Lentiviral formulations

Abstract

The present disclosure provides, at least in part, formulation buffers suitable for lentiviral vectors.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 381,107, filed on October 26, 2022. The contents of the foregoing application are hereby incorporated by reference in their entirety. Background Art

[0003] Viruses are very efficient at delivering nucleic acids to specific cell types while generally avoiding detection by the infected host's immune system. These features make certain viruses attractive candidates as gene delivery vehicles for use in gene therapy.

[0004] Viral vectors that can be used for gene therapy applications include lentiviral vectors. Such vectors include reconstructed viral vector systems derived from human immunodeficiency virus-1 (HIV-1) and are capable of introducing target genes 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 target gene is integrated into the genome of the host cell and is therefore replicated during cell division. Lentiviral vectors can effectively transduce non-dividing cells as well as those that are actively progressing in the cell cycle. Tissues and cells where chronic expression of target genes mediated by lentiviral vectors may occur include the brain, liver, muscle cells, retina, hematopoietic stem cells, bone marrow mesenchymal stem cells, and macrophages, among others.

[0005] Large-scale production of lentiviral vectors is hampered by several challenges, such as low titer of viral yield and low stability of the vector. In addition, lentiviral vectors are easily inactivated during the purification process, which may lead to reduced final quality and efficacy of the vector preparation, further creating another obstacle for large-scale production of purified lentiviral vectors. Therefore, there is still a need for formulation buffers that maintain vector stability. Summary of the invention

[0006] The present disclosure provides, at least in part, a method for producing a high titer lentiviral vector carrying a transgene of interest under satisfactory safety conditions. The present disclosure also provides, at least in part, a method for purifying such lentiviral particles, for example, from cell culture. The present disclosure also provides a formulation of a lentiviral preparation that maintains the structural integrity of the viral vector during purification, storage, and gene transfer events (e.g., ex vivo gene transfer).

[0007] In some aspects, the present disclosure provides an aqueous composition comprising:

[0008] a) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES); and

[0009] b) one or both of free positively charged amino acids (e.g., arginine, lysine, or histidine) and free non-polar amino acids (e.g., proline, methionine, or tryptophan).

[0010] In some embodiments, the aqueous composition is substantially free of (eg, free of) inorganic salts.

[0011] In some embodiments, the aqueous composition is substantially free of (e.g., free of) NaCl and MgCl. 2 One or both of the following.

[0012] In some embodiments, the aqueous composition comprises a total amount of less than 20, 10, 5, 2, or 1 mM inorganic salts.

[0013] In some embodiments, the concentration of HEPES is 10-200, 10-150, 10-100, 10-50, 10-40, 10-30, 15-25, or 20 mM.

[0014] In some embodiments, the concentration of HEPES is about 20 mM.

[0015] In some embodiments, the aqueous composition comprises a positively charged amino acid, wherein the positively charged amino acid comprises arginine (eg, L-arginine).

[0016] In some embodiments, the concentration of arginine is at least 25, 50, 75, 100, or 150 mM.

[0017] In some embodiments, the concentration of arginine is 25-50, 50-75, 75-100, 75-125, 100-200, 125-175, or 150 mM.

[0018] In some embodiments, the concentration of arginine is about 150 mM.

[0019] In some embodiments, the aqueous composition comprises a non-polar amino acid, wherein the non-polar amino acid comprises proline (eg, L-proline).

[0020] In some embodiments, the concentration of proline is 25-200, 50-200, 100-200, 125-175, or 150 mM.

[0021] In some embodiments, the concentration of proline is about 150 mM.

[0022] In some embodiments, the aqueous composition further comprises a cryoprotectant, eg, a carbohydrate, eg, a non-reducing carbohydrate, eg, sucrose.

[0023] In some embodiments, the aqueous composition further comprises sucrose.

[0024] In some embodiments, the concentration of sucrose is 25-200, 50-200, 100-200, 125-175, or 150 mM.

[0025] In some embodiments, the concentration of sucrose is about 150 mM.

[0026] In some embodiments, the aqueous composition further comprises a stabilizer (eg, HSA).

[0027] In some embodiments, the aqueous composition comprises HSA.

[0028] In some embodiments, HSA comprises recombinant HSA (rHSA) or human-derived HSA (eg, HSA isolated from human serum).

[0029] In some embodiments, HSA is present at 0.5%-3%, 0.5%-2%, 0.5%-1%, 1%-2%, 1.5%-2.5%, or 2% w / v.

[0030] In some embodiments, HSA is present at about 2% w / v.

[0031] In some embodiments, the aqueous composition is substantially free of (eg, free of) HSA.

[0032] In some embodiments, the pH of the aqueous composition is 6.0-7.5, 6.0-7.0, 6.0-6.5, 6.5-7.0, 6.2-6.8, 6.4-6.6, or 6.5.

[0033] In some embodiments, the pH of the aqueous composition is about 6.5.

[0034] In some embodiments, the aqueous composition is substantially free of (eg, free of) one, two, or three of PEG lipid, F108, and cholesterol.

[0035] In some embodiments:

[0036] The composition comprises a positively charged amino acid, wherein the positively charged amino acid comprises L-arginine;

[0037] The composition comprises a non-polar amino acid, wherein the non-polar amino acid comprises L-proline;

[0038] The composition further comprises sucrose; and

[0039] The composition is substantially free of inorganic salts.

[0040] In some embodiments:

[0041] The concentration of L-arginine is 100-200 mM;

[0042] The concentration of L-proline is 25-200 mM; and

[0043] The concentration of sucrose was 25-200 mM.

[0044] In some embodiments, the HEPES formulation buffer and / or storage buffer comprises L-arginine at a concentration of 100-200 mM, L-proline at a concentration of 25-200 mM, and sucrose at a concentration of 25-200 mM.

[0045] In some embodiments, the HEPES formulation buffer and / or storage buffer comprises 20 mM HEPES, 150 mM L-arginine, 150 mM L-proline, and 150 mM sucrose. In some embodiments, the HEPES formulation buffer and / or storage buffer further comprises 2% w / v HSA.

[0046] In some embodiments, the aqueous composition has an osmolality of about 400 mOsm / kg to about 700 mOsm / kg, for example, about 415 mOsm / kg to about 689 mOsm / kg.

[0047] In some embodiments, the present disclosure provides a composition comprising: the lentiviral vector as described in any one of the preceding embodiments and an aqueous composition.

[0048] In some embodiments, the composition comprises at least 1x10 6 , 1x10 7 , 5x10 7 , 1x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 or 6x10 9 transducing units / mL (TU / mL) of lentiviral vector.

[0049] In some embodiments, the lentiviral vector comprises a transgene.

[0050] In some embodiments, the transgene encodes a chimeric antigen receptor (CAR).

[0051] In some embodiments, the CAR comprises an antigen binding domain that binds a tumor antigen selected from the group consisting of TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostate enzyme, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-ab l, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumin, HPV E6, HPV E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein (prostein), survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and any combination thereof.

[0052] In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, one or more primary signaling domains, and / or one or more co-stimulatory signaling domains.

[0053] In some embodiments, the one or more primary signaling domains comprise a CD3-zeta stimulatory domain.

[0054] In some embodiments, the one or more costimulatory signaling domains comprise an intracellular domain selected from:

[0055] (a) a co-stimulatory protein selected from the group consisting of: OX40, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, 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(CD22 9), 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

[0056] (b) a ligand that specifically binds to CD83, for example, a 4-1BB (CD137) co-stimulatory domain or a CD28 co-stimulatory domain.

[0057] In some embodiments, the CAR comprises a transmembrane domain, wherein:

[0058] (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154;

[0059] (b) the transmembrane domain comprises the transmembrane domain of CD8;

[0060] (c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 90% sequence identity thereto; or

[0061] (d) the transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence having at least about 90% sequence identity thereto.

[0062] In some embodiments, the composition exhibits a level of subvisible particles greater than or equal to 25 μm of about 50-100 particles / mL or less.

[0063] In some embodiments, the composition exhibits a level of subvisible particles greater than or equal to 10 μm of about 1100-2000 particles / mL or less.

[0064] In some embodiments, the lentiviral vector in the composition retains a hydrodynamic diameter between about 85-200, 90-200, or 85-130 nm.

[0065] In some embodiments, the present disclosure provides a kit comprising: the lentiviral vector as described in any one of the preceding embodiments and an aqueous composition.

[0066] In some embodiments, the present disclosure provides a method for preparing a cell expressing CAR, the method comprising:

[0067] Providing a composition as described in any of the preceding embodiments; and

[0068] contacting the composition with immune effector cells under conditions that allow transduction of one or more of the immune effector cells,

[0069] Thereby, the CAR-expressing cells are prepared.

