purification process that does not include a purification step that includes adding arginine to the purified intermediate composition. In some embodiments, the addition of arginine reduces the processing time of the purification. In some embodiments, when the purification includes, e.g., adding arginine or a salt thereof, e.g., arginine-HCl, to the purified intermediate composition, the processing time of the purification 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 adding arginine to the purified intermediate composition. In some embodiments, the purified intermediate composition after the addition of arginine or a salt thereof, e.g., arginine-HCl, followed by purification steps, exhibits a total particle concentration of less than 400,000, 300,000, 200,000, or 100,000 particles per ml, as measured by microflow imaging. While not wishing to be bound by theory, in some embodiments, individual lentiviral particles (e.g., infectious viral particles) are substantially not detected by microflow imaging, but larger particles, including aggregates, e.g., aggregates of non-functional viruses, are detected. In some embodiments, the purified intermediate composition after the addition of arginine or a salt thereof, e.g., arginine-HCl, followed by purification steps, exhibits a concentration of less than about 5,000, 4,500, 4,000, 3,500, 3,000, or 2,500 particles ≧10 μm per ml, as measured by microflow imaging. In some embodiments, the purified intermediate composition after addition of arginine or a salt thereof, e.g., arginine-HCl, e.g., the purified intermediate composition after addition of arginine-HCl and subsequent purification steps, exhibits a concentration of ≧25 μm per ml of less than about 500, 400, 300, or 200 μm per ml, as measured by microflow imaging.In some embodiments, reduced aggregates result in less clogging of filtration membranes at a given time point. In some embodiments, arginine stabilizes lentiviral particles.

[0224] In some embodiments, the purified lentiviral composition comprises, for example, about 1 x 10 per milliliter. 7 Transducing units (TU / mL) ~ approx. 7 x 10 7 TU / mL (e.g., 1 x 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 x 10 7 Contains lentiviral vectors at a concentration of 1000 TU / mL.

[0225] Aqueous compositions for preserving lentiviruses In some embodiments, the present disclosure provides a preparation, e.g., an aqueous mixture, e.g., an aqueous solution or suspension, e.g., an aqueous composition, comprising a lentiviral vector disclosed herein and a buffer, e.g., a formulation buffer or storage buffer, e.g., a PIPES buffer, e.g., a PIPES buffer containing arginine, e.g., arginine-HCl. In some embodiments, lentiviral formulations comprising a formulation buffer or storage buffer, e.g., a PIPES buffer, e.g., a PIPES buffer containing arginine, e.g., arginine-HCl, exhibit improved biological properties compared to lentiviral formulations containing conventional lentiviral formulation buffers, such as HEPES. These improved biological properties include increased resistance to aggregation over a wide range of temperatures and salt concentrations, as disclosed in WO2017087861A1. In some embodiments, PIPES buffers exhibit improved transduction capacity at physiological and elevated temperatures (e.g., 42°C and 50°C), as well as increased resistance to loss of infectivity during multiple freeze / thaw cycles. Other buffers useful with the lentiviral preparations of the present disclosure include histidine buffer, phosphate buffer, sodium citrate buffer, MES buffer, and MOPS buffer. The lentiviral preparations of the present disclosure may optionally include a salt, such as sodium chloride, and may optionally include a carbohydrate, such as a non-reducing carbohydrate.

[0226] In some embodiments, the PIPES formulation buffer and / or storage buffer may contain a buffering agent, e.g., PIPES, at a concentration of about 10 mM to about 50 mM, about 15 mM to about 40 mM, about 20 mM to about 30 mM, e.g., about 20 mM. The lentiviral vector preparation may optionally contain a salt, such as sodium chloride, magnesium chloride, or calcium chloride. The salt may be present, for example, in an aqueous lentivirus preparation at a concentration 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 The soluble ... In some embodiments, the salt concentration is at least about 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, 225 mM, 240 mM, 250 mM, 260 mM, 275 mM, 280 mM, 295 mM, 300 mM, 310 mM, 325 mM, 330 mM, 340 mM, 350 mM, 360 mM, 375 mM, 380 mM, 395 mM, 400 mM, 450 mM, 460 mM, 475 mM, 480 mM, 495 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 1000 mM, 1250 mM, 1500 mM, 1750 mM, 2000 mM, 2250 mM, 2450 mM, 2500 mM, 2650 mM, 2750 mM, 2800 mM, 2950 mM, 3000 mM, 3100 mM, 3250 mM, 3300 mM, 3450 mM, 3500 mM, 3650 mM, 3750 mM, 3850 mM, 3950 mM, 40 , 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 1M.In some embodiments, the salt concentration is 1 to 2 mM, 2 to 3 mM, 3 to 4 mM, 4 to 5 mM, 5 to 6 mM, 6 to 7 mM, 7 to 8 mM, 8 to 9 mM, 9 to 10 mM, 10 to 15 mM, 15 to 20 mM, 20 to 25 mM, 25 to 30 mM, 30 to 35 mM, 35 to 40 mM, 40 to 45 mM, 45 to 50 mM, 50 to 65 mM, 55 to 60 mM, 56 to 70 mM, 57 to 71 mM, 58 to 72 mM, 59 to 80 mM, 60 to 61 mM, 61 to 73 mM, 62 to 74 mM, 63 to 75 mM, 64 to 76 mM, 65 to 77 mM, 66 to 78 mM, 67 to 79 mM, 68 to 80 mM, 69 to 81 mM, 70 to 82 mM, 71 to 72 mM, 72 to 73 mM, 73 to 74 mM, 75 to 76 mM, 76 to 77 mM, 77 to 78 mM, 78 to 80 mM, 79 to 81 mM, 80 to 82 mM, 81 to 83 mM, 82 to 84 mM, 83 to 85 mM, 84 to 85 mM, 85 to 86 mM, 86 to 87 mM, 87 to 88 mM, 88 to 89 mM, 89 to 90 mM, 90 to 91 mM, 91 to 92 mM, 92 to 93 mM, 93 to 94 mM, 94 to 95 mM, 95 to 96 0~55mM, 55~60mM, 60~65mM, 65~70mM, 70~75mM, 75~80mM, 80~85mM, 85~90mM, 90~100 mM, 100~125mM, 125~150mM, 150~175mM, 175~200mM, 200~225mM, 225~250mM, 250~27 5mM, 275~300mM, 300~325mM, 325~350mM, 350~375mM, 375~400mM, 400~450mM, 450~4 75mM, 475~500mM, 500~525mM, 525~575mM, 575~600mM, 600~625mM, 625~650mM, 650~ 675 mM, 675 to 700 mM, 700 to 725 mM, 725 to 750 mM, 750 to 775 mM, 775 to 800 mM, 800 to 825 mM, 825 to 850 mM, 850 to 875 mM, 875 to 900 mM, 900 to 925 mM, 925 to 950 mM, 950 to 975 mM, or 957 mM to 1M. 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 can be 50 mM or 75 mM, as appropriate.

[0227] Lentiviral vector preparations of the present disclosure can optionally contain carbohydrates, such as non-reducing carbohydrates described herein. Exemplary non-reducing carbohydrates include sucrose and trehalose, among others. When included in a lentiviral vector preparation, the carbohydrate can be present, for example, at a concentration of about 1% to about 10%, about 2.5% to about 10%, or about 2.5% to about 5% weight / volume (w / v) of the aqueous lentiviral preparation. For example, carbohydrates, such as the non-reducing carbohydrates described herein, may be present in the aqueous lentiviral preparation at a concentration 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%.

[0228] The lentiviral vector preparations disclosed herein may contain an amino acid or a salt thereof, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, or a salt thereof. When included in a lentiviral vector preparation, an amino acid, such as arginine-HCl, may be present at a concentration of about 25 to 50 mM (e.g., about 50 mM), 50 to 100 mM (e.g., about 75 mM), 100 to 200 mM (e.g., about 150 mM), or 200 to 400 mM (e.g., about 300 mM). In some embodiments, the lentiviral vector preparations disclosed herein may contain two or more amino acids or salts thereof, such as arginine or a salt thereof and histidine or a salt thereof.

[0229] The lentiviral vector preparations described herein can exhibit a pH of, for example, about 5.0 to about 8.0, e.g., 6.0 to about 7.0 (e.g., 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0). In some embodiments, the pH of the lentiviral vector preparation is 6.5.

[0230] In some embodiments, the PIPES formulation buffer and the PIPES storage buffer have the same composition, e.g., the same concentrations of PIPES, NaCl, sucrose, and optionally arginine, e.g., arginine-HCl. In some embodiments, the PIPES formulation buffer and the PIPES storage buffer have different compositions, e.g., different concentrations of PIPES, NaCl, sucrose, and optionally arginine, e.g., arginine-HCl.

[0231] Lentiviral vectors can be present in the lentiviral preparations of the present disclosure at various concentrations. For example, lentiviral vectors can be present in the lentiviral preparations at concentrations of, for example, about 1 x 10 7 Transducing units / milliliter (TU / mL) ~ approximately 1 x 10 9 TU / mL (e.g., 1 x 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 8 TU / 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 × 108 TU / mL, 9.5 × 10 8 TU / mL or 1 x 10 9 Optionally, the lentiviral preparation may be present at a concentration of approximately 3 x 10 8 TU / mL ~ approx. 5 x 10 8 TU / mL (e.g., 3 x 10 8 TU / mL, 3.5 × 10 8 TU / mL, 4 × 10 8 TU / mL, 4.5 × 10 8 TU / mL or 5 x 10 8 The medium may contain lentiviral vectors at a concentration of 1000 TU / mL.

[0232] The present disclosure also provides aqueous compositions containing 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 (e.g., sodium chloride, magnesium chloride, or calcium chloride). These compositions may further contain a carbohydrate, such as a non-reducing carbohydrate (e.g., sucrose or trehalose).

[0233] In some embodiments, aqueous compositions, e.g., aqueous compositions comprising a lentiviral vector described herein, can be stored at low temperatures, e.g., 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 period of time, e.g., 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, or 25 days. In some embodiments, the aqueous composition is stored at a temperature below 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. In some embodiments, purified lentiviral samples stored in PIPES storage buffer are stored frozen at −80° C. immediately after purification. In some embodiments, lentiviral preparations stored in this manner can be thawed and refrozen (e.g., freeze-thaw cycled) before use. In some embodiments, lentiviral 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, or at least nine freeze-thaw cycles without significant loss of stability and / or infectivity. In some embodiments, the preparations exhibit no more than 0.5%, no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 40%, no more than 50%, no more than 60%, no more than 70%, or no more than 80% loss of stability and / or infectivity compared to lentiviral preparations that have never undergone a freeze-thaw cycle.

[0234] In some embodiments, the lentiviral preparations disclosed herein can be stored at 4° C. for a period of time, such as 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, or 25 days. In some embodiments, the lentiviral preparations disclosed herein can be stored at −80° C. for a period of time, such as 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, or 25 days. In some embodiments, lentiviral preparations stored as disclosed herein (e.g., stored frozen) 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, a lentiviral preparation does not lose 0.5% or more, 1% or more, 2% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more 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 frozen) are 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% more stable than lentivirus that has never been frozen.In some embodiments, the lentiviral preparation is 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 prior to use to improve vector integrity.

[0235] The present disclosure further includes dried or lyophilized compositions prepared by drying or lyophilizing the aqueous compositions described herein, as well as aqueous compositions prepared by reconstituting such dried or lyophilized compositions in a buffer solution (or another standard vehicle for administration) described herein.

[0236] CAR target Described herein are viral vectors that transduce immune effector cells (e.g., T cells, NK cells) engineered to contain one or more chimeric antigen receptors (CARs), directing the immune effector cells to unwanted cells (e.g., cancer cells). This is achieved through an antigen-binding domain on the CAR that is specific for a cancer-associated antigen. 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 themselves intracellular, but in which fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC (major histocompatibility complex).

[0237] In some embodiments, the tumor antigen is CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also referred to as CRACC, SLAMF7, CD319, and 19A24); C-type 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 members; B-cell component mature (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 alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP) elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein (bcr-abl) consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral 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); globoH glycoceramide hexasaccharide moiety (Glo boH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis 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 cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen 1 recognized by T cells (Melan-A or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (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 avian myelocytomatosis 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 (brother of BORIS or regulator of imprinted sites), squamous cell carcinoma antigen 3 recognized by T cells (SART3); 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-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); 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); or immunoglobulin lambda-like polypeptide 1 (IGLL1);

[0238] The CARs described herein can comprise an antigen-binding domain (e.g., an antibody or antibody fragment, a TCR or a TCR fragment) that binds to a tumor-supporting antigen (e.g., a 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. Without wishing to be bound by theory, in some embodiments, CAR-expressing cells destroy tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival.

[0239] In some embodiments, the stromal cell antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), and tenascin. In one embodiment, the FAP-specific antibody is sibrotuzumab, competes for binding with sibrotuzumab, or has the same CDRs as sibrotuzumab. In some 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 activation protein (FAP), or tenascin, CD33, CD11b, C14, CD15, and CD66b.