[0070] In some embodiments, the method is an in vitro or ex vivo method.

[0071] In some embodiments, the immune effector cell population comprises one or both of T cells and NK cells.

[0072] In some embodiments, the composition comprises HSA, for example, at a concentration of about 0.5% to 3% w / v.

[0073] In some embodiments, HSA comprises recombinant HSA (rHSA) or human-derived HSA (eg, HSA isolated from human serum).

[0074] In some embodiments, the present disclosure provides a method of delivering a transgene to a subject, the method comprising administering to the subject the composition of any one of the preceding embodiments, wherein the lentiviral vector of the composition comprises a transgene.

[0075] In some embodiments, the transgene comprises a CAR, e.g., a CAR described herein.

[0076] In some embodiments, the CAR is a CD19 CAR, e.g., a CAR having the CDRs listed in Table 1.

[0077] In some embodiments, the composition further comprises particles, eg, silica particles, eg, mesoporous particles (MSP), optionally wherein the mesoporous silica particles are mesoporous silica rods.

[0078] In some embodiments:

[0079] (i) the retroviral vector is non-covalently, e.g., electrostatically, or covalently associated with the mesoporous silica particle; and / or

[0080] (ii) The cell activating agent is non-covalently or covalently associated with the mesoporous silica particles.

[0081] In some embodiments, the composition further comprises a cell activating agent.

[0082] In some embodiments, the cell activating agent:

[0083] (a) comprising an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule and / or a growth factor receptor;

[0084] (b) is a multispecific binding molecule comprising: (i) an anti-CD3 binding domain, and (ii) a co-stimulatory molecule binding domain (e.g., an anti-CD2 binding domain or an anti-CD28 binding domain); and / or

[0085] (c) conjugated to or adsorbed on particles (e.g., mesoporous silica particles).

[0086] In some aspects, the present disclosure provides a method of making a lentiviral vector, the method comprising:

[0087] a) providing a plurality of mammalian (e.g., human) cells,

[0088] b) contacting the plurality of mammalian cells with:

[0089] i) -AAV transfection reagent, and

[0090] ii) nucleic acid encoding a therapeutic effector, such as a therapeutic protein (e.g., CAR) and sufficient LTR sequences for packaging into viral particles, and optionally nucleic acid encoding a lentiviral packaging protein, a lentiviral envelope protein, and

[0091] under conditions that allow for introduction of the nucleic acid into at least one subpopulation of cells; and

[0092] c) culturing the cells under conditions suitable for producing the lentiviral vector.

[0093] In some embodiments, when the plurality of mammalian cells is in 50 L culture, the number of transducing units per ml of culture produced is no less than 50%, 60%, 70%, or 80% of the number of transducing units per ml of culture in an otherwise similar 100 ml culture.

[0094] In some embodiments, when used under the conditions described in Example 5, the method produces at least 1x10 7 or 3x10 7 or at least 1x10 8 transduction units.

[0095] In some embodiments, the method produces a ratio of PP (physical particles): IP (infectious particles) equal to or less than 1188:1, 953:1, and 1800:1.

[0096] In some embodiments, the mammalian cell is a 293 cell, e.g., an Expi293F cell.

[0097] In some embodiments, 0.3–0.6 μl - AAV / million cells (e.g., about 0.4 μl / million cells) was used at a concentration -AAV.

[0098] In some embodiments, the nucleic acid is used at a concentration of 0.3-0.6 μg nucleic acid / million cells (eg, about 0.4 μg / million cells).

[0099] In some embodiments, - the ratio of AAV:DNA (for transfection) is 1:0.5 to 1:2, for example, about 1:1 (wherein the DNA optionally used for transfection comprises DNA encoding a therapeutic effector, DNA encoding one or more retroviral packaging proteins and DNA encoding a retroviral envelope protein).

[0100] In some embodiments, -AAV transfection reagent complexed with nucleic acid.

[0101] In some embodiments, the method further comprises before step b) - AAV transfection reagent mixed with nucleic acid.

[0102] In some embodiments, the complexed volume of the transfection reagent and nucleic acid is between about 1% and about 15%, e.g., about 1% and about 10% (e.g., about 5%-7.5% or 7.5%-10%).

[0103] In some embodiments, the composite volume is 3%-7%, 4%-6%, or about 5%.

[0104] In some embodiments, - The AAV transfection reagent and nucleic acid are incubated for a sufficient time to allow complexation to occur, e.g., about 10-90 minutes, e.g., 15-60 minutes, e.g., 15-30, 30-45 or 45-60 minutes.

[0105] In some aspects, the present disclosure provides a method of making a lentiviral vector, the method comprising:

[0106] a) culturing a plurality of mammalian (e.g., human) cells at a pH above about 6.9 or about 6.9-7.3, such as about 7.0-7.1;

[0107] b) following step a), adjusting the pH of the culture to about 6.0-6.8, e.g., 6.6-6.8, e.g., about 6.7;

[0108] c) Following step b), the culture is contacted with a transfection reagent and DNA.

[0109] In some embodiments, the transfection reagent comprises -AAV transfection reagent.

[0110] In some embodiments, the DNA encodes one or more retroviral packaging proteins, retroviral envelope proteins, and a therapeutic effector such as a therapeutic protein (e.g., a CAR).

[0111] In some embodiments, a) comprises culturing the cells for about 2-4 days, e.g., about 3 days.

[0112] In some embodiments, the method further comprises the additional step of culturing the cells between steps b) and c).

[0113] In some embodiments, the method further comprises the additional step of culturing the cells after step c).

[0114] In some embodiments, step b) comprises lowering the pH by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.

[0115] In some embodiments, prior to step a), the plurality of mammalian cells are cultured at 0.1×10 6 cells / mL and 0.3x10 6 cells / mL (e.g., about 0.15 x 10 6 cells / mL or about 0.2x10 6 cells / mL) in a final volume of medium (e.g., FreeStyle TM culture medium).

[0116] In some embodiments, the plurality of mammalian cells is seeded between 50 and 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).

[0117] In some embodiments, the cell density is appropriate to allow cells to grow and expand until transfection (e.g., at about 1.0×10 6 cells / mL and about 3.0x10 6 cells / mL (e.g., between 1.5x10 6 cells / mL and 2.5x10 6 Multiple mammalian cells can be cultured under conditions ranging from 10 cells / mL to 20 cells / mL.

[0118] In some aspects, the present disclosure provides a method of making a lentiviral vector, the method comprising:

[0119] a) providing a composition comprising the lentiviral vector and at least one impurity (e.g., wherein the composition comprises a clarified cell harvest or filtrate), and

[0120] b) contacting the composition with arginine or a salt thereof.

[0121] In some embodiments, one or more of the following:

[0122] i) the arginine is at a concentration of about 25-50 mM (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; or

[0123] ii) the concentration of arginine is sufficient to increase the transducing unit level of the lentiviral vector by about 10%-300%, about 20%-180%, about 30%-160%, about 50%-150%, about 75%-125%, or about 100%, compared to an otherwise similar composition, e.g., in an assay according to Example 7;

[0124] iii) after step b), the composition exhibits a total particle concentration per ml of less than 400,000, less than 300,000, less than 200,000 or less than 100,000 as measured by microfluidic imaging, wherein optionally the particles comprise aggregated lentiviruses;

[0125] iv) after step b), the composition exhibits a concentration of less than about 5,000, about 4,500, about 4,000, about 3,500, about 3,000, or about 2,500 particles per ml ≥ 10 μm as measured by microfluidic imaging, wherein optionally the particles comprise aggregated lentiviruses;

[0126] v) after step b), the composition exhibits a concentration of less than about 500, about 400, about 300, or about 200 particles per ml ≥ 25 μm as measured by microfluidic imaging, wherein optionally the particles comprise aggregated lentiviruses;

[0127] vi) after step b), the composition exhibits reduced aggregation of the lentiviral vector compared to an otherwise similar filtrate without the addition of the arginine or a salt thereof;

[0128] vii) the recovery of transducing units of the lentiviral vector is greater, e.g., at least about 10%, about 20%, about 50%, about 100% or about 200% greater than an otherwise similar control without added arginine, e.g., as measured in an assay according to Example 7.

[0129] In some embodiments, b) comprises contacting the composition with a solution comprising arginine and a buffer, wherein optionally the buffer is PIPES, wherein optionally the concentration of PIPES in the solution is about 10 mM to about 50 mM, e.g., about 20 mM. In certain embodiments, the pH of the solution is about 6.0 to about 7.0, e.g., about 6.5.

[0130] In some embodiments, the solution further comprises a salt, wherein optionally the salt is selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride, eg, sodium chloride.

[0131] In some embodiments, the salt is present in the solution at a concentration of about 25-150 mM, such as about 50-100 mM, such as about 75 mM.