[0240] CD19 A non-limiting exemplary tumor antigen is CD19. CARs that bind to CD19 are known in the art. For example, those disclosed in International Publication Nos. 2012 / 079000 and 2014 / 153270 can be used according to the present disclosure. Any known CD19 CAR in the art, for example, the CD19 antigen binding domain of any known CD19 CAR, can be used according to the present 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.

[0241] Non-limiting exemplary CD19 CARs include, for example, the CD19 CARs described herein or those described in Xu et al. Blood 123.24(2014):3750-9; Kochenderfer et al. Blood 122.25(2013):4129-39, Cruz et al. Blood 122.17(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, 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.

[0242] 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 comprises the scFv fragment described in Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997).

[0243] In some embodiments, the antigen binding domain (e.g., a humanized antigen binding domain) binds to CD19 and comprises a sequence from Table 3 of WO 2014 / 153270 (incorporated herein by reference). WO 2014 / 153270 also describes methods for assaying the binding and efficacy of various CAR constructs.

[0244] Humanization of murine CD19 antibodies is desirable in clinical settings, where murine-specific residues can elicit a human anti-mouse antigen (HAMA) response in patients undergoing CART19 therapy, i.e., treatment with T cells transduced with a CAR19 construct. The production, characterization, and efficacy of humanized CD19 CAR sequences are described in WO 2014 / 153270, which is incorporated by reference in its entirety, including Examples 1-5 (pp. 115-159).

[0245] In some embodiments, the antigen binding domain comprises the parent murine scFv sequence of the CAR19 construct provided in WO 2012 / 079000 (incorporated herein by reference). In some embodiments, the antigen binding domain binds to CD19 and comprises the scFv described in WO 2012 / 079000.

[0246] In some embodiments, the CD19 CAR comprises the fusion polypeptide sequence provided as SEQ ID NO: 12 in WO 2012 / 079000, which provides an scFv fragment of murine origin that specifically binds to human CD19.

[0247] In some embodiments, the CD19 CAR comprises the amino acid sequence provided as SEQ ID NO: 12 in WO 2012 / 079000.

[0248] In some embodiments, the CD19 CAR comprises: [ka] or a sequence substantially homologous thereto.

[0249] In some embodiments, the CD19 CAR comprises: [ka] It contains the amino acid sequence of

[0250] In some embodiments, the CD19 CAR has the amino acid sequence: [ka] and a humanized CD19 CAR comprising:

[0251] In some embodiments, the CD19 CAR comprises a sequence, such as a CDR, VH, VL, scFv or full-length CAR sequence disclosed in Table 1 below, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0252] [Table 1]

[0253] [Table 2]

[0254] [Table 3]

[0255] [Table 4]

[0256] [Table 5]

[0257] [Table 6]

[0258] [Table 7]

[0259] [Table 8]

[0260] Exemplary CD19 CAR A In some embodiments, the CD19 CAR comprises a binding domain of a single-chain variable fragment (scFv) derived from the FMC63 monoclonal antibody, an IgG4 hinge region, a CD28 transmembrane domain, a 4-1BB (CD137) costimulatory domain, and a CD3ζ activation domain. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence in Table 25, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence in Table 25, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the CD19 CAR comprises a polypeptide sequence of Table 25, or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 25. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 25 according to Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 25 according to Chothia. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence in Table 25. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence in Table 25 according to Kabat. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 of the sequence in Table 25 by Chothia. In some embodiments, the CD19 CAR comprises light chain CDR1-3 of the sequence in Table 25 by Kabat. In some embodiments, the CD19 CAR comprises light chain CDR1-3 of the sequence in Table 25 by Chothia.

[0261] [Table 9]

[0262] [Table 10]

[0263] [Table 11]

[0264] [Table 12]

[0265] Exemplary CD19 CAR B In some embodiments, the CD19 CAR comprises a murine anti-CD19 single-chain variable fragment (scFv) linked to CD28 and CD3-zeta costimulatory domains. In a specific embodiment, the anti-CD19 single-chain variable fragment comprises the FMC63 antibody (e.g., the antibody described in Nicholson et al., Molecular Immunology, 34(16-17):1157-1165, 1997; the entire contents of which are incorporated herein by reference). In some embodiments, the CD19 CAR is encoded by a nucleotide sequence in Table 26, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence of Table 26, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutations thereto. In some embodiments, the CD19 CAR comprises a polypeptide sequence of Table 26, or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutations thereto. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence of Table 26. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence of Table 26 according to Kabat. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequences in Table 26 according to Chothia. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 of the sequences in Table 26 according to Kabat. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 of the sequences in Table 26 according to Chothia. In some embodiments, the CD19 CAR comprises light chain CDR1-3 of the sequences in Table 26.In some embodiments, the CD19 CAR comprises light chain CDR1-3 of the sequences in Table 26 according to Kabat. In some embodiments, the CD19 CAR comprises light chain CDR1-3 of the sequences in Table 26 according to Chothia.

[0266] [Table 13]

[0267] [Table 14]

[0268] Exemplary CD19 CAR C In some embodiments, the CD19 CAR comprises a murine anti-CD19 single-chain variable fragment (scFv) linked to CD28 and CD3-zeta costimulatory domains. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence in Table 27, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence in Table 27, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the CD19 CAR comprises a polypeptide sequence of Table 27 or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 27. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 27 according to Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 27 according to Chothia. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence in Table 27. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence in Table 27 according to Kabat. In some embodiments, the CD19 CAR comprises heavy chain CDR1-3 of the sequence in Table 27 by Chothia. In some embodiments, the CD19 CAR comprises light chain CDR1-3 of the sequence in Table 27 by Kabat. In some embodiments, the CD19 CAR comprises light chain CDR1-3 of the sequence in Table 27 by Chothia.

[0269] [Table 15]

[0270] [Table 16]

[0271] [Table 17]

[0272] Exemplary CD19 CAR F In some embodiments, the CD19 CAR is encoded by a nucleotide sequence of Table 27, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutations thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence of Table 34, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutations thereto. In some embodiments, the CD19 CAR comprises a polypeptide sequence of Table 34, or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutations thereto. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 34 according to Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 34 according to Chothia. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 34 according to Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence in Table 34 according to Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence in Table 34 according to Chothia. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 of a sequence in Table 34. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 of a sequence in Table 34 according to Kabat. In some embodiments, the CD19 CAR comprises light chain CDR1-3 of the sequences in Table 34 by Chothia.

[0273] [Table 18]

[0274] [Table 19]

[0275] Exemplary CD19-CD20 CAR G In some embodiments, the CD19 CAR is a bispecific CAR. In specific embodiments, the CD19 bispecific CAR comprises a light chain variable domain that targets CD19 and a heavy chain variable domain that targets 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 in Table 35, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the bispecific CAR comprises a polypeptide encoded by a nucleotide sequence in Table 35, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the bispecific CAR comprises a polypeptide sequence of Table 35 or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation therein. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 35. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 35 according to Kabat. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence in Table 35 according to Chothia. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 of a sequence in Table 35. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 of a sequence in Table 35 according to Kabat. In some embodiments, the bispecific CAR comprises heavy chain CDR1-3 of a sequence in Table 35 according to Chothia. In some embodiments, the bispecific CAR comprises light chain CDR1-3 of a sequence in Table 35 according to Kabat. In some embodiments, the bispecific CAR comprises light chain CDR1-3 of a sequence in Table 35 according to Chothia.

[0276] Table 20

[0277] Table 21

[0278] Table 22

[0279] BCMA A non-limiting exemplary tumor antigen is BCMA. CARs that bind to BCMA are known in the art. For example, those disclosed in WO 2016 / 014565 or WO 2019 / 241426 can be used in accordance with the present disclosure. Any known BCMA CAR in the art, for example, the BCMA antigen-binding domain of any known BCMA CAR, can be used in accordance with the present 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 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.

[0280] In some embodiments, the BCMA CAR is selected from the group consisting of 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-C2, BCMA_EBB-C3, BCMA_EBB-C4, BCMA_EBB-C5, BCMA_EBB-C6, BCMA_EBB-C7, BCMA_EBB-C8, BCMA_EBB-C9, BCMA_EBB-C10, BCMA_EBB-C11, BCMA_EBB-C12, BCMA_EBB-C13, BCMA_EBB-C14, BCMA_EBB-C15, BCMA_EBB-C16, BCMA_EBB-C17, BCMA_EBB-C18, BCMA_EBB-C19 ... The antibody comprises one or more CDRs, VH, VL, scFV or full-length sequences of 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, or a sequence substantially (e.g., 95 to 99%) identical thereto.

[0281] Exemplary antigen binding domains that bind to BCMA are described in WO 2012 / 0163805, WO 2017 / 021450, WO 2017 / 011804, WO 2017 / 025038, WO 2016 / 090327, WO 2016 / 130598, WO 2016 / 210293, WO 2016 / 090320, WO 2016 / 014789, US Patent Application Publication Nos. 2016 / 094304, 2016 / 154055, 2015 / 166073, 2015 / 188119, 2015 / 158671, U.S. Patent Nos. 9,243,058, 8,920,776, 9,273,141, 7,083,785, 9,034,324, U.S. Patent Application Publication Nos. 2007 / 0049735, 2 Nos. 015 / 0284467, 2015 / 0051266, 2015 / 0344844, 2016 / 0131655, 2016 / 0297884, 2016 / 0297885, 2017 / 0051308, 2017 / 0051252, 2016 / 020332, WO 2016 / 087531, WO 2016 / 079177, WO 2016 / 087531 Nos. 5 / 172800, 2017 / 008169, U.S. Pat. No. 9,340,621, U.S. Patent Application Publication Nos. 2013 / 0273055, 2016 / 0176973, 2015 / 0368351, 2017 / 0051068, 2016 / 0368988, and 2015 / 0232557, which are fully incorporated by reference herein in their entireties, in some embodiments one or more of the BCMA antigen binding domains disclosed therein.

[0282] 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) LC CDR1, LC CDR2, and LC CDR3 of a human anti-BCMA binding domain described herein (e.g., in Tables 2-14) and / or one or more (e.g., all three) HC CDR1, HC CDR2, and HC CDR3 of a human anti-BCMA binding domain described herein (e.g., in Tables 2-14). In some embodiments, the human anti-BCMA binding domain comprises a human VL described herein (e.g., in Tables 2, 6, and 10) and / or a human VH described herein (e.g., in Tables 2, 6, and 10). In some embodiments, the antigen-binding domain is an scFv comprising a VL and VH of the amino acid sequences in Tables 2, 6, and 10. In some embodiments, the antigen binding domain (e.g., scFv) comprises: a VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of an amino acid sequence provided in Tables 2, 6, and 10, but not more than 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence with 95-99% identity to an amino acid sequence of Tables 2, 6, and 10; and / or a VH comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions) of an amino acid sequence provided in Tables 2, 6, and 10, but not more than 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence with 95-99% identity to an amino acid sequence of Tables 2, 6, and 10.

[0283] In certain embodiments, the antigen binding domain described herein comprises: (1) one, two, or three light chain (LC) CDRs selected from the following: (i) an LC CDR1 of SEQ ID NO: 54, an LC CDR2 of SEQ ID NO: 55, and an LC CDR3 of SEQ ID NO: 56; and / or (2) one, two, or three heavy chain (HC) CDRs from any one of the following: (i) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 84; (ii) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 46; (iii) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 68; or (iv) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 76.

[0284] In certain embodiments, the antigen binding domain described herein comprises: (1) one, two, or three light chain (LC) CDRs 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 from any one of the following: (i) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 130, and HC CDR3 of SEQ ID NO: 88; (ii) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 87, and HC CDR3 of SEQ ID NO: 88; or (iii) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 109, and HC CDR3 of SEQ ID NO: 88.

[0285] In certain embodiments, the antigen binding domain described herein comprises: (1) one, two, or three light chain (LC) CDRs 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 from any one of the following: (i) HC CDR1 of SEQ ID NO: 179, HC CDR2 of SEQ ID NO: 180, and HC CDR3 of SEQ ID NO: 181; (ii) HC CDR1 of SEQ ID NO: 137, HC CDR2 of SEQ ID NO: 138, and HC CDR3 of SEQ ID NO: 139; or (iii) HC CDR1 of SEQ ID NO: 160, HC CDR2 of SEQ ID NO: 161, and HC CDR3 of SEQ ID NO: 162.

[0286] In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 45, 84, 54, 55, and 56, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 45, 46, 54, 55, and 56, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 45, 68, 54, 55, and 56, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55, and 56, respectively.

[0287] In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 47, 48, 84, 57, 58, and 59, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 47, 48, 46, 57, 58, and 59, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 47, 48, 68, 57, 58, and 59, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 47, 48, 76, 57, 58, and 59, respectively.

[0288] In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 49, 50, 85, 60, 58, and 56, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 49, 50, 51, 60, 58, and 56, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 49, 50, 69, 60, 58, and 56, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 49, 50, 77, 60, 58, and 56, respectively.