[0132] In certain embodiments, the pH of the solution is about 6.5.

[0133] In some embodiments, the solution further comprises a carbohydrate, eg, a non-reducing carbohydrate, eg, sucrose or trehalose.

[0134] In certain embodiments, the carbohydrate is present in the solution at a concentration of about 1% to about 10% by weight per volume of solution, such as about 2% to about 5% by weight per volume of solution, about 2.5% by weight per volume of solution.

[0135] In certain embodiments, the carbohydrate is present in the solution at a concentration of about 30-150 mM (about 73 mM) or about 150-300 (eg, about 220) mM.

[0136] In some embodiments, the solution further comprises one or both of NaCl (e.g., about 25-150 mM, e.g., about 50-100 mM, e.g., about 75 mM) and sucrose (e.g., about 30-150 mM, e.g., about 73 mM, or e.g., about 150-300 mM, e.g., about 220 mM, or about 2.5% by weight / volume of solution).

[0137] In some embodiments, the solution comprises 20 mM PIPES, 75 mM sodium chloride, and 2.5% sucrose by weight / volume solution, and wherein the pH of the solution is about 6.5. In certain embodiments, the solution comprises about 20 mM PIPES, about 75 mM sodium chloride, and about 2.5% sucrose by weight / volume solution, and wherein the pH of the solution is about 6.5.

[0138] In certain embodiments, the solution comprises 20 mM PIPES, 75 mM sodium chloride, and 73 mM sucrose, and wherein the pH of the solution is about 6.5. In certain embodiments, the solution comprises about 20 mM PIPES, about 75 mM sodium chloride, and about 73 mM sucrose, and wherein the pH of the solution is about 6.5.

[0139] In some embodiments, the solution comprises 20 mM PIPES, 75 mM sodium chloride, and 220 mM sucrose, and wherein the pH of the solution is about 6.5. In some embodiments, the solution comprises about 20 mM PIPES, about 75 mM sodium chloride, and about 220 mM sucrose, and wherein the pH of the solution is about 6.5.

[0140] In some embodiments, the solution further comprises 20 mM PIPES, 75 mM arginine, e.g., arginine-HCl, and wherein the pH of the solution is about 6.5. In some embodiments, the solution further comprises about 20 mM PIPES, about 75 mM arginine, e.g., arginine-HCl, and wherein the pH of the solution is about 6.5.

[0141] In some embodiments, the osmolality of the solution is about 270 mOsm / kg to about 330 mOsm / kg, e.g., about 275 mOsm / kg to about 300 mOsm / kg, e.g., about 285 mOsm / kg.

[0142] In certain embodiments, the method further comprises: c) performing a purification step, for example, a filtration step on the composition of b), thereby producing a semi-purified composition comprising the lentiviral vector.

[0143] In certain embodiments, the method further comprises, after step c), contacting the semi-purified composition with arginine or a salt thereof.

[0144] In some embodiments, arginine encapsulates the lentiviral vector.

[0145] In certain embodiments, arginine stabilizes the lentiviral vector.

[0146] 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.

[0147] In certain embodiments, the cell density at the time of transfection is about 1.0 x 10 6 cells / mL and about 3.0x10 6 cells / mL (e.g., 1.5x10 6 cells / mL and 2.5x10 6 cells / mL).

[0148] In some embodiments, the viability of the cells at the time of transfection is or is assessed to be at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0149] In certain embodiments, the viability of the cells is measured at or about the time of transfection (eg, within 30 minutes prior to transfection).

[0150] In some embodiments, the method is used for a process with two or more nucleic acids (eg, two or more plasmids, eg, two plasmids, three plasmids, four plasmids, or five plasmids).

[0151] In some aspects, the present disclosure provides a method of making a lentiviral vector, the method comprising:

[0152] a) providing a population of human cells (e.g., 293 cells);

[0153] b) introducing into these cells nucleic acids encoding retroviral packaging proteins, retroviral envelope proteins, and therapeutic effectors such as therapeutic proteins (e.g., CAR),

[0154] c) contacting the cells with benzonatase at a time of about 6-40, 10-40, 10-30, or about 20 hours after step b); and

[0155] d) culturing the cells under conditions suitable for production of the lentiviral vector.

[0156] In some aspects, the present disclosure provides a method of making a lentiviral vector, the method comprising:

[0157] a) providing a population of human cells (e.g., 293 cells);

[0158] b) introducing into these cells nucleic acids encoding retroviral packaging proteins, retroviral envelope proteins, and therapeutic effectors such as therapeutic proteins (e.g., CAR),

[0159] c) contacting the cell with benzonatase;

[0160] d) culturing the cells under conditions suitable for producing the lentiviral vector;

[0161] e) harvesting the lentiviral vectors from the cells 6-10 hours, 10-20 hours, 20-30 hours, 30-40 hours or 40-50 hours after step c).

[0162] In some aspects, the present disclosure provides a method of making a lentiviral vector, the method comprising:

[0163] a) providing a plurality of mammalian (e.g., human) cells, wherein the plurality of cells (e.g., wherein the cells are fibroblasts, e.g., embryonic kidney fibroblasts, e.g., Expi293F cells), wherein the cells comprise a 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),

[0164] b) culturing the cells under conditions suitable for producing the lentiviral vector.

[0165] In some aspects, the present disclosure provides an aqueous composition comprising a lentiviral vector, arginine, 1,4-piperazinediethanesulfonic acid (PIPES) buffer, and a salt.

[0166] In certain embodiments, the concentration of arginine in the aqueous composition is about 25-50 mM (about 50 mM), about 50-100 mM (e.g., about 75 mM), about 100-200 mM (e.g., about 150 mM), or about 200-400 (e.g., about 300) mM arginine, wherein optionally the concentration of PIPES in the aqueous composition is about 10 mM to about 50 mM, e.g., about 20 mM.

[0167] In some embodiments, the pH of the aqueous composition is from about 6.0 to about 7.0, for example, about 6.5.

[0168] In certain embodiments, the aqueous composition further comprises a salt, wherein optionally the salt is selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride.

[0169] In some embodiments, the salt is sodium chloride (NaCl).

[0170] In certain embodiments, the salt in the aqueous composition is about 25 mM to about 150 mM, for example, about 50 mM to about 75 mM.

[0171] In some embodiments, the aqueous composition comprises 20 mM PIPES and 75 mM sodium chloride, and wherein the pH of the aqueous composition is about 6.5.

[0172] In certain embodiments, the aqueous composition further comprises a carbohydrate, eg, a non-reducing carbohydrate, eg, sucrose or trehalose.

[0173] In some embodiments, the carbohydrate is present in the aqueous composition at a concentration of about 1% to about 10% by weight per volume of solution, e.g., about 2% to about 5% by weight per volume of aqueous composition, about 2.5% by weight per volume of aqueous composition.

[0174] In one embodiment, the carbohydrate is present in the aqueous composition at a concentration of about 30-150 mM (about 73 mM) or about 150-300 (eg, about 220) mM.

[0175] In some embodiments, the aqueous composition comprises one or both of NaCl (e.g., about 25-150 mM, e.g., about 50-100 mM, e.g., about 75 mM) and sucrose (e.g., about 30-150 mM, e.g., about 73 mM, or e.g., about 150-300 mM, e.g., about 220 mM, or about 2.5% by weight / volume of the aqueous composition).

[0176] In one embodiment, the aqueous composition comprises 20 mM PIPES, 75 mM sodium chloride, and 2.5 wt% sucrose / volume of the aqueous composition, and wherein the pH of the aqueous composition is about 6.5.

[0177] In certain embodiments, the aqueous composition comprises 20 mM PIPES, 75 mM sodium chloride, and 73 mM sucrose, and wherein the pH of the aqueous composition is about 6.5.

[0178] In one embodiment, the aqueous composition comprises 20 mM PIPES, 75 mM sodium chloride, and 220 mM sucrose, and wherein the pH of the aqueous composition is about 6.5.

[0179] In certain embodiments, the aqueous composition has an osmolality of about 270 mOsm / kg to about 330 mOsm / kg, e.g., about 275 mOsm / kg to about 300 mOsm / kg, e.g., about 285 mOsm / kg.

[0180] In one embodiment, the lentiviral vector of any of the preceding claims is present in an amount of about 3×10 8 TU / mL to about 5x10 8 The concentration of TU / mL exists.

[0181] In certain embodiments, the aqueous composition is free of 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.

[0182] 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).

[0183] In certain embodiments, the CAR comprises, in the N-terminal to C-terminal direction, an antigen binding domain, a transmembrane domain, and one or more signaling domains.

[0184] In some embodiments, the signaling domain comprises one or more primary signaling domains and / or one or more co-stimulatory signaling domains.