[0289] In some embodiments, the BCMA CAR comprises a sequence, such as a CDR, VH, VL, scFv or full-length CAR sequence disclosed in Tables 2-14, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0290] [Table 23]

[0291] [Table 24]

[0292] [Table 25]

[0293] [Table 26]

[0294] [Table 27]

[0295] [Table 28]

[0296] [Table 29]

[0297] [Table 30]

[0298] [Table 31]

[0299] Table 32

[0300] Table 33

[0301] Table 34

[0302] Table 35

[0303] Table 36

[0304] Table 37

[0305] Table 38

[0306] Table 39

[0307] Table 40

[0308] Table 41

[0309] Table 42

[0310] Table 43

[0311] Table 44

[0312] Table 45

[0313] Table 46

[0314] Table 47

[0315] Table 48

[0316] Table 49

[0317] Table 50

[0318] Table 51

[0319] Table 52

[0320] Table 53

[0321] Table 54

[0322] Table 55

[0323] Table 56

[0324] Table 57

[0325] Table 58

[0326] Table 59

[0327] Table 60

[0328] Table 61

[0329] Table 62

[0330] [Table 63]

[0331] [Table 64]

[0332] [Table 65]

[0333] [Table 66]

[0334] [Table 67]

[0335] [Table 68]

[0336] In some embodiments, a BCMA CAR may be generated using the VH and VL sequences from WO 2012 / 0163805, the contents of which are incorporated herein by reference in their entirety. In some embodiments, a BCMA CAR may be generated using the CDR, VH, VL, scFv or full-length CAR sequences from WO 2019 / 241426, the contents of which are incorporated herein by reference in their entirety.

[0337] Exemplary BCMA CAR D In some embodiments, the BCMA CAR comprises a murine extracellular single-chain variable fragment (scFv) specific for recognition of the B-cell maturation antigen (BCMA), followed in tandem by a human CD8α hinge and transmembrane domain fused to the T-cell cytoplasmic signaling domains of the CD137 (4-1BB) and CD3ζ chains. Binding of the BCMA CAR D to a BCMA-expressing target cell results in signal transduction initiated by the CD3ζ and 4-1BB domains, followed by activation of the CAR-positive T cell. Antigen-specific activation of the BCMA CAR D results in proliferation of the CAR-positive T cell, cytokine secretion, and subsequent cytolytic death of the BCMA-expressing cell. In some embodiments, the BCMA CAR is encoded by a nucleotide sequence in Table 28, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% thereof, or 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In some embodiments, the BCMA CAR comprises a polypeptide encoded by a nucleotide sequence of Table 28, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutation thereto. In some embodiments, the BCMA CAR comprises a polypeptide sequence having a polypeptide sequence of Table 28, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutation thereto. In some embodiments, the BCMA CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of the sequence of Table 28. In some embodiments, the BCMA CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of the sequence of Table 28 according to Kabat. In some embodiments, the BCMA CAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence in Table 28 according to Chothia. In some embodiments, the BCMA CAR comprises heavy chain CDR1-3 of the sequence in Table 28. In some embodiments, the BCMA CAR comprises heavy chain CDR1-3 of the sequence in Table 28 according to Kabat.In some embodiments, the BCMA CAR comprises heavy chain CDR1-3 of a sequence in Table 28 according to Chothia. In some embodiments, the BCMA CAR comprises light chain CDR1-3 of a sequence in Table 28 according to Kabat. In some embodiments, the BCMA CAR comprises light chain CDR1-3 of a sequence in Table 28 according to Chothia.

[0338] [Table 69]

[0339] [Table 70]

[0340] 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-zeta 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 as set forth 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% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 mutation thereto. In certain embodiments, the second anti-BCMA sdAb comprises the CDR1, CDR2 and CDR3 set forth in SEQ ID NO: 381, an amino acid sequence comprising a peptide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutations thereto. 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 their entirety. In some embodiments, the CD19 CAR is encoded by the nucleotide sequence of Table 29, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutations thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence of Table 29, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof, or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutation therein.In some embodiments, the CD19 CAR comprises a polypeptide sequence of Table 29 or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% thereof or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 mutations thereto. In some embodiments, the BCMA CAR comprises a heavy chain CDRl-3 and a light chain CDRl-3 of a sequence in Table 29 according to Kabat. In some embodiments, the BCMA CAR comprises a heavy chain CDRl-3 and a light chain CDRl-3 of a sequence in Table 29 according to Chothia. In some embodiments, the BCMA CAR comprises a heavy chain CDRl-3 of a sequence in Table 29 according to Kabat. In some embodiments, the BCMA CAR comprises a heavy chain CDRl-3 of a sequence in Table 29 according to Kabat. In some embodiments, the BCMA CAR comprises heavy chain CDR1-3 of a sequence in Table 29 according to Chothia. In some embodiments, the BCMA CAR comprises light chain CDR1-3 of a sequence in Table 29 according to Kabat. In some embodiments, the BCMA CAR comprises light chain CDR1-3 of a sequence in Table 29 according to Chothia.

[0341] [Table 71]

[0342] [Table 72]

[0343] [Table 73]

[0344] Other Exemplary Targets Further non-limiting exemplary tumor antigens include CD20, CD22, EGFR, CD123, and CLL-1.

[0345] CARs that bind to CD20 are known in the art. For example, those disclosed in WO 2018 / 067992 or WO 2016 / 164731, which are incorporated herein by reference, can be used in accordance with the present disclosure. Any CD20 CAR known in the art, such as the CD20 antigen-binding domain of any known CD20 CAR, can be used in accordance with the present disclosure. Exemplary CD20 binding sequences or CD20 CAR sequences are disclosed, for example, in Tables 1-5 of WO 2018 / 067992, which are incorporated herein by reference. In some embodiments, the CD20 CAR comprises the CDRs, variable regions, scFv, or full-length sequence of a CD20 CAR disclosed in WO 2018 / 067992 or WO 2016 / 164731, both of which are incorporated herein by reference. In some embodiments, the CD20CAR comprises a sequence disclosed in Table 23 below, such as a CDR, VH, VL, scFv or entire CAR sequence, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0346] Exemplary antigen-binding domains that bind to CD20 are described in WO 2016 / 164731 and WO 2018 / 067992, which are incorporated herein by reference, and in some embodiments are one or more of the CD20 antigen-binding domains disclosed therein.

[0347] Exemplary antigen binding domains that bind to CD22 are described in WO 2016 / 164731 and WO 2018 / 067992, which are incorporated herein by reference.

[0348] In some embodiments, the antigen-binding domain comprises the HC CDR1, HC CDR2, and HC CDR3 of any heavy chain binding domain amino acid sequence listed in Table 15. In embodiments, the antigen-binding domain further comprises the LC CDR1, LC CDR2, and LC CDR3. In embodiments, the antigen-binding domain comprises the LC CDR1, LC CDR2, and LC CDR3 amino acid sequences listed in Table 16.

[0349] In some embodiments, the antigen binding domain comprises one, two, or all of LC CDR1, LC CDR2, and LC CDR3 of any light chain binding domain amino acid sequence listed in Table 16 and one, two, or all of HC CDR1, HC CDR2, HC CDR3 of any heavy chain binding domain amino acid sequence listed in Table 15.

[0350] Exemplary antigen binding domains that bind to EGFRvIII are described in WO 2014 / 130657.

[0351] Exemplary antigen binding domains that bind to CD123 are described in WO 2014 / 130635 and WO 2016 / 028896, which are incorporated herein by reference.

[0352] In some embodiments, the antigen binding domain comprises a sequence from Tables 1-2 of WO 2014 / 130635, which is incorporated herein by reference.

[0353] In some embodiments, the antigen binding domain comprises a sequence from Tables 2, 6, and 9 of WO 2016 / 028896, which is incorporated herein by reference.

[0354] Exemplary antigen-binding domains that bind to CLL-1 are disclosed in WO 2016 / 014535, which is incorporated herein by reference.

[0355] In some embodiments, the antigen-binding domain comprises one, two, or three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3, from an antibody described herein (e.g., an antibody described in WO 2015 / 142675, U.S. Patent Application Publication Nos. 2015-0283178-A1, 2016-0046724-A1, 2014 / 0322212A1, 2016 / 0068601A1, 2016 / 0051651A1, 2016 / 0096892A1, 2014 / 0322275A1, or WO 2015 / 090230, which are incorporated herein by reference). and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3, from an antibody described herein (e.g., an antibody described in WO 2015 / 142675, U.S. Patent Application Publication Nos. 2015-0283178-A1, 2016-0046724-A1, 2014 / 0322212A1, 2016 / 0068601A1, 2016 / 0051651A1, 2016 / 0096892A1, 2014 / 0322275A1, or WO 2015 / 090230, which are incorporated herein by reference). In some embodiments, the antigen binding domain comprises the heavy chain variable region and / or the light chain variable region of an antibody listed above.

[0356] In embodiments, the antigen-binding domain is an antigen-binding domain described in WO 2015 / 142675, U.S. Patent Application Publication Nos. 2015-0283178-A1, 2016-0046724-A1, 2014 / 0322212A1, 2016 / 0068601A1, 2016 / 0051651A1, 2016 / 0096892A1, 2014 / 0322275A1, or WO 2015 / 090230, which are incorporated herein by reference.

[0357] 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, among others, as described in, for example, WO 2014 / 153270, WO 2014 / 130635, WO 2016 / 028896, WO 2014 / 130657, WO 2016 / 014576, WO 2015 / 090230, WO 2016 / 014565, WO 2016 / 014535, and WO 2016 / 025880, each of which is incorporated herein by reference in its entirety.

[0358] 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, three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3, from an antibody listed above and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3, from an antigen binding domain listed above. In some embodiments, the antigen binding domain comprises a heavy chain variable region and / or a variable light chain region of an antibody listed or described above.

[0359] In some embodiments, the antigen-binding domain comprises one, two, three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3, of an antibody listed above and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3, from an antibody listed above. In some embodiments, the antigen-binding domain comprises a heavy chain variable region and / or a variable light chain region of an antibody listed or described above.

[0360] [Table 74]

[0361] Table 75

[0362] Table 76

[0363] Table 77

[0364] Table 78

[0365] Table 79

[0366] Table 80

[0367] Table 81

[0368] Table 82

[0369] Table 83

[0370] Table 84

[0371] Table 85

[0372] Table 86

[0373] Table 87

[0374] Table 88

[0375] Table 89

[0376] Table 90

[0377]

Table 91

[0378] Table 92

[0379] Table 93

[0380] Table 94

[0381]

Table 95

[0382] Table 96

[0383] Table 97

[0384] Table 98

[0385]

Table 99

[0386]

Table 100

[0387] Table 101

[0388] Table 102

[0389] Table 103

[0390] Table 104

[0391] Table 105

[0392] CARs that bind to CD22 are known in the art. For example, those disclosed in WO2018 / 067992 or WO2016 / 164731 can be used in accordance with the present disclosure. Any known CD22 CAR in the art, such as the CD22 antigen-binding domain of any known CD22 CAR, can be used in accordance with the present disclosure.

[0393] 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 WO2016164731 and Tables 6-10 of WO2018067992. In some embodiments, the CD22 CAR sequence comprises the CDRs, variable regions, scFv, or full-length sequence of a CD22 CAR disclosed in WO2018067992 or WO2016164731.

[0394] In some embodiments, the CAR comprises an antigen-binding domain that binds to CD22 (CD22 CAR). In some embodiments, the antigen-binding domain targets human CD22. In some embodiments, the antigen-binding domain comprises a single-chain Fv sequence as described herein.

[0395] The sequence of the 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 (SEQ ID NO: 755)

[0396] In some embodiments, the CD22CAR comprises a sequence disclosed in Tables 15-16 and Table 24 below, such as a CDR, VH, VL, scFv or entire CAR sequence, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0397] [Table 106]

[0398] [Table 107]

[0399] [Table 108]

[0400] [Table 109]

[0401] [Table 110]

[0402] [Table 111]

[0403] [Table 112]

[0404] Table 113

[0405] Table 114

[0406] Table 115

[0407] Table 116

[0408] Table 117

[0409] Table 118

[0410] Table 119

[0411] Table 120

[0412] Table 121

[0413] Table 122

[0414] [Table 123]

[0415] CARs that bind to EGFR are known in the art. For example, those disclosed in WO 2014 / 130657, which is incorporated herein by reference, can be used in accordance with the present disclosure. Any known EGFR CAR in the art, such as the EGFR antigen-binding domain of any known EGFR CAR, can be used in accordance with the present disclosure. An exemplary EGFRvIII CAR can include the CDRs, variable regions, scFv, or full-length CAR sequences disclosed in WO 2014 / 130657, which is incorporated herein by reference, for example, in Table 2 of WO 2014 / 130657.