[0185] In certain embodiments, one of the one or more primary signaling domains comprises a CD3-zeta stimulatory domain.

[0186] In some embodiments, one or more of the costimulatory signaling domains comprises an intracellular domain selected from a costimulatory protein selected from the group consisting of OX40, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF 1), 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, ITG AX, 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, CD10 0 (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, for example, 4-1BB (CD137) costimulatory domain or CD28 costimulatory domain.

[0187] In some embodiments, one or more of the costimulatory signaling domains comprises an intracellular domain selected from a costimulatory protein 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, 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, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD2 29), 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.

[0188] In certain embodiments, the antigen binding domain is a scFv.

[0189] In some embodiments, the antigen binding domain binds to an antigen 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 (EGFRvlll); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD1 17); interleukin-13 receptor subunit alpha-2; mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); proteinase serine 21; 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); prostatic enzyme; 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 (pro, megalin factor) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight-melanoma associated antigen (HM WMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related protein (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK);Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycosylceramide (GloboH); mammary differentiation antigen (NY-BR-1); urine protein 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pan-nexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); olfactory receptor 51E2 (OR51 E2); 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 mutants; prostate-specific protein; survivin; telomerase; prostate cancer tumor antigen-1, melanoma antigen recognized by T cells 1; rat sarcoma (Ras) mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma cell apoptosis inhibitor (ML- IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytoma viral oncogene neuroblastoma homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1 B1); CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); pre-acrosomal protein binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); legumin; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutated heat shock protein 70-2 (muthsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1);Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 with an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), for example, in combination with CD19, CD22, mesothelin, or CD123. ;

[0190] In certain embodiments, the CAR comprises an anti-CD19 antibody or fragment thereof, a 4-1BB (CD137) transmembrane domain, and a CD3-zeta signaling domain.

[0191] 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).

[0192] In one aspect, the present disclosure provides a method for producing a lentiviral vector, the method comprising:

[0193] a) providing a population of human cells (e.g., 293 cells);

[0194] b) introducing into these cells nucleic acids encoding retroviral packaging proteins, retroviral envelope proteins, and therapeutic effectors such as therapeutic proteins (e.g., CAR),

[0195] c) contacting the cells with benzonatase about 2-6 (e.g., about 3), 4-10 (e.g., about 6), 6-40, 10-40, 10-30 (e.g., about 24), or about 20 hours after step b); and

[0196] d) culturing the cells under conditions suitable for production of the lentiviral vector.

[0197] In certain embodiments, benzonatase is added 6-10 hours, 10-20 hours, 20-30 hours, 30-40 hours, or 40-50 hours prior to harvesting the lentiviral vector from the cells.

[0198] In one aspect, the present disclosure provides a method for producing a lentiviral vector, the method comprising:

[0199] a) providing a population of human cells (e.g., 293 cells);

[0200] b) introducing into these cells nucleic acids encoding retroviral packaging proteins, retroviral envelope proteins, and therapeutic effectors such as therapeutic proteins (e.g., CAR),

[0201] c) contacting the cells with benzonatase (e.g., 3-24 hours after step b);

[0202] d) culturing the cells under conditions suitable for producing the lentiviral vector;

[0203] e) harvesting the lentiviral vectors from the cells 6-10 hours, 10-20 hours, 20-30 hours, 30-40 hours or 40-50 hours after step c).

[0204] In some embodiments, the concentration of benzonatase is about 10-40 U / mL, eg, 20-30 U / mL, eg, about 25 U / mL.

[0205] In certain embodiments, the concentration of benzonatase is about 3-60 U / mL, 3-10 U / mL, 3-7 U / mL, 4-6 U / mL, or about 5 U / mL.

[0206] In one embodiment, the concentration of benzonatase is 5-50, 5-15, 15-25, or 25-50 U / mL.

[0207] In certain embodiments, the method further comprises, prior to step c), mixing benzonatase with, for example, about 1-5 mM, about 1-3 mM, or about 2 mM MgCl 2 touch.

[0208] In one aspect, the present disclosure provides a method for producing a lentiviral vector, the method comprising:

[0209] 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., wherein the cells are fibroblasts, e.g., embryonic kidney fibroblasts, e.g., Expi293F cells), wherein the plurality of mammalian cells comprise a nucleic acid (e.g., DNA) encoding one or more retroviral packaging proteins, retroviral envelope proteins, and a therapeutic effector, such as a therapeutic protein (e.g., CAR),

[0210] b) culturing the cells under conditions suitable for producing the lentiviral vector.

[0211] In certain embodiments, a) comprises introducing the nucleic acid into a plurality of mammalian cells.

[0212] In some embodiments, the method further comprises at least partially isolating the lentiviral vector from the plurality of mammalian cells.

[0213] In one embodiment, the one or more retroviral packaging proteins comprise lentiviral gag, lentiviral pol, or lentiviral rev, or any combination thereof.

[0214] In certain embodiments, the retroviral envelope protein comprises VSV-G.

[0215] In some aspects, the present disclosure provides a lentiviral vector formulation comprising:

[0216] A plurality of lentiviral vectors, the plurality of lentiviral vectors comprising:

[0217] a) a lentiviral genome encoding a therapeutic effector such as a therapeutic protein (e.g., CAR), and

[0218] b) an envelope encapsulating the lentiviral genome (wherein optionally the envelope comprises VSV-G);

[0219] The preparation contains at least 5x10 7 , 1x10 8 , 1x10 9 or 1x10 10 transduction units;

[0220] The preparation contains less than 90% of SV40 large T antigen or less than 10 μg / ml, 1 μg / ml of nucleic acid (eg, DNA) encoding SV40 large T antigen.

[0221] In some embodiments, the plurality of lentiviral vectors comprises at least 1x10 9 , at least 2x10 9 , at least 5x10 9 , at least 1x10 10 , at least 2x10 10 , at least 5x10 10 , at least 1x10 11 , at least 2x10 11 , at least 5x10 1 or at least 1x10 12 cells.

[0222] In certain embodiments, the plurality of mammalian cells are in a culture volume of at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, or at least 500 L.

[0223] In one embodiment, the method comprises culturing a plurality of mammalian cells in a serum-free medium.

[0224] In certain embodiments, the plurality of mammalian cells are grown in suspension.

[0225] In some embodiments, the CAR comprises a CD19 CAR (e.g., a humanized CD19 CAR, e.g., as described in WO2014153270A1).

[0226] In certain embodiments, the CAR comprises a dual CAR (e.g., a humanized CD19-CD22 CAR, e.g., as described in WO2016164731A2).

[0227] In some embodiments, the nucleic acid encoding the CAR further encodes a shRNA (e.g., as described in WO 2017049166A).

[0228] In one embodiment, the lentiviral vector is produced in cells cultured in the absence of serum.

[0229] In certain embodiments, the lentiviral vector is characterized by a hydrodynamic radius of 100 ± 25 nm as measured by dynamic light scattering (DLS).

[0230] In certain embodiments, the lentiviral vector maintains a hydrodynamic radius of 100 ± 25 nm over 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).

[0231] 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%).

[0232] In one embodiment, the lentiviral vector maintains 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%) over 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).

[0233] 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 relative to the concentration of the lentiviral vector in the aqueous composition before the freeze / thaw cycle, wherein each freeze / thaw cycle comprises freezing the aqueous composition and then allowing the aqueous composition to thaw at room temperature.

[0234] In some embodiments, the lentiviral vector maintains a concentration of about 70% to about 100% (e.g., about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 100%) after 6-10 freeze / thaw cycles, e.g., after 6-9 freeze / thaw cycles.

[0235] In some aspects, the present disclosure provides an aqueous composition comprising a lentiviral vector, a buffer selected from the group consisting of a phosphate buffer, a sodium citrate buffer, a 2-(N-morpholino)ethanesulfonic acid (MES) buffer, a 3-morpholinepropane-1-sulfonic acid (MOPS) buffer, and a salt.

[0236] In some embodiments, the salt is selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride.

[0237] In one embodiment, the aqueous composition further comprises a non-reducing carbohydrate selected from the group consisting of sucrose and trehalose.

[0238] In some aspects, the disclosure provides scalable processes for producing large quantities of viral vectors (e.g., lentiviral vectors), e.g., for prophylactic, diagnostic, immunotherapeutic, or therapeutic uses. These processes can be performed using suspension cells (e.g., HEK293 cells, e.g., Expi293F cells). In some embodiments, substantially all suspension cells do not express large T antigens, e.g., SV40 T antigens. In some embodiments, the process can be performed using a bioreactor.

[0239] In some aspects, the present disclosure provides highly reproducible, efficient, scalable processes for producing large quantities of viral vectors (eg, lentiviral vectors) with either or both high viral titers or high viral yields.

[0240] In some aspects, the present disclosure provides highly reproducible, efficient, scalable processes for purifying large quantities of viral vectors (eg, lentiviruses) with either or both high viral titers or high viral yields.

[0241] In another aspect, the present disclosure provides compositions and methods for stabilizing viral vectors, such as lentiviral vectors, during a purification process. BRIEF DESCRIPTION OF THE DRAWINGS

[0242] Figure 1 is a schematic diagram showing the overall study design.

[0243] Figure 2 is a graph showing the particle size distribution (PSD; Dh) of LVV products before and after incubation with HEPES or XV15 supplemented with sucrose at 37°C for 12 hours.

[0244] Figure 3 is a graph showing the concentration of LVV product particles before and after incubation with HEPES or XV15 supplemented with sucrose at 37°C for 12 hours.

[0245] Figure 4 is a graph showing the TU titer of LVV products before and after incubation with HEPES or XV15 supplemented with sucrose at 37°C for 12 hours.

[0246] Figure 5 is a graph showing total p24 titers of LVV products before and after incubation with HEPES or XV15 supplemented with sucrose for 12 hours at 37°C.

[0247] Figure 6 is a graph showing the zeta potential (ZP) distribution of LVV in HEPES and PIPES buffers.

[0248] Figure 7 It is a figure which shows the phase diagram of LVV in HEPES and PIPES buffers.

[0249] Figure 8 is a graph showing the relationship between pH and the particle size (left y-axis) or PDI (right y-axis) of the carrier.

[0250] Fig. 9 is a graph showing the relationship between pH and the zeta potential of the carrier. The dashed line (-30 mV) represents the potential minimum ZP for stable particles in suspension.

[0251] Fig.10 is a Pareto plot of TU titers with the effects of individual excipients ranked in descending order. The vertical dashed line indicates a statistically significant effect (α=0.15). The dotted bars are non-significant terms removed from the model. The striped bars are terms that have a main effect on the response.

[0252] Fig.11is a Pareto plot of MADLS with the effects of individual excipients listed in descending order. The vertical dashed line indicates a statistically significant effect (α=0.15). The dotted bars are non-significant terms removed from the model. The striped bars are terms with a main effect on the response.

[0253] Fig.12 is a Pareto plot of p24 titers with the effects of the individual excipients ranked in descending order. The vertical dashed line indicates a statistically significant effect (α=0.15). The dotted bars are non-significant terms removed from the model. Fig.13 is a Pareto plot of TU titers obtained from combination 1.

[0254] Fig.14 is a graph showing the relationship between arginine and TU titer obtained from combination 1.

[0255] Fig.15 is a graph showing the relationship between recombinant HSA (rHSA) and TU titer obtained from combination 1.

[0256] Fig.16 is a graph showing the relationship between proline and TU titer obtained from combination 1.

[0257] Fig.17 is a graph showing the relationship between lactose and TU titer obtained from combination 1.

[0258] Fig.18 is a Pareto plot of p24 titers obtained from combination 1.

[0259] Fig.19 is a graph showing the relationship between glycerol and p24 titer obtained from combination 1.

[0260] Fig. 20 is a graph showing the relationship between recombinant HSA (rHSA) and p24 titer obtained from combination 1.

[0261] Fig.21 is a graph showing the relationship between proline and p24 titer obtained from combination 1.

[0262] Fig. 22 is a graph showing the relationship between lactose and p24 titer obtained from combination 1.

[0263] Fig.23 is a Pareto plot of TU titers obtained from combination 2.

[0264] Fig.24 is a graph showing the relationship between arginine and the TU titer obtained from combination 2.

[0265] Fig.25 is a graph showing the relationship between glutamate and TU titer obtained from combination 2.

[0266] Fig.26 is a graph showing the relationship between recombinant HSA (HSA) and TU titer obtained from combination 2.

[0267] Fig. 27 is a graph showing the relationship between arginine and the TU titer obtained from combination 2.

[0268] Fig.28 Is to show MgC 2 Plot of the relationship between TU titers obtained from combination 2.

[0269] Fig.29 is a Pareto plot of p24 titers obtained from combination 2.

[0270] Fig.30 is a graph showing a factorial designed particle analysis of LVV product formulated in PIPES buffer. The concentration of particles greater than 10 μm is plotted against the Z-mean. The dashed line represents the LVV particle size distribution (between 85 nm and 130 nm) and the number of subvisible particles greater than 10 μm (1300).

[0271] Fig.31 is a graph showing a factorial designed particle analysis of LVV product formulated in PIPES buffer. The concentration of particles greater than 25 μm is plotted against the Z-mean. The dashed line represents the LVV particle size distribution (between 85 nm and 130 nm) and the number of subvisible particles greater than 25 μm (150).

[0272] Fig.32 is a graph showing the TU titer of LVV products formulated with HEPES-F1 after buffer exchange (ABE) and spiked with different HSA sources using 2.0% HSA in the formulation, according to the number of freeze-thaw cycles (FT; white, 0FT; stripes, 3FT).

[0273] Fig.33 is a graph showing the TU titer of LVV products formulated with HEPES-F1 after buffer exchange (ABE) and spiked with different HSA sources using 0.5% HSA in the formulation, according to the number of freeze-thaw cycles (FT; white, 0FT; stripes, 3FT).

[0274] Fig.34 is a graph showing the p24 titer of LVV products formulated with HEPES-F1 after buffer exchange (ABE) and spiked with different HSA sources using 2.0% HSA in the formulation, according to the number of freeze-thaw cycles (FT; white, 0FT; stripes, 3FT).

[0275] Fig.35 is a graph showing the p24 titer of LVV products formulated with HEPES-F1 after buffer exchange (ABE) and spiked with different HSA sources using 0.5% HSA in the formulation, according to the number of freeze-thaw cycles (FT; white, 0FT; stripes, 3FT).

[0276] Fig.36 is a graph showing the total number of subvisible particles compared to the excipient range by freeze-thaw cycle (FT; white, 0FT; stripes, 3FT).

[0277] Fig.37 is a graph showing the TU titers of three batches of LVV product formulated with HEPES-F1 after buffer exchange (ABE), HSA incorporation (+HSA), and three freeze-thaw cycles (+3FT).

[0278] Fig.38 is a graph showing p24 ELISA results for three batches of LVV product formulated with HEPES-F1 after buffer exchange (ABE), HSA incorporation (+HSA), and three freeze-thaw cycles (+3FT).

[0279] Fig.39 is a graph showing the results of total p24 titer of three batches of LVV product formulated with HEPES-F1 after buffer exchange (ABE), HSA incorporation (+HSA), and three freeze-thaw cycles (+3FT).

[0280] Fig.40 is a graph showing the total number of subvisible particles larger than 10 μm observed in three batches of LVV product formulated with HEPES-F1 after buffer exchange (ABE), HSA incorporation (+HSA), and three freeze-thaw cycles (+3FT).

[0281] Fig.41 is a graph showing the total number of subvisible particles larger than 25 μm observed in three batches of LVV product formulated with HEPES-F1 after buffer exchange (ABE), HSA incorporation (+HSA), and three freeze-thaw cycles (+3FT).

[0282] Fig.42 is a graph showing the TU titers of three batches of LVV product formulated with HEPES-F2 after buffer exchange (ABE), HSA incorporation (+HSA), and three freeze-thaw cycles (+3FT).

[0283] Fig.43is a graph showing the results of total p24 titer of three batches of LVV product formulated with HEPES-F2 after buffer exchange (ABE), HSA incorporation (+HSA), and three freeze-thaw cycles (+3FT).

[0284] Fig.44 is a graph showing the total number of subvisible particles larger than 10 μm observed in three batches of LVV product formulated with HEPES-F2 after buffer exchange (ABE), HSA incorporation (+HSA), and three freeze-thaw cycles (+3FT).

[0285] Fig.45 is a graph showing the total number of subvisible particles larger than 25 μm observed in three batches of LVV product formulated with HEPES-F2 after buffer exchange (ABE), HSA incorporation (+HSA), and three freeze-thaw cycles (+3FT).

[0286] Fig.46 is a graph showing the particle size distribution by intensity for three batches of LVV product in PIPES and HEPES buffers.

[0287] Fig.47 is a graph showing particle concentration in LVV product measured by MADLS after single (VS) and double (VS-2SF) sterile filtration (to compare the effect of HSA formulation).

[0288] Fig.48 is a graph showing TU titers in LVV products after single (VS) and double (VS-2SF) sterile filtration to compare the effect of HSA formulations.

[0289] Fig.49 is a graph showing TU titers of LVV products formulated in PIPES buffer after storage at different temperatures (25°C, 4°C, -20°C and -80°C) for a period of up to 9 months.