[0416] CARs that bind to CD123 are known in the art. For example, those disclosed in WO 2014 / 130635 or WO 2016 / 028896 can be used in accordance with the present disclosure. Any known CD123 CAR in the art, for example, the CD123 antigen-binding domain of any known CD123 CAR, can be used in accordance with the present disclosure. For example, CAR1 to CAR8 disclosed in WO 2014 / 130635; or CAR123-1 to CAR123-4 and hzCAR123-1 to hzCAR123-32 disclosed in WO 2016 / 028896. The amino acid and nucleotide sequences encoding the CD123 CAR molecule and antigen binding domain (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2014 / 130635 and WO 2016 / 028896.

[0417] CAR that binds to CLL-1 is known in the art.For example, it is disclosed in US 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, can be used according to the present disclosure.

[0418] In some embodiments, the CAR comprises a CLL-1 CAR or antigen binding domain according to Table 2 of WO 2016 / 014535, which is incorporated herein by reference. Amino acid and nucleotide sequences encoding CLL-1 CAR molecules and antigen binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 014535.

[0419] CARs that bind to CD33 are known in the art. For example, those disclosed in U.S. Patent Application Publication No. 2016 / 0096892A1 and WO 2016 / 014576, which are incorporated herein by reference, can be used in accordance with the present disclosure. Any known CD33 CAR in the art, for example, the CD33 antigen-binding domain of any known CD33 CAR, can be used in accordance with the present disclosure. For example, CAR33-1 to CAR33-9 disclosed in WO 2016 / 014576 can be used in accordance with the present disclosure.

[0420] In some embodiments, the CAR comprises a CD33 CAR or antigen binding domain according to Table 2 or 9 of WO 2016 / 014576, which is incorporated herein by reference. Amino acid and nucleotide sequences encoding CD33 CAR molecules and antigen binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 014576.

[0421] In one embodiment, the antigen binding domain against CD33 is selected from the group consisting of, 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 2012:683065 (2012) and Pizzitola et al., Leukemia doi:10.1038 / Lue.2014.62 (2014). In one embodiment, the antigen-binding domain against CD33 is an antigen-binding portion, e.g., a CDR, of an antibody, antigen-binding fragment, or CAR described in WO 2016 / 014576.

[0422] In one embodiment, the antigen-binding domain against GD2 is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, 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). In some embodiments, the antigen-binding domain against GD2 is an 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 e.g., WO2012033885, WO2013040371, WO2013192294, WO2013061273, WO2013123061, WO2013074916, and WO201385552. In some embodiments, the antigen binding domain against GD2 is an antigen binding portion of an antibody described in US Patent Application Publication No. 20100150910 or WO2011160119.

[0423] In one embodiment, the antigen-binding domain against the Tn antigen is an antigen-binding portion, e.g., a CDR, of an antibody described in, e.g., U.S. Pat. No. 8,440,798, Brooks et al., PNAS 107(22):10056-10061 (2010), and Stone et al., OncoImmunology 1(6):863-873 (2012).

[0424] In one embodiment, the antigen-binding domain against PSMA is the antigen-binding portion, e.g., CDRs, of an antibody and single-chain antibody fragment (scFv A5 and D7) described in, for example, 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); WO 2006125481 (mAbs 3 / A12, 3 / E7, and 3 / F11).

[0425] In one embodiment, an antigen-binding domain against ROR1 is an antigen-binding portion, e.g., CDRs, of an antibody described in, e.g., Hudecek et al., Clin Cancer Res 19(12):3153-3164 (2013); WO2011159847; and U.S. Patent No. 20130101607.

[0426] In one embodiment, the antigen-binding domain against FLT3 is an antigen-binding portion, e.g., a CDR, of an antibody described in, for example, WO2011076922, U.S. Pat. No. 5,777,084, EP0754230, U.S. Pat. No. 20090297529, and several commercially available catalog antibodies (R&D, ebiosciences, Abcam).

[0427] In one embodiment, an antigen binding domain against TAG72 is an antigen binding portion, e.g., CDRs, of an antibody described in, for example, Hombach et al., Gastroenterology 113(4):1163-1170 (1997); and Abcam ab691.

[0428] In one embodiment, an antigen-binding domain against a FAP is an antigen-binding portion, e.g., a CDR, of an antibody described in, e.g., Ostermann et al., Clinical Cancer Research 14:4584-4592(2008) (FAP5), U.S. Patent Application Publication No. 2009 / 0304718; sibrotuzumab (see, e.g., Hofheinz et al., Oncology Research and Treatment 26(1), 2003); and Tran et al., J Exp Med 210(6):1125-1135(2013).

[0429] In one embodiment, the antigen-binding domain against CD38 is an antigen-binding portion, e.g., CDRs, of an antibody described in daratumumab (see, e.g., Groen et al, Blood 116(21):1261-1262 (2010)); MOR202 (see, e.g., U.S. Pat. No. 8,263,746); or U.S. Pat. No. 8,362,211.

[0430] In one embodiment, an antigen binding domain against CD44v6 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Casucci et al., Blood 122(20):3461-3472 (2013).

[0431] In one embodiment, an antigen-binding domain against CEA is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Chmielewski et al., Gastoenterology 143(4):1095-1107 (2012).

[0432] In one embodiment, the antigen-binding domain against EPCAM is an antigen-binding portion, e.g., CDR, of an antibody selected from MT110, an EpCAM-CD3 bispecific Ab (see, e.g., clinicaltrials.gov / ct2 / show / NCT00635596); edrecolomab; 3622W94; ING-1; and adecatumumab (MT201).

[0433] In one embodiment, an antigen-binding domain against PRSS21 is an antigen-binding portion, such as a CDR, of an antibody described in US Pat. No. 8,080,650.

[0434] In one embodiment, an antigen binding domain against B7H3 is an antigen binding portion, eg, CDRs, of the antibody MGA271 (Macrogenics).

[0435] In one embodiment, an antigen binding domain against KIT is an antigen binding portion, e.g., a CDR, of an antibody described in, e.g., U.S. Pat. No. 7,915,391, U.S. Patent Application Publication No. 20120288506, as well as several commercially available catalog antibodies.

[0436] In one embodiment, an antigen binding domain against IL-13Ra2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2008 / 146911, WO2004087758, several commercial catalog antibodies, and WO2004087758.

[0437] In one embodiment, an antigen-binding domain against CD30 is an antigen-binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 7,090,843 B1 and EP 0,805,871.

[0438] In one embodiment, an antigen-binding domain against GD3 is an antigen-binding portion, e.g., CDR, of an antibody described in, e.g., U.S. Pat. Nos. 7,253,263; 8,207,308; U.S. Patent Application Publication No. 20120276046; EP 1013761; WO 2005035577; and U.S. Pat. No. 6,437,098.

[0439] In one embodiment, the antigen-binding domain against CD171 is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Hong et al., J Immunother 37(2):93-104(2014).

[0440] In one embodiment, the antigen-binding domain against IL-11Ra is an antigen-binding portion, e.g., CDR, of an antibody available from Abcam (cat#ab55262) ​​or Novus Biologicals (cat#EPR5446). In another embodiment, the antigen-binding domain against IL-11Ra is a peptide; see, e.g., Huang et al., Cancer Res 72(1):271-281 (2012).

[0441] In one embodiment, the antigen-binding domain against PSCA is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Morgenroth et al., Prostate 67(10):1121-1131(2007) (scFv 7F5); Nejatollahi et al., J of Oncology 2013(2013), Article ID 839831 (scFv C5-II); and U.S. Patent Application Publication No. 20090311181.

[0442] In one embodiment, an antigen-binding domain against VEGFR2 is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Chinnasamy et al., J Clin Invest 120(11):3953-3968(2010).

[0443] In one embodiment, an antigen-binding domain against Lewis Y is an antigen-binding portion, e.g., CDRs, of an antibody described in, e.g., 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).

[0444] In one embodiment, an antigen-binding domain against CD24 is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Maliar et al., Gastroenterology 143(5):1375-1384 (2012).

[0445] In one embodiment, an antigen binding domain against PDGFR-β is an antigen binding portion, eg, CDRs, of the antibody Abeam ab32570.

[0446] In one embodiment, an antigen binding domain against SSEA-4 is the antigen binding portion, eg, CDRs, of the antibody MC813 (Cell Signaling) or other commercially available antibodies.

[0447] In one embodiment, an antigen-binding domain against CD20 is an antigen-binding portion, e.g., CDRs, of the antibody rituximab, ofatumumab, ocrelizumab, veltuzumab, or GA101; or an antibody described in WO 2016 / 164731.

[0448] In one embodiment, an antigen binding domain against folate receptor alpha is an antigen binding portion, e.g., CDRs, of the antibody IMGN853 or the antibodies described in US Patent Application Publication No. 20120009181; US ​​Patent No. 4,851,332; LK26: US Patent No. 5,952,484.

[0449] In one embodiment, the antigen binding domain against ERBB2 (Her2 / neu) is an antigen binding portion, eg, CDRs, of the antibodies: trastuzumab or pertuzumab.

[0450] In one embodiment, an antigen binding domain against MUC1 is an antigen binding portion, e.g., CDRs, of the antibody SAR566658.

[0451] In one embodiment, the antigen binding domain against EGFR is an antigen binding portion, e.g., CDRs, of the antibodies: cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.

[0452] In one embodiment, the antigen-binding domain against NCAM is the antigen-binding portion, e.g., CDRs, of the antibody clone 2-2B:MAB5324 (EMD Millipore).

[0453] In one embodiment, the antigen-binding domain against EphrinB2 is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Abengozar et al., Blood 119(19):4565-4576 (2012).

[0454] In one embodiment, the antigen-binding domain against IGF-I receptor is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, U.S. Pat. No. 8,344,112 B2; European Patent Application Publication No. 2,322,550 A1; WO 2006 / 138315; or PCT / US2006 / 022995.

[0455] In one embodiment, the antigen binding domain against CAIX is the antigen binding portion, eg, CDRs, of antibody clone 303123 (R&D Systems).

[0456] In one embodiment, an antigen binding domain against LMP2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 7,410,640 or U.S. Patent Application Publication No. 20050129701.

[0457] In one embodiment, an antigen-binding domain against gp100 is an antigen-binding portion, e.g., CDRs, of the antibody HMB45, NKIβB, or an antibody described in WO2013165940 or US20130295007.

[0458] In one embodiment, an antigen binding domain against tyrosinase is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 5,843,674; or U.S. Pat. No. 19950504048.

[0459] In one embodiment, the antigen-binding domain against EphA2 is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Yu et al., Mol Ther 22(1):102-111 (2014).

[0460] In one embodiment, an antigen-binding domain against GD3 is an antigen-binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 7,253,263; U.S. Pat. No. 8,207,308; U.S. Patent Application Publication No. 20120276046; European Patent Application Publication No. 1013761 A3; 20120276046; WO 2005035577; or U.S. Pat. No. 6,437,098.

[0461] In one embodiment, an antigen-binding domain against Fucosyl-GM1 is an antigen-binding portion, e.g., CDRs, of an antibody described in, e.g., US Patent Application Publication No. 20100297138; or WO 2007 / 067992.

[0462] In one embodiment, the antigen binding domain against sLe is the antigen binding portion, e.g., CDRs, of the antibody G193 (for Lewis Y) (see Scott AM et al., Cancer Res 60:3254-61 (2000) and as described in Neeson et al., J Immunol May 2013 190 (Meeting Abstract Supplement) 177.10).

[0463] In one embodiment, an antigen-binding domain against GM3 is an antigen-binding portion, e.g., CDRs, of the antibody CA 2523449 (mAb 14F7).

[0464] In one embodiment, an antigen-binding domain against HMWMAA is an antigen-binding portion, e.g., CDRs, of an antibody described in, e.g., Kmiecik et al., Oncoimmunology 3(1):e27185(2014) (PMID:24575382) (mAb9.2.27); U.S. Patent No. 6,528,481; WO 2010033866; or U.S. Patent Application Publication No. 20140004124.

[0465] In one embodiment, an antigen binding domain against o-acetyl-GD2 is an antigen binding portion, eg, CDRs, of the antibody 8B6.

[0466] In one embodiment, the antigen-binding domain against TEM1 / CD248 is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Marty et al., Cancer Lett 235(2):298-308(2006); Zhao et al., J Immunol Methods 363(2):221-232(2011).

[0467] In one embodiment, an antigen binding domain against CLDN6 is an antigen binding portion, e.g., CDRs, of the antibody IMAB027 (Ganymed Pharmaceuticals) (see, e.g., clinicaltrial.gov / show / NCT02054351).

[0468] In one embodiment, an antigen-binding domain against TSHR is an antigen-binding portion, eg, CDRs, of an antibody described in, eg, US Pat. Nos. 8,603,466; 8,501,415; or 8,309,693.

[0469] In one embodiment, an antigen-binding domain against GPRC5D is an antigen-binding portion, eg, CDRs, of the antibody: FAB6300A (R&D Systems); or LS-A4180 (Lifespan Biosciences).

[0470] In one embodiment, an antigen-binding domain against CD97 is an antigen-binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 6,846,911; de Groot et al., J Immunol 183(6):4127-4134 (2009); or an antibody from R&D:MAB3734.

[0471] In one embodiment, an antigen-binding domain against ALK is an antigen-binding portion, e.g., CDR, of an antibody described in, e.g., Mino-Kenudson et al., Clin Cancer Res 16(5):1561-1571 (2010).

[0472] In one embodiment, the antigen-binding domain against polysialic acid is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Nagae et al., J Biol Chem 288(47):33784-33796(2013).

[0473] In one embodiment, an antigen-binding domain against PLAC1 is an antigen-binding portion, e.g., CDR, of an antibody described in, for example, Ghods et al., Biotechnol Appl Biochem 2013 doi:10.1002 / bab.1177.

[0474] In one embodiment, the antigen-binding domain against GloboH is the antigen-binding portion of the antibody VK9; or an antibody described, for example, in 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).

[0475] In one embodiment, an antigen-binding domain against NY-BR-1 is an antigen-binding portion, such as the CDRs, of an antibody described in Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007).

[0476] In one embodiment, the antigen-binding domain against WT-1 is an antigen-binding portion, e.g., CDRs, of an antibody described in, for example, Dao et al., Sci Transl Med 5(176):176ra33(2013); or WO 2012 / 135854.

[0477] In one embodiment, an antigen-binding domain against MAGE-A1 is an antigen-binding portion, e.g., CDR, of an antibody described in, e.g., Willemsen et al., J Immunol 174(12):7853-7858(2005) (TCR-like scFv).

[0478] In one embodiment, the antigen-binding domain against sperm protein 17 is an antigen-binding portion, e.g., a CDR, of an antibody described in, for example, Song et al., Target Oncol 2013 Aug 14 (PMID: 23943313); Song et al., Med Oncol 29(4):2923-2931 (2012).

[0479] In one embodiment, the antigen binding domain against Tie2 is an antigen binding portion, e.g., CDRs, of the antibody AB33 (Cell Signaling Technology).

[0480] In one embodiment, an antigen-binding domain against MAD-CT-2 is an antigen-binding portion, such as a CDR, of an antibody described in, for example, PMID: 2450952; US Pat. No. 7,635,753.

[0481] In one embodiment, an antigen-binding domain against Fos-related antigen 1 is an antigen-binding portion, e.g., CDRs, of antibody 12F9 (Novus Biologicals).

[0482] In one embodiment, an antigen-binding domain against MelanA / MART1 is an antigen-binding portion, e.g., CDRs, of an antibody described in EP 2514766A2; or U.S. Pat. No. 7,749,719.

[0483] In one embodiment, the antigen-binding domain against a sarcoma translocation breakpoint is an antigen-binding portion, e.g., a CDR, of an antibody described in, for example, Luo et al., EMBO Mol. Med. 4(6):453-461 (2012).

[0484] In one embodiment, an antigen-binding domain against TRP-2 is an antigen-binding portion, e.g., CDRs, of an antibody described in, e.g., Wang et al., J Exp Med. 184(6):2207-16 (1996).

[0485] In one embodiment, an antigen-binding domain against CYP1B1 is an antigen-binding portion, e.g., CDRs, of an antibody described in, for example, Maecker et al., Blood 102(9):3287-3294 (2003).

[0486] In one embodiment, the antigen binding domain against RAGE-1 is the antigen binding portion, eg, CDRs, of the antibody: MAB5328 (EMD Millipore).

[0487] In one embodiment, an antigen binding domain against human telomerase reverse transcriptase is an antigen binding portion, e.g., CDRs, of the antibody: cat no: LS-B95-100 (Lifespan Biosciences).

[0488] In one embodiment, an antigen binding domain against intestinal carboxylesterase is an antigen binding portion, e.g., CDRs, of the antibody: 4F12: cat no: LS-B6190-50 (Lifespan Biosciences).

[0489] In one embodiment, an antigen-binding domain against mut hsp70-2 is an antigen-binding portion, e.g., CDRs, of the antibody: Lifespan Biosciences: Monoclonal: cat no: LS-C133261-100 (Lifespan Biosciences).

[0490] In one embodiment, an antigen-binding domain against CD79a is an antigen-binding portion, e.g., CDRs, of the following antibodies: anti-CD79a antibody [HM47 / A9] (ab3121), available from Abcam; CD79A antibody #3351, available from Cell Signalling Technology; or antibody HPA017748 (an anti-CD79A antibody produced in rabbit), available from Sigma Aldrich.

[0491] In one embodiment, the antigen binding domain against CD79b is selected from the group consisting of the following antibodies: polatuzumab vedotin, anti-CD79b, from Dornan et al., “Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma” Blood. 2009 Sep 24;114(13):2721-9. doi:10.1182 / blood-2009-02-205500. Epub 2009 Jul 24; 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 The antigen-binding portion, e.g., CDR, of the bispecific antibody Anti-CD79b / CD3 described in 2014 is an antigen-binding portion, e.g., CDR.

[0492] In one embodiment, the antigen-binding domain against CD72 is an antigen-binding portion, e.g., CDRs, of the following antibodies: J3-109 described in Myers and Uckun, "An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia," Leuk Lymphoma. 1995 June;18(1-2):119-22; or 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.

[0493] In one embodiment, an antigen-binding domain against LAIR1 is an antigen-binding portion, e.g., CDRs, of the following antibodies: ANT-301 LAIR1 antibody available from ProSpec; or anti-human CD305 (LAIR1) antibody available from BioLegend.

[0494] In one embodiment, the antigen-binding domain against FCAR is an antigen-binding portion, e.g., CDRs, of the antibody CD89 / FCAR Antibody available from Sino Biological Inc. (catalog #10414-H08H).

[0495] In one embodiment, an antigen binding domain against LILRA2 is an antigen binding portion, e.g., CDRs, of the following antibodies: LILRA2 monoclonal antibody (M17), clone 3C7, available from Abnova, or murine anti-LILRA2 antibody, monoclonal (2D7), available from Lifespan Biosciences.

[0496] In one embodiment, the antigen-binding domain against CD300LF is an antigen-binding portion, e.g., CDRs, 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.

[0497] In one embodiment, the antigen-binding domain against CLEC12A is an antigen-binding portion, e.g., CDR, of the following antibodies: a bispecific T cell derivative (BiTE) scFv-antibody and an 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, and MCLA-117 (Merus).

[0498] In one embodiment, the antigen-binding domain against BST2 (also referred to as CD317) is the antigen-binding portion, e.g., CDRs, of the following antibodies: murine anti-CD317 antibody, monoclonal [3H4] available from Antibodies-Online or murine anti-CD317 antibody, monoclonal [696739] available from R&D Systems.

[0499] In one embodiment, the antigen binding domain against EMR2 (also referred to as CD312) is the antigen binding portion, e.g., CDRs, 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.

[0500] In one embodiment, an antigen binding domain against LY75 is an antigen binding portion, e.g., CDRs, 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.

[0501] In one embodiment, the antigen-binding domain against GPC3 is an antigen-binding portion, e.g., CDR, of the following antibodies: hGC33, which is 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, or MDX-1414, HN3, or YP7 (all three of which 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).

[0502] In one embodiment, the antigen-binding domain against FCRL5 is an antigen-binding portion, e.g., CDR, of the 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. In one embodiment, the antigen-binding domain against FCRL5 is an antigen-binding portion, e.g., CDR, of the anti-FcRL5 antibody described in, for example, WO 2001 / 038490, WO 2005 / 117986, WO 2006 / 039238, WO 2006 / 076691, WO 2010 / 114940, WO 2010 / 120561, or WO 2014 / 210064.

[0503] In one embodiment, the antigen binding domain against IGLL1 is an antigen binding portion, e.g., CDRs, of mouse anti-immunoglobulin lambda-like polypeptide 1 antibody, monoclonal [AT1G4] available from Lifespan Biosciences, or mouse anti-immunoglobulin lambda-like polypeptide 1 antibody, monoclonal [HSL11] available from BioLegend.

[0504] In one embodiment, the antigen-binding domain comprises one, two, three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3, from an antibody listed above, and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3, from an antibody listed above. In one embodiment, the antigen-binding domain comprises the heavy chain variable region and / or the variable light chain region of an antibody listed above.

[0505] In another aspect, the antigen-binding domain comprises a humanized antibody or antibody fragment. In some aspects, a non-human antibody is humanized, whereby specific sequences or regions of the antibody are modified to increase similarity to antibodies or fragments thereof naturally produced in humans. In one aspect, the antigen-binding domain is humanized.

[0506] CARs that bind to mesothelin are known in the art, such as those disclosed in WO2015090230 and WO2017112741, which are incorporated herein by reference, that bind to human mesothelin, e.g., Tables 2, 3, 4, and 5 of WO2017112741. Any mesothelin CAR known in the art, e.g., the mesothelin antigen-binding domain of any known mesothelin CAR, can be used in accordance with the present disclosure.

[0507] CARs that bind to GFR ALPHA-4 are known in the art. For example, those disclosed in WO 2016 / 025880 can be used in accordance with the present disclosure. Any known GFR ALPHA-4 CAR in the art, such as the GFR ALPHA-4 antigen-binding domain of any known GFR ALPHA-4 CAR, can be used in accordance with the present disclosure. Amino acid and nucleotide sequences encoding GFR ALPHA-4 CAR molecules and antigen-binding domains (e.g., comprising one, two, or three VH CDRs and one, two, or three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 025880.

[0508] Antigen-binding domain structure In some embodiments, the antigen binding domain of the encoded CAR molecule comprises 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)).

[0509] In some instances, scFvs 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 VH and VL domains together using a flexible polypeptide linker. The scFv molecule contains a linker (e.g., a Ser-Gly linker) with optimal length and / or amino acid composition. Linker length can greatly affect how the variable regions of an scFv fold and interact. In fact, when short polypeptide linkers are used (e.g., 5-10 amino acids), intrachain folding is prevented. Intrachain folding is also required for the two variable regions to unite and form a functional epitope-binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and WO 2006 / 020258 and WO 2007 / 024715, which are incorporated herein by reference.

[0510] An scFv can include a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence can include any naturally occurring amino acid. In some embodiments, the linker sequence includes the amino acids glycine and serine. In another embodiment, the linker sequence includes a series of glycine and serine repeats, such as (Gly4Ser)n, where n is a positive integer of 1 or more (SEQ ID NO: 22). In some embodiments, the linker can be (Gly4Ser)4 (SEQ ID NO: 29) or (Gly4Ser)3 (SEQ ID NO: 30). Varying the linker length can maintain or enhance activity, resulting in superior efficacy in activity tests.

[0511] 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 generating 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) (references incorporated herein in their entireties). For example, scTCRs can be engineered that contain Vα and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is highly useful for cancer-associated targets that are themselves intracellular, but fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC.

[0512] In certain embodiments, the encoded antigen binding domain is -4 M~10 -8 It has a binding affinity KD of M.

[0513] In some embodiments, the encoded CAR molecule is 10 -4 M~10 -8 M, e.g. 10 -5 M~10 -7 M, e.g. 10 -6 M or 10 -7 Binding affinity K of M D In some embodiments, the antigen-binding domain has a binding affinity that is at least 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or 1000-fold less than a reference antibody, e.g., an antibody described herein. In some embodiments, the encoded antigen-binding domain has a binding affinity that is at least 5-fold less than the reference antibody (e.g., the antibody from which the antigen-binding domain is derived). In some aspects, such antibody fragments are functional in that they provide a biological response, including, but not limited to, immune response activation, inhibition of signaling initiation from its target antigen, as will be understood by one of skill in the art.

[0514] In some embodiments, the antigen binding domain of the CAR is an scFv antibody fragment that is humanized relative to the murine sequence of the scFv from which it is derived.

[0515] In some embodiments, the antigen-binding domain (e.g., scFv) of a CAR described herein is encoded by a nucleic acid molecule whose sequence has been codon-optimized for expression in mammalian cells. In some embodiments, the entire CAR construct is encoded by a nucleic acid molecule whose entire sequence has been codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons that encode the same amino acid) in coding DNA is biased across different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Various codon optimization methods are known in the art, including, for example, those disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

[0516] Particular antigen-antibody pairs are known in the art. Non-limiting exemplary embodiments of antigen-antibody pairs and their components are provided herein above in the section entitled "Target" and below.

[0517] Bispecific CAR In certain embodiments, the antigen-binding domain is a bi- or multispecific molecule (e.g., a multispecific antibody molecule). In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or 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, a bispecific antibody molecule comprises heavy chain and light chain variable domain sequences that have binding specificity for a first epitope and heavy chain and light chain variable domain sequences that have binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody that has binding specificity for a first epitope and a half antibody that has binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody or fragment thereof that has binding specificity for a first epitope and a half antibody or fragment thereof that has binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises an scFv or fragment thereof that has binding specificity for a first epitope and an scFv or fragment thereof that has binding specificity for a second epitope.