[0290] Fig.50 is a graph showing TU titers of LVV products formulated in HEPES (F1) buffer after storage at different temperatures (25°C, 4°C, -20°C and -80°C) for a period of up to 9 months.

[0291] Fig.51 is a graph showing TU titers of LVV products formulated in HEPES (F2) buffer after storage at different temperatures (25°C, 4°C, -20°C and -80°C) for a period of up to 9 months. DETAILED DESCRIPTION

[0292] The present disclosure is based, at least in part, on a method for producing high titer lentiviral vectors carrying a transgene of interest and 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 formulations of lentiviral preparations that maintain the structural integrity of the viral vector during purification, storage, and ex vivo gene transfer events.

[0293] definition

[0294] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings.

[0295] As used herein, the singular form "a" or "an" includes plural referents unless otherwise indicated.

[0296] The term "or" is intended herein to mean and is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0297] "About" and "approximately" generally mean an acceptable degree of error for a measured quantity given the nature or precision of the measurements. Exemplary degrees of error are within 10%, within 5%, or within 2% of a given value or range of values.

[0298] 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 the naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, γ-carboxyglutamate, and O-o-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an α-carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as naturally occurring amino acids, for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refers to compounds that have a structure that is different from the general chemical structure of an amino acid but functions in a manner similar to that of a naturally occurring amino acid.

[0299] 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, "N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer" refers to a mixture comprising N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid anions. Due to the chemical equilibrium established between the weak acid and its conjugate base, a solution containing a buffer can resist sudden changes in pH when a small amount of acid or base is added to the solution.

[0300] As used herein, the term "inorganic salt" refers to a chemical component (e.g., a component of a solution) that lacks carbon-hydrogen bonds, is composed of positively charged cations and negatively charged anions, and has no net charge. Exemplary inorganic salts include sodium chloride (NaCl) and magnesium chloride (MgCl 2 ). In some embodiments, the salt is present in the aqueous solution in a dissociated form.

[0301] As used herein, the term "freeze / thaw cycle" refers to exposing a liquid mixture (such as an aqueous solution or suspension) to a temperature equal to or lower than its freezing point until the mixture is frozen, followed by thawing the mixture at a temperature higher than its freezing point. The freezing step can be performed, for example, by placing the mixture in an environment where the temperature is about -80 ° C to about -20 ° C. The mixture can remain frozen before thawing, for example, for one or more days, weeks, months or years. The thawing step can be performed by exposing the mixture to a temperature of about 2 ° C to about 8 ° C, or by storing the mixture at room temperature (e.g., the ambient temperature of the laboratory, or about 25 ° C). Alternatively, a water bath (e.g., at 37 ° C) can be used for thawing.

[0302] As used herein, the term "hydrodynamic radius" refers to the apparent radius (Rh, in nm) of a particle in solution, as inferred from the diffusion characteristics of the particle. The hydrodynamic radius of a viral particle is a factor that determines the diffusion rate of the viral particle in aqueous solution and the ability of the particle to migrate in a macromolecular gel. The hydrodynamic radius of a viral particle depends in part on the mass and molecular structure of each component of the particle, as well as its hydration state. Methods for determining the hydrodynamic radius of a viral particle are well known in the art and include the use of dynamic light scattering and size exclusion chromatography.

[0303] As used herein, the term "non-reducing carbohydrate" refers to a carbohydrate that does not exist in a chemical equilibrium state with an aldehyde, and therefore lacks the ability to be oxidized to a carboxylic acid by a transition metal cation (such as silver (Ag+) and copper (Cu2+). Exemplary non-reducing carbohydrates include, but are not limited to, disaccharides such as sucrose, trehalose, and palatinol, trisaccharides such as raffinose and melezitose, and tetrasaccharides such as stachyose. Non-reducing carbohydrates additionally include monosaccharide derivatives such as sorbitol, mannitol, erythritol, and xylitol, disaccharide derivatives such as lactitol and maltitol, aldonic acids and their lactones such as gluconic acid, glucono gamma-lactone, uronic acids and their lactones such as libaric acid, arabinonic acid, and galactaric acid, uronic acids such as glucuronic acid, galacturonic acid, and oxymethoxylic acid, ester derivatives such as trehalose octaacetate, sucrose octaacetate, and cellobiose octaacetate, and ether derivatives in which the hydroxyl group is O-alkylated. Non-reducing carbohydrates include carbohydrates with a D or L stereochemical orientation.

[0304] As used herein, the term "osmolality" refers to a measure of the osmotic pressure of dissolved solute particles in an aqueous solution. Solute particles include both ions and non-ionic molecules. Osmolality is expressed as the concentration of osmotically active particles (i.e., osmoles) dissolved in 1 kg of solvent (i.e., water). Osmolality is expressed herein in units of milliosmoles per 1 kg of water (mOsm / kg).

[0305] As used herein, the term "percent by weight per volume" or "% w / v" means the weight percentage (in grams) of a single component relative to the total volume (in milliliters) of the mixture containing the 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.

[0306] As used herein, the term "polydispersity" refers to the degree of uniformity of the size of particles (such as lentiviral particles) in a sample. A higher polydispersity indicates a lower uniformity, and a lower polydispersity indicates a higher level of uniformity. For example, when the level of uniformity is very high, the lentiviral particles can be considered to be close to the same size and therefore monodisperse. As will be understood by one of ordinary skill in the art, as polydispersity decreases, the level of uniformity increases. Therefore, a lower polydispersity indicates a higher level of uniformity. For example, a formulation with a polydispersity of 15% has a lower uniformity than a formulation with a polydispersity of 10%. When the level of uniformity is low, the particle population can be considered to contain significantly different sizes and therefore be polydisperse.

[0307] As used herein, the term "binding domain" or "antibody molecule" refers to a protein comprising at least one immunoglobulin variable domain sequence, e.g., an immunoglobulin chain or a fragment thereof. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence in the plurality has binding specificity to a first epitope and the second immunoglobulin variable domain sequence in the plurality has binding specificity to a second epitope. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for no more than two antigens. The bispecific antibody molecule is characterized in that a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope.

[0308] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the naturally occurring conformation of an antibody molecule, and generally determines the class to which the antibody belongs.

[0309] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in the naturally occurring conformation of an antibody molecule. κ (kappa) and λ (lambda) light chains refer to the two major antibody light chain isotypes.

[0310] As used herein, the term "antigen-binding fragment" 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 Fv (scFv), camelid antibodies, disulfide-linked Fv (sdFv), Fab fragments, F(ab') fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; F(ab)2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementarity determining regions (CDRs) or other epitope-binding fragments of antibodies.

[0311] The portion of the described CAR comprising an antibody or antibody fragment thereof can exist in a variety of forms, wherein the antigen binding domain is expressed as part of a continuous 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, New York; 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 aspects, the antigen binding domain of the CAR composition comprises an antibody fragment. In another aspect, CAR comprises an antibody fragment containing scFv. The precise amino acid sequence boundaries of a given CDR can be determined using any of many well-known schemes, including those described by the following documents: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest" [Protein sequences with immunological importance], 5th edition, National Institutes of Health, Public Health Division, Bethesda, Maryland ("Kabat (Kabat)" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia (Chothia)" numbering scheme) or a combination thereof.

[0312] In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, recombinant methods can be used to connect the two domains through a synthetic linker that enables them to form a single protein chain, wherein the VL region and the VH region pair to form a monovalent molecule (referred to as a single-chain Fv ("scFv"); see, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al. Proc. Natl. Acad. Sci. 85:5879-5883, 1988). The term "antigen-binding fragment" is also intended to encompass such single-chain antibodies. 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.

[0313] 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 comprising a functional signaling domain derived from a stimulatory molecule as defined below (also referred to herein as an "intracellular signaling domain"). In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, e.g., as provided in RCAR, the domains in the CAR polypeptide construct are discontinuous with each other, e.g., in different polypeptide chains.

[0314] In some aspects, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-ζ). In some aspects, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule defined as follows. In some aspects, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS and / or CD28. In some aspects, CAR comprises a chimeric fusion protein (which comprises an extracellular antigen recognition domain), a transmembrane domain and an intracellular signaling domain (which comprises a functional signaling domain derived from a stimulatory molecule). In some aspects, CAR comprises a chimeric fusion protein (which comprises an extracellular antigen recognition domain), a transmembrane domain and an intracellular signaling domain (which comprises a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule). In some aspects, CAR comprises a chimeric fusion protein (which comprises an extracellular antigen recognition domain), a transmembrane domain and an intracellular signaling domain (which comprises two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule). In some aspects, CAR comprises a chimeric fusion protein (which comprises an extracellular antigen recognition domain), a transmembrane domain, and an intracellular signaling domain (which comprises at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule). In some aspects, CAR comprises an optional leader sequence at the amino-terminus (N-term) of the CAR fusion protein. In some aspects, CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cut from the antigen recognition domain (e.g., scFv) during cell processing and CAR localization to the cell membrane.