[0518] In some embodiments, the antibody molecule is a multispecific (e.g., bispecific or trispecific) antibody molecule. Such molecules include, for example, bispecific fusion proteins, such as expression constructs comprising two scFvs with a hydrophilic helical peptide linker between them and a complete constant region, e.g., as described in U.S. Pat. No. 5,637,481; minibody constructs with linked VL and VH chains further linked by a peptide spacer to the antibody hinge and CH3 regions that can dimerize to form bispecific / multivalent molecules, e.g., as described in U.S. Pat. No. 5,837,821; and antibody constructs with linked VL and VH chains further linked by a peptide spacer to the antibody hinge and CH3 regions that can dimerize to form bispecific / multivalent molecules, such as those described in U.S. Pat. No. 5,837,821; and single-chain binding polypeptides having both VH and VL domains linked by a peptide linker combined into multivalent structures by non-covalent or chemical cross-linking, using both scFV or bispecific antibody-type formats to form, for example, homobivalent, heterobivalent, trivalent, and tetravalent structures, such as those described in U.S. Pat. No. 5,869,620. The contents of the above-referenced applications are incorporated herein by reference in their entireties.

[0519] Within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH1) upstream of its VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall bispecific antibody molecule has the configuration VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL1) upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the configuration VL1-VH1-VH2-VL2. Optionally, a linker is placed between two antibodies or antibody fragments (e.g., scFvs), for example, between VL1 and VL2 when the construct is arranged as VH1-VL1-VL2-VH2, or between VH1 and VH2 when the construct is arranged as VL1-VH1-VH2-VL2. The linker can be a linker 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). Generally, the linker between two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. Optionally, the linker is placed between the VL and VH of the first scFv. Optionally, the linker is placed between the VL and VH of the second scFv. In constructs with multiple linkers, any two or more of the linkers can be the same or different. Thus, in some embodiments, the bispecific CAR comprises a VL, a VH and optionally one or more linkers in an arrangement as described herein.

[0520] 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 connects to the extracellular domain of the chimeric molecule. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In some embodiments, the transmembrane domain is associated with one of the other domains of the chimeric protein (e.g., CAR); for example, in some embodiments, the transmembrane domain can be derived from the same protein from which the signaling domain, costimulatory domain, or hinge domain is derived. In another embodiment, the transmembrane domain is not derived from the same protein from which any other domain of the chimeric protein (e.g., CAR) is derived. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In some embodiments, the transmembrane domain can homodimerize with another CAR on the cell surface of a CAR-expressing cell. In a different embodiment, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interactions with the binding domain of a natural binding partner present on the same CAR-expressing cell.

[0521] The transmembrane domain can be derived from natural or recombinant sources. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain can transmit a signal to the intracellular domain whenever the CAR binds to the target. Particularly useful transmembrane domains in the present invention can include at least the transmembrane region of, for example, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain is selected from the group consisting of, 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, ITG It may comprise at least the transmembrane domain of AM, 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.

[0522] In some examples, the transmembrane domain can be attached 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 can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge or an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge. In some embodiments, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 4. In some aspects, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO: 12.

[0523] In some embodiments, the encoded transmembrane domain comprises the amino acid sequence of a CD8 transmembrane domain having at least one, two, or three modifications but not more than 20, 10, or 5 modifications to the amino acid sequence of SEQ ID NO: 12, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the encoded transmembrane domain comprises the sequence of SEQ ID NO: 12.

[0524] In other embodiments, the nucleic acid encoding the CAR molecule comprises a nucleotide sequence of a CD8 transmembrane domain comprising, for example, the sequence of SEQ ID NO: 13 or a sequence having 95-99% identity thereto.

[0525] In some embodiments, the encoded antigen-binding domain is connected to the transmembrane domain by a hinge region. In some embodiments, the encoded hinge region comprises the amino acid sequence of a CD8 hinge, e.g., SEQ ID NO: 4; or the amino acid sequence of an IgG4 hinge, e.g., SEQ ID NO: 6, or a sequence having 95 to 99% identity to SEQ ID NO: 4 or 6. In other embodiments, the nucleic acid sequence encoding the hinge region comprises the sequence of SEQ ID NO: 5 or SEQ ID NO: 7, or a sequence having 95 to 99% identity to SEQ ID NO: 5 or 7, corresponding to a CD8 hinge or an IgG4 hinge, respectively.

[0526] In some embodiments, the hinge or spacer comprises an IgG4 hinge. For example, in some embodiments, the hinge or spacer comprises the amino acid sequence [ka] In some embodiments, the hinge or spacer comprises a hinge of: [ka] and a hinge encoded by the nucleotide sequence of

[0527] In some embodiments, the hinge or spacer comprises an IgD hinge. For example, in some embodiments, the hinge or spacer comprises the amino acid sequence [ka] In some embodiments, the hinge or spacer comprises a hinge of: [ka] and a hinge encoded by the nucleotide sequence of

[0528] In some embodiments, the transmembrane domain can be recombinant, in which case it contains predominantly hydrophobic residues such as leucine and valine, hi some embodiments, triplets of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.

[0529] Optionally, a short oligo- or polypeptide linker, 2-10 amino acids in length, can form the bond between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. 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.

[0530] In some embodiments, the hinge or spacer comprises a KIR2DS2 hinge.

[0531] Signaling domains In embodiments of the invention having an intracellular signaling domain, such domain can comprise, for example, one or more of a primary signaling domain and / or a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a sequence encoding a primary signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain.

[0532] The intracellular signaling sequences within the cytoplasmic portion of the CAR of the present invention can be linked to each other randomly or in a specific order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length, can form the linkage 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, e.g., alanine, glycine, can be used as a suitable linker.

[0533] In some embodiments, the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In some embodiments, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule described herein. In some embodiments, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0534] Primary signaling domain The primary signaling domain controls the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. In CARs, such domains are used for the same purpose.

[0535] Examples of ITAM-containing primary intracellular signaling domains that are particularly useful in the present invention include those of CD3ζ, common FcRγ (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In some embodiments, a CAR of the invention comprises an intracellular signaling domain, e.g., the primary signaling domain of CD3ζ.

[0536] In some embodiments, the encoded primary signaling domain comprises a functional signaling domain of CD3ζ. The encoded CD3ζ primary signaling domain can comprise an amino acid sequence having at least one, two, or three modifications but not more than 20, 10, or 5 modifications to SEQ ID NO: 18 or SEQ ID NO: 20, or a sequence with 95-99% identity to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, the encoded primary signaling domain comprises the sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In other embodiments, the nucleic acid sequence encoding the primary signaling domain comprises the sequence of SEQ ID NO: 19 or SEQ ID NO: 21, or a sequence with 95-99% identity thereto.

[0537] Costimulatory signaling domain In some embodiments, the encoded intracellular signaling domain comprises a costimulatory signaling domain. For example, the intracellular signaling domain can comprise a primary signaling domain and a costimulatory signaling domain. In some embodiments, the encoded costimulatory signaling domain is selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, 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, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD22 6), 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.

[0538] In some embodiments, the encoded costimulatory signaling domain comprises an amino acid sequence having at least one, two, or three modifications but not more than 20, 10, or 5 modifications to SEQ ID NO: 14 or SEQ ID NO: 16, or a sequence with 95-99% identity to SEQ ID NO: 14 or SEQ ID NO: 16. In some embodiments, the encoded costimulatory signaling domain comprises the sequence of SEQ ID NO: 14 or SEQ ID NO: 16. In other embodiments, the nucleic acid sequence encoding the costimulatory signaling domain comprises the sequence of SEQ ID NO: 15 or SEQ ID NO: 17, or a sequence with 95-99% identity thereto.

[0539] In other embodiments, the encoded intracellular domain comprises 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 comprising these intracellular signaling domains are expressed in the same frame and as a single polypeptide chain.

[0540] In some embodiments, the nucleic acid sequence encoding the intracellular signaling domain comprises the sequence of SEQ ID NO: 15 or SEQ ID NO: 17, or a sequence having 95 to 99% identity thereto, and the sequence of SEQ ID NO: 19 or SEQ ID NO: 21, or a sequence having 95 to 99% identity thereto.

[0541] In some embodiments, the nucleic acid molecule further encodes a leader sequence. In some embodiments, the leader sequence comprises the sequence of SEQ ID NO:2.

[0542] In some embodiments, the intracellular signaling domain is designed to comprise the signaling domain of CD3ζ and the signaling domain of CD28. In some embodiments, the intracellular signaling domain is designed to comprise the signaling domain of CD3ζ and the signaling domain of 4-1BB. In some embodiments, the signaling domain of 4-1BB is the signaling domain of SEQ ID NO: 14. In some embodiments, the signaling domain of CD3ζ is the signaling domain of SEQ ID NO: 18.

[0543] In some embodiments, the intracellular signaling domain is designed to comprise the signaling domain of CD3ζ and the signaling domain of CD27. In some embodiments, the signaling domain of CD27 comprises the amino acid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 16). In some embodiments, the signaling domain of CD27 comprises the amino acid sequence of [ka] It is encoded by the nucleic acid sequence of

[0544] Inhibitory domain In some embodiments, the vector comprises a nucleic acid sequence encoding a CAR, e.g., a CAR described herein, and a nucleic acid sequence encoding an inhibitory molecule comprising an inhKIR cytoplasmic domain; a transmembrane domain, e.g., a KIR transmembrane domain; and an inhibitor cytoplasmic domain, e.g., an ITIM domain, e.g., an inhKIR ITIM domain. In some embodiments, the inhibitory 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.

[0545] In some embodiments, the nucleic acid sequence encoding the inhibitory molecule comprises a SLAM family cytoplasmic domain; a transmembrane domain, e.g., a SLAM family transmembrane domain; and an inhibitor 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 is 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.

[0546] In some embodiments, the vector is an in vitro transcription vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. In some embodiments, the nucleic acid sequence of the vector further comprises a poly(A) tail, e.g., a polyA tail. In some embodiments, the nucleic acid sequence of the vector further comprises a 3'UTR, e.g., a 3'UTR comprising at least one repeat of a 3'UTR derived from, e.g., human β-globulin, e.g., as described herein. In some embodiments, the nucleic acid sequence of the vector further comprises a promoter, e.g., a T2A promoter.

[0547] promoter In some embodiments, the vector further comprises a promoter. In some embodiments, the promoter is selected from the group consisting of an EF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, a ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter. In some embodiments, the promoter is an EF-1 promoter. In some embodiments, the EF-1 promoter comprises the sequence of SEQ ID NO: 1.

[0548] In some embodiments of the present invention, immune effector cells, such as T cells, can be obtained from a unit of blood collected from a subject using any technique known to those skilled in the art, such as Ficoll™ separation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains T cells, monocytes, granulocytes, B cells, lymphocytes, including other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction, and the cells can optionally be suspended in a buffer or medium for subsequent processing. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, may lack magnesium, or may lack many, if not all, divalent cations.

[0549] [Table 124]

[0550] [Table 125]

[0551] [Table 126]

[0552] [Table 127]

[0553] [Table 128]

[0554] [Table 129]

[0555] Manufacturing method The lentiviral vectors described herein (e.g., those generated using the methods described herein) can be used, for example, for the in vitro production of CAR-T cells.

[0556] The CARTs disclosed herein can be produced ex vivo by any method known in the art. For example, the methods described in WO 2012 / 079000 or WO 2020 / 047452 (both of which are incorporated herein by reference) can be used. The CARTs disclosed herein can also be produced in vivo by any method known in the art. For example, the methods described in WO 2020 / 176397 (both of which are incorporated herein by reference). Immune effector cells (e.g., T cells or NK cells) can express one CAR or two or more CARs.

[0557] In some embodiments, the methods disclosed herein can produce immune effector cells engineered to express one or more CARs in less than 24 hours. Without 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 an undifferentiated phenotype may persist longer and / or expand better in vivo after infusion. 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.

[0558] In some embodiments, CART cells produced by the manufacturing methods provided herein comprise a higher percentage of effector T cells compared to CART cells produced by conventional manufacturing processes, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein better preserve T cell stemness compared to CART cells produced by conventional manufacturing processes, e.g., as measured 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, e.g., as measured 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, e.g., as measured using scRNA-seq. In some embodiments, immune effector cells are engineered to contain nucleic acid molecules encoding one or more CARs disclosed herein.

[0559] 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 include a de-beading step. In some embodiments, CART cells produced by the methods disclosed herein can be administered to a subject with minimal ex vivo expansion, 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 no ex vivo expansion. Thus, the methods described herein provide a high-throughput manufacturing process for producing improved CAR-expressing cell products for use in treating disease in a subject.

[0560] In some embodiments, the present disclosure provides a method of producing a population of cells (e.g., T cells) that express 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 a frozen or fresh leukapheresis product) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface; (ii) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR, thereby providing a population of cells (e.g., T cells) that comprises the nucleic acid molecule; and (iii) harvesting the 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) can be performed simultaneously with step (i) or within 20 hours after initiation of step (i), e.g., within 12, 13, 14, 15, 16, 17, or 18 ... (b) step (ii) is carried out 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 within 18 hours after the start of step (i), and step (iii) is carried out within 26 hours after the start of step (i), for example within 22, 23 or 24 hours after the start of step (i), for example within 24 hours after the start of step (i); and step (iii) is performed within 30 hours after the initiation of step (ii), e.g., within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the initiation of step (ii); or (c) the population of cells from step (iii) has not proliferated or has proliferated by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, e.g., no more than 10%, relative to the population of cells at the initiation of step (i), e.g., as assessed by the number of viable cells. 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 present on a viral vector, e.g., a viral vector selected from a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule of step (ii) is on a non-viral vector. In some embodiments, the nucleic acid molecule of step (ii) is present on a plasmid. In some embodiments, the nucleic acid molecule of step (ii) is not present on any vector. In some embodiments, step (ii) comprises transducing a population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule encoding a CAR.

[0561] In some embodiments, the population of cells (e.g., T cells) is collected from an apheresis sample (e.g., a leukapheresis sample) from a subject.

[0562] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and transported as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The frozen apheresis sample is then thawed, and T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorter (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then seeded for CART manufacturing 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-thawing before seeding for CART manufacturing.

[0563] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and shipped 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® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then seeded for CART manufacturing 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-thawing before seeding for CART manufacturing.

[0564] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject. T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorter (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then transported as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then thawed and seeded for CART manufacturing using the activation process described herein.

[0565] In some embodiments, cells (e.g., T cells) are contacted with anti-CD3 and anti-CD28 antibodies for, e.g., 12 hours, and then transduced (e.g., one or more CARs) with a vector (e.g., a lentiviral vector) encoding a CAR (e.g., one or more vectors). After 24 hours of culture, the cells are washed and formulated for storage or administration.

[0566] Without wishing to be bound by theory, brief CD3 and CD28 stimulation may promote efficient transduction of self-renewing T cells. Compared to conventional CART manufacturing approaches, the activation process provided herein does not involve prolonged ex vivo expansion. Similar to cytokine processes, the activation process provided herein also preserves undifferentiated T cells during CART manufacturing.

[0567] In some embodiments, the population of cells is contacted with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface.

[0568] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28. In some embodiments, the agent that stimulates the CD3 / TCR complex is selected from an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a natural ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the agent that stimulates the CD3 / TCR complex is an antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex is an anti-CD3 antibody. In some embodiments, the agent that stimulates a costimulatory molecule is selected from an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a natural ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the agent that stimulates a costimulatory molecule is an antibody. In some embodiments, the agent that stimulates a costimulatory molecule is an anti-CD28 antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule does not comprise beads. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently bound to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™.

[0569] In some embodiments, the matrix comprises or is composed of a polymer, e.g., a biodegradable or biocompatible inert material, e.g., these materials are non-toxic to cells. In some embodiments, the matrix is ​​composed of hydrophilic polymer chains, which achieve maximum mobility in aqueous solution through chain hydration. In some embodiments, the mobile matrix can be composed of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. Polysaccharides can include, for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan, guar gum, or alginate. Other polymers include polyesters, polyethers, polyacrylates, polyacrylamides, polyamines, polyethyleneimines, polyquaternium polymers, polyphosphazenes, polyvinyl alcohols, polyvinyl acetates, polyvinylpyrrolidones, block copolymers, or polyurethanes. In some embodiments, the mobile matrix is ​​a polymer of dextran.

[0570] In some embodiments, the population of cells is contacted with a nucleic acid molecule (e.g., one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, the population of cells is transduced with a DNA molecule (e.g., one or more DNA molecules) encoding a CAR (e.g., one or more CARs).

[0571] In some embodiments, in the case of co-transduction of two nucleic acid molecules (e.g., lentiviral vectors), each encoding a CAR disclosed herein, each of the vectors comprising the nucleic acid molecule encoding a CAR can be added to a reaction mixture (e.g., comprising a cell population) at a different multiplicity of infection (MOI).

[0572] Without wishing to be bound by theory, it is believed that in some embodiments, using different MOIs for vectors containing nucleic acid molecules encoding different CAR molecules can affect the final composition of the cell population. For example, in the case of co-transduction of a lentiviral vector encoding one CAR with a lentiviral vector encoding another CAR targeting a different target, different MOIs can be used to maximize the percentage of desirable mono-CART and dual-CART cells while simultaneously reducing undesirable mono-CART and non-transduced cells.

[0573] 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 batch of viral vector, the characteristics of the cells being transduced, and transduction efficiency. In some embodiments, the step of contacting the population of cells with the nucleic acid molecule encoding a CAR is performed simultaneously with the step of contacting the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface, as described above. In some embodiments, the contacting of the population of cells with the nucleic acid molecule encoding a CAR 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 initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface, as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 20 hours after initiating contact of the cell population with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 19 hours after initiating contact of the cell population with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 18 hours after initiating contact of the cell population with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 17 hours after initiating contact of the cell population with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above.

[0574] In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 16 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 15 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 14 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 14 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 13 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 12 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 11 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 10 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 9 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface, as previously described.In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 8 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 7 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 6 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 5 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 4 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 3 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 2 hours after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 1 hour after initiating contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding a CAR is performed within 30 minutes of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface, as previously described.

[0575] In some embodiments, the population of cells is harvested for storage or administration.

[0576] In some embodiments, the population of cells is harvested for storage or administration within 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after initiation of contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface. In some embodiments, the population of cells is harvested for storage or administration within 26 hours after initiation of contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface. In some embodiments, the population of cells is harvested for storage or administration within 25 hours after initiation of contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface. In some embodiments, the population of cells is harvested for storage or administration within 24 hours after initiation of contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface. In some embodiments, the population of cells is harvested for storage or administration within 23 hours after initiation of contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface. In some embodiments, the population of cells is harvested for storage or administration within 22 hours after initiation of contact of the population of cells with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the cell surface.

[0577] In some embodiments, the population of cells is not expanded ex vivo.

[0578] In some embodiments, the population of cells is expanded by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 45, 50, 55, or 60% compared to the population of cells prior to contact with the aforementioned agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface, e.g., as assessed by the number of viable cells. In some embodiments, the population of cells is expanded by no more than 5% compared to the population of cells prior to contact with the aforementioned agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface, e.g., as assessed by the number of viable cells. In some embodiments, the population of cells is expanded by no more than 10% compared to the population of cells prior to contact with the aforementioned agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface, e.g., as assessed by the number of viable cells. In some embodiments, the population of cells is expanded by no more than 15% compared to the population of cells prior to contact with the aforementioned agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface, e.g., as assessed by the number of viable cells. In some embodiments, the population of cells is expanded by no more than 20% compared to the population of cells prior to contact with the aforementioned agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface, e.g., as assessed by the number of viable cells. In some embodiments, the population of cells is expanded by no more than 25% compared to the population of cells prior to contact with the aforementioned agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface, e.g., as assessed by the number of viable cells. In some embodiments, the population of cells is expanded by no more than 30% compared to the population of cells prior to contact with the aforementioned agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface, e.g., as assessed by the number of viable cells. In some embodiments, the population of cells is expanded by no more than 35% compared to the population of cells prior to contact with the aforementioned agents that stimulate the CD3 / TCR complex and / or agents that stimulate costimulatory molecules on the cell surface, as assessed, for example, by the number of viable cells.In some embodiments, the population of cells is expanded by no more than 40% compared to the population of cells prior to contact with the aforementioned agents that stimulate the CD3 / TCR complex and / or agents that stimulate costimulatory molecules on the cell surface, as assessed, for example, by the number of viable cells.

[0579] In some embodiments, the population of cells is expanded for no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours compared to the population of cells prior to contact with one or more of the aforementioned cytokines, e.g., as assessed by the number of viable cells.

[0580] In some embodiments, the activation process is carried out in a serum-free cell culture medium. In some embodiments, the activation process is carried out in a cell culture medium containing one or more cytokines selected from IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), or IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, hetIL-15 is [ka] In some embodiments, hetIL-15 comprises an amino acid sequence having at least about 70, 75, 80, 85, 90, 95, or 99% identity to SEQ ID NO:309.

[0581] In some embodiments, the activation process is carried out in a cell culture medium containing an LSD1 inhibitor.

[0582] In some embodiments, the activation process is carried out in a cell culture medium containing a MALT1 inhibitor. In some embodiments, the serum-free cell culture medium contains a serum replacement. In some embodiments, the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR). In some embodiments, the level of ICSR can be, for example, up to 5%, such as about 1%, 2%, 3%, 4%, or 5%. Without wishing to be bound by theory, the use of a cell culture medium containing ICSR, for example, 2% ICSR, such as the Rapid Media shown in Table 21 or Table 25, can improve cell viability during the manufacturing processes described herein.

[0583] In some embodiments, the present disclosure provides a method of generating a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), comprising: (a) providing an apheresis sample (e.g., a fresh or cryopreserved leukapheresis sample) collected from a subject; (b) selecting T cells from the apheresis sample (e.g., using negative selection, positive selection, or beadless selection); (c) selecting the isolated T cells from a population of cells, e.g., 1 x 10 6 ~1×10 7 (d) contacting the T cells with an agent that stimulates the T cells, e.g., an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the cell surface (e.g., contacting the T cells with an anti-CD3 and / or anti-CD28 antibody, e.g., contacting the T cells with TransAct); (e) contacting the T cells with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR, e.g., for 6 to 48 hours, e.g., for 20 to 28 hours (e.g., contacting the T cells with a virus comprising a nucleic acid molecule encoding a CAR); and (f) washing and harvesting the T cells for storage (e.g., reconstitution of the T cells in cryopreservation medium) or administration. In some embodiments, step (f) is performed within 30 hours after the initiation of step (d) or (e), e.g., within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the initiation of step (d) or (e).

[0584] In some embodiments, provided herein are populations of cells (e.g., immune effector cells, e.g., T cells or NK cells) produced by any of the manufacturing processes described herein.

[0585] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of a cytokine process or activation process described herein) is (1) the same, (2) differs by no more than, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, or (3) is increased, e.g., by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells at the start of the manufacturing process (e.g., at the start of a cytokine process or activation process described herein). In some embodiments, the population of cells at the end of the manufacturing process (e.g., at the end of a cytokine process or activation process described herein) persists for, e.g., more than 26 hours (e.g., persists for more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or exhibits a higher (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% higher) percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, compared to cells made by a similar method but including expanding the population of cells in vitro, e.g., for 3 days or more (e.g., expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0586] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of the cytokine process or activation process described herein) is 20, 25, 30, 35, 40, 45, 50, 55, or 60% or greater.

[0587] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of a cytokine process or activation process described herein) is (1) the same, (2) differs by no more than, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, or (3) is reduced, e.g., by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of the manufacturing process (e.g., at the start of a cytokine process or activation process described herein). In some embodiments, the population of cells at the end of the manufacturing process (e.g., at the end of a cytokine process or an activation process described herein) exhibits a lower (e.g., at least 5, 6, 7, 8, 9, 10, 11, or 12 days) percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, compared to cells produced by otherwise similar methods, including, e.g., persisting for 26 hours or more (e.g., persisting for 5, 6, 7, 8, 9, 10, 11, or 12 days or more), or expanding the population of cells in vitro, e.g., for 3 days or more (e.g., expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0588] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of the cytokine process or activation process described herein) is 40, 45, 50, 55, 60, 65, 70, 75, or 80% or less.

[0589] In some embodiments, after administration in vivo, the population of cells at the end of the manufacturing process (e.g., at the end of a cytokine process or an activation process described herein) persists longer or proliferates at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) than cells otherwise produced by similar methods, including, for example, for more than 26 hours (e.g., persists for more than 5, 6, 7, 8, 9, 10, 11, or 12 days), or expanded for more than 3 days (e.g., expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0590] In some embodiments, the population of cells is enriched for IL6R-expressing cells (e.g., cells that are positive for IL6Ra and / or IL6Kb) prior to the start of the manufacturing process (e.g., prior to the start of a cytokine process or activation process described herein). In some embodiments, the population of cells comprises 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% or more IL6R-expressing cells (e.g., cells that are positive for IL6Ra and / or IL6Kb), e.g., at the start of the manufacturing process (e.g., at the start of a cytokine process or activation process described herein).

[0591] In vitro CAR-T manufacturing The lentiviral vectors described herein (e.g., those produced using the methods described herein) can be used, for example, for the in vitro production of CAR-T cells.

[0592] In some embodiments, cells transduced with a viral vector described herein are expanded, for example, by the methods described herein. In some embodiments, the cells are expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, or 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In some embodiments, the cells are expanded for a period of 4 to 9 days. In some embodiments, the cells are expanded for a period of 8 days or less, e.g., 7, 6, or 5 days. In some embodiments, cells are expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. Potency can be defined, for example, by various T cell functions, such as proliferation, target cell killing, cytokine production, activation, migration, or a combination thereof. In some embodiments, the cells are grown for 5 days and exhibit at least a 1-fold, 2-fold, 3-fold, or 4-fold increase in cell doublings upon antigen stimulation compared to the same cells grown in culture for 9 days under the same culture conditions. In some embodiments, the cells are grown in culture for 5 days and the resulting cells exhibit increased proinflammatory cytokine production, e.g., IFN-γ and / or GM-CSF levels, compared to the same cells grown in culture for 9 days under the same culture conditions. In some embodiments, the cells grown for 5 days exhibit at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in proinflammatory cytokine production, e.g., IFN-γ and / or GM-CSF levels in pg / ml, compared to the same cells grown in culture for 9 days under the same culture conditions.