[0315] A CAR comprising an antigen binding domain (e.g., scFv, single domain antibody, or TCR (e.g., TCRα binding domain or TCRβ binding domain)) targeting a specific tumor marker X (where X can be a tumor marker as described herein) is also referred to as an X CAR. For example, a CAR comprising an antigen binding domain targeting BCMA is referred to as a BCMA CAR. CAR can be expressed in any cell, e.g., an immune effector cell (e.g., T cell or NK cell) as described herein.

[0316] The term "signaling domain" refers to a functional part of a protein that acts by transmitting information within the cell to act as an effector by producing a second messenger or by responding to such a messenger, and regulating cell activity via a defined signaling pathway. The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides one or more cytoplasmic signaling sequences that regulate immune cell activation in a stimulating manner for at least some aspects of the immune cell signaling pathway. In some aspects, the signal is a primary signal that is initiated by, for example, the binding of a TCR / CD3 complex to a peptide-carrying MHC molecule, and results in mediating T cell responses, including but not limited to proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulating manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of cytoplasmic signaling sequences containing ITAM include, but are not limited to, those derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In some CARs, the intracellular signaling domain in any one or more CARs described herein includes an intracellular signaling sequence, for example, a primary signaling sequence of CD3-ζ. In some CARs disclosed herein, the primary signaling sequence of CD3-ζ is a human sequence or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

[0317] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates signals that promote immune effector functions of cells containing CAR (e.g., CART cells). Examples of immune effector functions, for example, in CART cells, include cytolytic activity and auxiliary activity (including secreted cytokines).

[0318] In some embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent simulation. In some embodiments, the intracellular signaling domain may include a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may include a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may include a cytoplasmic sequence from a co-receptor or a costimulatory molecule.

[0319] The primary intracellular signaling domain may include 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 CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12.

[0320] The term "ζ" or alternatively "ζ chain", "CD3-ζ" or "TCR-ζ" refers to CD247. Swiss-Prot accession number P20963 provides an exemplary human CD3 ζ amino acid sequence. "ζ stimulatory domain" or alternatively "CD3-ζ stimulatory domain" or "TCR-ζ stimulatory domain" refers to the stimulatory domain of CD3-ζ 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 ζ comprises residues 52 to 164 of GenBank accession number 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 term "zeta" or alternatively "zeta chain", "CD3-zeta" (or "CD3zeta, CD3zeta or CD3z) or "TCR-zeta" is defined as the protein provided as GenBank Accession No. BAG36664.1, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), and a "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or a functional derivative thereof, which are sufficient to functionally transmit the initial signal necessary for T cell activation. In some aspects, the cytoplasmic domain of zeta comprises residues 52 to 164 of GenBank Accession No. BAG36664.1, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) of a functional ortholog thereof.

[0321] The term "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell to mediate a costimulatory response (such as but not limited to proliferation) of a T cell. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, and CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10 and DAP12 contribute to an effective immune response. Costimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137). Additional examples of such co-stimulatory molecules include ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, 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.

[0322] The costimulatory intracellular signal transduction domain can be the intracellular part of a costimulatory molecule.Costimulatory molecules can be represented in the following protein families: TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein) and activated NK cell receptor. The example of such a molecule includes CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3 and the ligand specifically bound to CD83, etc.

[0323] The intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0324] "Complementarity determining domain" or "complementarity determining region" ("CDR") refers interchangeably to the hypervariable regions of VL and VH. CDR is the target protein binding site of the antibody chain, which carries the specificity for this target protein. There are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in each human VL or VH, constituting about 15%-20% of the variable domain. The CDRs are structurally complementary to the epitope of the target protein and are therefore directly responsible for the binding specificity. The remaining VL or VH segments (the so-called framework regions) show less amino acid sequence variation (Kuby, Immunology, 4th ed., Chapter 4, WH Freeman & Co., New York, 2000).

[0325] The positions of CDRs and framework regions can be determined using various definitions well known in the art, e.g., Kabat, Chothia, the International Immunogenetics Database (IMGT) (available on the World Wide Web at imgt.org), and AbM (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). The definition of antigen binding sites is also described in the following literature: Ruiz et al., Nucleic Acids Res. [Nucleic Acids Research], 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res. [Nucleic Acids Research], 29:207-209 (2001); MacCallum et al., J. Mol. Biol. [Journal of Molecular Biology], 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 86:9268-9272 (1989); Martin et al., Methods Enzymol. [Methods in Enzymology], 203:121-153 (1991); and Rees et al., in Sternberg MJE (ed.), Protein Structure Prediction [Protein Structure Prediction], Oxford University Press. Oxford University Press, Oxford, 141-172 (1996).

[0326] As used herein, the term "contaminating polynucleotide" refers to a polynucleotide that is not derived from a lentiviral vector. Contaminating polynucleotides may include, for example, non-lentiviral polynucleotides derived from cells that produce lentiviral vectors, such as chromosomal mammalian DNA (e.g., human DNA) that is not included in the transgene or other components of the lentiviral vector.

[0327] "Derived from" (when the term is used herein) refers to the relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first molecule and the second molecule, and does not imply or include a limitation on the process or source of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include a limitation on a particular process for generating the intracellular signaling domain, e.g., it does not mean that in order to provide the intracellular signaling domain, one must start with the CD3 zeta sequence and delete unwanted sequences, or impose mutations, to arrive at the intracellular signaling domain.

[0328] 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.

[0329] As used herein, the term "recognition" refers to an antibody or antigen-binding fragment thereof that finds and interacts with (e.g., binds to) its epitope, whether or not the epitope is linear or conformational. The term "epitope" refers to a site on an antigen that specifically binds to an antibody or antigen-binding fragment of the present disclosure. An epitope can be formed from continuous amino acids or non-continuous amino acids juxtaposed due to the three-dimensional folding of a protein. The epitope formed from continuous amino acids is generally retained when exposed to a denaturing solvent, while the epitope formed due to the three-dimensional folding is generally lost when treated with a denaturing solvent. 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. The method for determining the spatial conformation of an epitope includes techniques in the art, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology [epitope mapping protocol in the method in molecular biology], Vol. 66, GE Morris ed. (1996)).

[0330] As used herein, the term "retroviral packaging protein" refers to a protein derived from a retrovirus or a variant thereof that helps package a 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 with at least 80%, 90%, or 95% sequence identity. In some embodiments, one or more retroviral packaging proteins are provided as polyproteins.

[0331] As used herein, the term "retroviral envelope protein" refers to a protein derived from a retrovirus or a variant thereof that can be assembled into an envelope around a nucleic acid (e.g., a viral genome). An exemplary retroviral envelope protein is env, e.g., wild type or a variant thereof. In some embodiments, the retroviral envelope protein is a lentiviral envelope protein, e.g., wild type or a variant thereof. In some embodiments, the retroviral envelope protein is VSV-G, e.g., wild type or a variant. In some embodiments, the retroviral protein is a pseudotype. In some embodiments, the retroviral envelope protein is from a virus different from one or more retroviral packaging proteins or LTRs of the nucleic acid to be packaged.

[0332] As used herein, the phrases "specific binding" and "binding" refer to a binding reaction that determines the presence of a specific protein in a heterogeneous population of proteins and other biomolecules (e.g., proteins and other biomolecules recognized by a ligand having a specificity). A ligand (e.g., a protein, proteoglycan, or glycosaminoglycan) that specifically binds to a protein will bind to the protein with a KD of less than 500 nM. For example, a ligand that specifically binds to a protein will bind to the protein with a KD of up to 500 nM (e.g., between 1 pM and 500 nM). A ligand that does not exhibit specific binding to a protein or its domain will exhibit a KD greater than 500 nM (e.g., greater than 600 nm, 700 nM, 800 nM, 900 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for the specific protein or its domain. A variety of assay formats can be used to determine the affinity of a ligand for a specific protein. For example, solid phase ELISA assays are commonly used to identify ligands that specifically bind to a target protein. 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), for descriptions of assay formats and conditions that can be used to determine binding of a specific protein.

[0333] The term "subject" includes humans 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. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

[0334] As used herein, the term "therapeutic effector" refers to a molecule (eg, RNA or polypeptide) that can produce a therapeutic effect on a subject at an effective level.

[0335] The terms "therapeutically acceptable amount" or "therapeutically effective dose" interchangeably refer to an amount sufficient to achieve the 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 cause undesirable side effects. In some embodiments, a therapeutically acceptable amount induces or causes side effects, but only those that are acceptable to the patient's condition by a healthcare provider. A therapeutically acceptable amount can be determined by first administering a low dose and then increasing the dose incrementally until the desired effect is achieved. "Prophylactically effective dose" and "therapeutically effective dose" can, in some embodiments, respectively prevent the onset of disease symptoms or cause a reduction in severity, including symptoms associated with cancer.