[0593] The initial activation process in the absence of calcium can lead to enhanced activation. As will be readily recognized by those skilled in the art, the washing step can be accomplished by semi-automated "flow-through" centrifugation (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or saline solution with or without other buffering agents. Alternatively, undesirable elements of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0594] It is recognized that the in vitro methods of the present application can utilize culture medium conditions containing 5% or less, e.g., 2%, human AB serum, and can use known culture medium conditions and compositions, such as those described in Smith et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement," Clinical & Translational Immunology (2015) 4, e31; doi:10.1038 / cti.2014.31.

[0595] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation. The isolated T cells can be further used in the methods described herein.

[0596] The methods described herein can include selection of a specific subpopulation of immune effector cells, e.g., T cells, e.g., a T regulatory cell-depleted population, CD25+ depleted cells, e.g., using, e.g., negative selection techniques described herein. Preferably, the T regulatory cell-depleted cell population comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

[0597] In some embodiments, T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody or fragment thereof, or a CD25-binding ligand, IL-2. In some embodiments, the anti-CD25 antibody or fragment thereof, or CD25-binding ligand is conjugated to or otherwise coated on a substrate, e.g., a bead. In some embodiments, the anti-CD25 antibody or fragment thereof is conjugated to a substrate described herein.

[0598] In some embodiments, T regulatory cells, e.g., CD25+ T cells, are removed from the population using a CD25 depletor from Miltenyi™. In some embodiments, the ratio of cells to CD25 depletor is 1×10 7 cells per 20 μL, or 1 x 10 7 cells per 15 μL, or 1 x 10 7 cells per 10 μL, or 1 x 10 7 5 μL of cells, or 1 x 10 7 2.5 μL, or 1 x 10 cells 7 In some embodiments, 500 million cells / ml are used, e.g., for T regulatory cell, e.g., CD25+ depletion. In further embodiments, cell concentrations of 600, 700, 800, or 900 million cells / ml are used.

[0599] In some embodiments, the immune effector cell population to be depleted comprises about 6×10 9 In other embodiments, the immune effector cell population to be depleted comprises about 1 x 10 9 ~1×10 10 (and any integer value therebetween) CD25+ T cells. In some embodiments, the resulting T regulatory depleted cell population comprises 2×10 9 T regulatory cells, e.g., CD25+ cells or less (e.g., 1 x 10 9 , 5×10 8 , 1×10 8 , 5×10 7 , 1×10 7 or fewer CD25+ cells).

[0600] In some embodiments, T regulatory cells, e.g., CD25+ cells, are removed from a population using the CliniMAC system with a depletion tubing set, e.g., tubing 162-01. In some embodiments, the CliniMAC system is run in a depletion setting, e.g., DEPLETION2.1.

[0601] Without wishing to be bound by a particular theory, it is believed that a reduction in the levels of negative regulators of immune cells (e.g., unwanted immune cells, such as T cells) in a subject prior to apheresis or during the manufacture of a CAR-expressing cell product may contribute to the reduction of immune cell negative regulators (e.g., unwanted immune cells, such as T cells). REG A reduction in the number of T cells may reduce a subject's risk of recurrence. REG Methods for depleting cells are known in the art. For example, T REG Methods for reducing cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25 depletion, and combinations thereof.

[0602] In some embodiments, the method of production involves the use of T cells prior to CAR-expressing cell production. REG For example, manufacturing methods include contacting a sample, e.g., an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or fragment thereof or a CD25-binding ligand) to reduce (e.g., deplete) the number of CAR-expressing cells (e.g., T cells, NK cells) prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product. REG This involves depleting the cells.

[0603] In some embodiments, the subject is treated with T REG In some embodiments, the subject has been pre-treated with one or more therapies that reduce the T REGMethods for reducing cells include, but are not limited to, administering to a subject one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof can occur before, during, or after infusion of a CAR-expressing cell product.

[0604] In some embodiments, the subject is pretreated with cyclophosphamide prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of the subject relapsing from CAR-expressing cell treatment. In some embodiments, the subject is pretreated with an anti-GITR antibody prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of the subject relapsing from CAR-expressing cell treatment.

[0605] In some embodiments, the cell population to be removed is not regulatory T cells or tumor cells, but cells that otherwise negatively affect CAR T cell proliferation and / or function, such as cells expressing CD14, CD11b, CD33, CD15, or other markers expressed by potentially immunosuppressive cells. In some embodiments, it is intended that such cells be removed simultaneously with the regulatory T cells and / or tumor cells, or after said depletion, or in a different order.

[0606] The methods described herein can include two or more selection steps, e.g., two or more depletion steps. Enrichment of a T cell population by negative selection can be achieved, for example, by a combination of antibodies directed against surface markers unique to the cells to be negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the cells to be negatively selected. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail can include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0607] The methods described herein further include depleting cells from the population that express a tumor antigen, e.g., a tumor antigen that does not contain CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, thereby providing a T regulatory-depleted, e.g., CD25+-depleted and tumor antigen-depleted cell population suitable for expression of a CAR, e.g., a CAR described herein. In some embodiments, tumor antigen-expressing cells are depleted simultaneously with T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-tumor antigen antibody or fragment thereof can be attached to the same substrate, e.g., beads, which can be used to deplete the cells, or an anti-CD25 antibody or fragment thereof and an anti-tumor antigen antibody or fragment thereof can be attached to separate beads, a mixture of which can be used to deplete the cells. In other embodiments, depletion of T regulatory cells, e.g., CD25+ cells, and depletion of tumor antigen-expressing cells are sequential, e.g., can occur in either order.

[0608] Also provided are methods that include removing from a population one or more cells that express a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, e.g., PD1+ cells, LAG3+ cells, and TIM3+ cells, thereby providing a T regulatory-depleted, e.g., CD25+-depleted, and checkpoint inhibitor-depleted, cell population, e.g., PD1+, LAG3+, and / or TIM3+-depleted cells. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA, and LAIR1. In some embodiments, checkpoint inhibitor-expressing cells are removed simultaneously with T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-check point inhibitor antibody or fragment thereof can be attached to the same bead, which can be used to remove the cells, or an anti-CD25 antibody or fragment thereof and an anti-check point inhibitor antibody or fragment thereof can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of checkpoint inhibitor-expressing cells are sequential, e.g., can occur in either order.

[0609] The methods described herein may include a positive selection step. For example, T cells can be isolated by incubating with anti-CD3 / anti-CD28 (e.g., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient to positively select the desired T cells. In some embodiments, the time is approximately 30 minutes. In further embodiments, the time ranges from 30 minutes to 36 hours or longer, and any integer value therebetween. In further embodiments, the time is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet other embodiments, the time is 10 to 24 hours, e.g., 24 hours. Longer incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, the use of longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, simply by shortening or lengthening the time that T cells are allowed to bind to the CD3 / CD28 beads and / or increasing or decreasing the bead-to-T cell ratio (as further described herein), subpopulations of T cells can be preferentially selected for at the beginning of the culture or at other time points during the process. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for at the beginning of the culture or at other desired time points. In some embodiments, T cell populations can be selected that express one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other suitable molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, for example, by the methods described in WO 2013 / 126712.

[0610] To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) to ensure maximum contact between the cells and beads. For example, in some embodiments, a concentration of 10 billion cells / ml, 9 billion cells / ml, 8 billion cells / ml, 7 billion cells / ml, 6 billion cells / ml, or 5 billion cells / ml is used. In some embodiments, a concentration of 1 billion cells / ml is used. In further embodiments, a concentration of 75, 80, 85, 90, 95, or 100 million cells / ml of cells is used. In further embodiments, a concentration of 125 or 150 million cells / ml can be used.

[0611] Using a high concentration can increase cell yie...

Claims

1. 1. A method for producing lentiviral particles, comprising: a) culturing a plurality of mammalian cells that do not express large T antigen at a first pH; b) the plurality of mammalian cells; i) FectoVIR®-AAV transfection reagent, and ii) a nucleic acid encoding a transgene and sufficient LTR sequences for packaging into a viral particle; and contacting the cell with a transfection complex comprising: c) culturing said plurality of mammalian cells under conditions suitable for production of said lentiviral particles; d) harvesting the lentivirus particles; e) purifying the lentivirus particles to produce a filtrate; The method includes:

2. The method of claim 1, wherein the transfection complex comprises one or more nucleic acids encoding retroviral envelope proteins and retroviral packaging proteins.

3. The method described in claim 1, wherein the introduced gene encodes a CAR comprising an antigen-binding domain, a transmembrane domain and one or more signaling domains.

4. The method of claim 1, wherein the transfection complex contains FectoVIR®-AAV at a concentration of 0.3 to 0.6 μl of FectoVIR®-AAV per million cells.

5. The method of claim 1, wherein the transfection complex contains the nucleic acid at a concentration of 0.3 to 0.6 μg / million cells.

6. The method of claim 1, wherein the transfection complex comprises a ratio of FectoVIR®-AAV:nucleic acid of 1:0 to 1:

2.

7. The method of claim 1, wherein the first pH is lowered to a second pH prior to contacting the cell with the transfection complex.

8. The method of claim 1, wherein the second pH is 0.1-1.0 pH units lower than the first pH.

9. The method of claim 1, wherein the second pH is 0.5 pH units lower than the first pH.

10. The method of claim 1, wherein a nuclease is added prior to step d).

11. The method of claim 10, wherein the nuclease is benzonase.

12. The method of claim 1, wherein a solution containing arginine is added prior to step e).

13. The method of claim 12, wherein the arginine concentration prior to step e) is about 25-50 mM or about 50-100 mM.

14. The method of claim 12, wherein the solution further comprises a buffer, a salt, a non-reducing carbohydrate, and / or a pH of about 6.0 to about 7.

0.

15. The method of claim 14, wherein the buffer is PIPES at a concentration of about 10 mM to about 50 mM.

16. The method of claim 14, wherein the salt is at a concentration of about 25 to 150 mM.

17. The method of claim 16, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.

18. The method of claim 14, wherein the non-reducing carbohydrate is present in the solution at a concentration of about 1 to about 10% weight / volume of the solution, or at a concentration of about 30 to 150 mM or 150 to 300 mM.

19. The method of claim 18, wherein the non-reducing carbohydrate is selected from the group consisting of sucrose and trehalose.

20. The method of claim 1, wherein after step e), a solution containing arginine is added.

21. The method described in claim 20, wherein the arginine concentration in the filtrate is 100 to 200 mM or 200 to 400 mM.

22. The method of claim 1, resulting in at least 1 x 107 lentiviral particles.

23. The method of claim 1, wherein the filtrate has reduced aggregation of the lentiviral particles.

24. The method of claim 1, wherein the filtrate has a total particle concentration of less than 400,000 per ml as measured by microflow imaging.

25. The method of claim 1 , wherein the mammalian cell is an Expi293F cell.

26. An aqueous composition comprising a lentiviral vector, arginine, a 1,4-piperazinediethanesulfonic acid (PIPES) buffer, and a salt.

27. The method of claim 27, wherein the arginine in the aqueous composition is at a concentration of about 25-50 mM, 50-100 mM, 100-200 mM, or 200-400 mM arginine. ii) the PIPES buffer is at a concentration of about 10 mM to about 50 mM; iii) the aqueous composition has a pH of about 6.0 to about 7.0; iv) the salt is at a concentration of about 25 mM to about 150 mM in the aqueous composition; and / or v) the salt is selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride; 27. The aqueous composition of claim 26.

28. 27. The aqueous composition of claim 26, further comprising a carbohydrate.

29. i) the carbohydrate is a non-reducing carbohydrate. ii) the carbohydrate is selected from sucrose or trehalose, and / or iii) the carbohydrate is present in the aqueous composition at a concentration of about 1 to about 10% weight / volume of the aqueous composition, or at a concentration of about 30 to 150 mM or 150 to 300 mM; 29. The aqueous composition of claim 28.

30. A composition comprising: i) 20 mM PIPES, 75 mM sodium chloride, and 2.5% sucrose by weight / volume of the aqueous composition, and having a pH of about 6.

5. ii) containing 20 mM PIPES, 75 mM sodium chloride, and 73 mM sucrose and having a pH of about 6.5; and / or iii) 20 mM PIPES, 75 mM sodium chloride, and 220 mM sucrose, having a pH of about 6.5; 27. The aqueous composition of claim 26.

31. The lentiviral vector comprises about 3 x 10 ８ TU / mL ~ approx. 5×10 ８ 27. The aqueous composition of claim 26, wherein the composition is present in a concentration of 1000 TU / mL.

32. An aqueous composition comprising a lentiviral vector, a salt, and 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.