[0336] As used herein, the term "transfection" refers to the introduction of DNA into eukaryotic cells. Transfection can be accomplished by a variety of means, including but not limited to calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, liposome infection, protoplast fusion, retroviral infection, and gene gun.

[0337] As used herein, the terms "treat, treating or treatment" of any disease or disorder, in one embodiment, refer to the alleviation of the disease or disorder (i.e., slowing or stagnating or reducing the development of the disease or at least one clinical symptom thereof). In another embodiment, "treat, treating or treatment" refers to the alleviation or reduction of at least one physical parameter, including those that cannot be discerned by the patient. In yet another embodiment, "treat, treating or treatment" refers to regulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both.

[0338] As used herein, the term "viral titer" refers to the number of infectious vector particles or "transduction units" that result in the transfer of a given nucleic acid sequence from the particles into target cells. Viral titer can be measured by functional assays, such as those described in Xiao et al., Exp. Neurobiol. [Experimental Neurobiology] 144: 1 13-124, 1997, or Fisher et al., J. Virol. [Journal of Virology] 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 that has been integrated into the host cell genome, for example, using polymerase chain reaction (PCR) techniques known in the art.

[0339] As used herein, the term "viral vector" refers to a viral particle with the ability to introduce a nucleic acid molecule into a host. Viral vectors carrying one or more foreign genes are usually packaged into infectious viral particles by viral packaging using packaging plasmids of a specific cell line. Infectious viral particles infect cells to achieve the expression of foreign genes. "Recombinant" viral vectors refer to viral vectors constructed by genetic recombination technology. Any suitable method can be used to construct recombinant viral vectors, such as by transducing or transfecting a packaging cell line with a nucleic acid encoding a viral genome and then isolating newly packaged viral particles. It is understood that recombinant technology can be performed in the upstream stage of the production of the viral vector itself. For example, recombinant technology can be used to produce plasmids, which can then be produced on a larger scale, and finally the plasmids can be introduced into a cell line for packaging to produce a viral vector.

[0340] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, for example, including the self-inactivating lentiviral vector provided by Milone et al., Mol. Ther. [Molecular Therapy] 17(8): 1453–1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, for example, those from Oxford BioMedica. Gene delivery technology, LENTIMAX from Lentigen TM Vector Systems, etc. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

[0341] Methods for producing lentivirus

[0342] The present disclosure provides, among other things, an improved method for making a lentiviral vector. The following general steps can be used. First, a host cell can be cultured. Exemplary types of host cells, such as human cells lacking large T antigens, are described in more detail in the section entitled "Host Cells" herein. Without wishing to be bound by theory, it is believed that host cells lacking large T antigens can bring manufacturing advantages compared to host cells containing large T antigens (see, e.g., Example 1 of PCT / IB2022 / 053880, which is hereby incorporated by reference in its entirety).

[0343] In some embodiments, to produce large numbers of cells, host cells are cultured in successively larger vessels (eg, bioreactors) until sufficiently large numbers of cells are produced.

[0344] Once a sufficient number of host cells are obtained, the desired nucleic acid can be introduced into the host cells. These nucleic acids can be introduced by transfection, for example, using -AAV transfection reagent, for example, as described in the section entitled "Transfection" herein. The transfected nucleic acid may include a viral genome to be packaged, wherein the viral genome includes a therapeutic gene of interest and sufficient LTR sequences for packaging into viral particles. Additional nucleic acids that may be introduced into the host cell include plasmids that facilitate packaging, for example, plasmids encoding viral gag, pol, env, and rev. In some embodiments, the pH of the culture medium may be lowered prior to transfection, for example, from about 7.1 to about 6.7, for example, as described in the section entitled "Culture Conditions and Transfection Conditions" herein. The cells then begin to produce lentivirus.

[0345] After transfection, a nuclease such as benzonatase can be added to the culture medium, for example, as described in the section entitled "Culture Medium". Without wishing to be bound by theory, in some embodiments, the cell culture medium is a source of contaminating nucleic acids in the final lentiviral preparation, for example, the culture medium may contain host cell DNA from lysed host cells. Therefore, adding benzonatase to the cell culture medium can degrade contaminating nucleic acids, thereby improving the purification of lentivirus.

[0346] Next, the lentivirus can be harvested from the host cell culture to begin purification of the lentivirus. In some embodiments, the harvesting of the lentivirus includes separating the supernatant or cell culture medium from the cells. In some embodiments, the cells are not lysed before clarification. In some embodiments, the cells can be lysed and the lysate can be clarified.

[0347] Purification of lentivirus from cell culture medium or cell lysate typically involves several continuous purification steps. The purification step may include filtration (e.g., ultrafiltration) and chromatography steps. In some embodiments, arginine may be added during the purification process, for example, before or after a filtration step or chromatography step. For example, the addition of arginine is described in a chapter entitled "Purification". Without wishing to be bound by theory, in some embodiments, arginine stabilizes lentiviral vectors and / or reduces their aggregation.

[0348] Purified lentivirus can be used for a variety of applications. For example, lentivirus can be used to deliver genes to ex vivo cells, for example, to generate CART cells from immune effector cells in a single sample. As another example, lentivirus can be administered to a subject to deliver genes in situ to the subject's cells. For example, lentivirus can be used for in vivo CART production. In some embodiments, lentivirus is suitable for administration in human subjects, for example, a lentivirus encoding CAR can be administered to a subject, thereby allowing the nucleic acid encoding CAR to be introduced into immune effector cells in the subject.

[0349] Naturally occurring lentiviruses are a genus of viruses in the family Retroviridae, characterized by a long incubation period. Slow viruses can usually deliver a large amount of genetic information to the DNA of host cells. Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), the pathogen of human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes sheep encephalitis (visna) or pneumonia (maedi), caprine arthritis-encephalitis virus, which causes goat immunodeficiency, arthritis and encephalopathy; equine infectious anemia virus, which can cause 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 possible central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in subhuman primates. The diseases caused by these viruses are characterized by a long incubation period and a long course of disease. Typically, the viruses latently infect monocytes and macrophages, which they then spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T cells).

[0350] Lentivirus production methods are described, for example, in International Application PCT / IB2022 / 053880 (e.g., Examples 1-13 therein), which is incorporated by reference in its entirety, including the Examples.

[0351] genetically modified

[0352] In some embodiments, the lentivirus or lentiviral vector disclosed herein may include a nucleic acid, e.g., a transgene, such as a protein-encoding transgene. The nucleic acid may include a transgene, e.g., as described in the section entitled "Transgene" herein. The transgene may be operably linked to a promoter sequence. The nucleic acid may also include one or more (e.g., two) LTR sequences. Without wishing to be bound by theory, LTR may facilitate insertion of transgenes and promoters into the host cell genome. The LTR sequence may include a wild-type lentiviral LTR sequence or a variant thereof. For example, 3'LTR may include a deletion that causes the virus to self-inactivate after integration. In addition, 5'LTR may be a chimeric LTR. In some embodiments, the transgene may be integrated into the chromosomal DNA of the target cell.

[0353] Exemplary transgenes include those encoding chimeric antigen receptors (CARs). CAR may include several domains, such as antigen binding domains, transmembrane domains, and one or more signaling domains. In these cases, the signaling domain may contain one or more primary signaling domains (e.g., CD3-ζ stimulatory domains) and / or one or more costimulatory signaling domains (e.g., CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, or a ligand specifically bound to CD83).

[0354] In some embodiments, a transgene (e.g., a transgene comprising a CAR) can encode an antigen binding domain (such as a scFv) that binds 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 (EGFRvlll), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG 72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD1 17), interleukin-13 receptor subunit alpha-2, mesothelin, interleukin 1 1 receptor alpha (IL-1 1Ra), prostate stem cell antigen (PSCA), protease serine 21, 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), prostate enzyme, prostatic acid phosphatase (PAP), mutated elongation factor 2 (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (pro, megalin factor) subunit, beta, 9 (LMP2), glycoprotein 1 00 (gp100), oncogene fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight-melanoma associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related protein (TEM7R), claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C 5 group,Member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide portion of globoH glycosylceramide (GloboH), breast differentiation antigen (NY-BR-1), urine protein 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor β3 (ADRB3), pan-nexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K 9 (LY6K), olfactory receptor 51E2 (OR51 E2), 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, prostate-specific protein, survivin, telomerase, prostate cancer tumor antigen-1, melanoma antigen recognized by T cells 1, 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 myelocytoma viral oncogene neuroblastoma homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1 B1), CCCTC binding factor (zinc finger protein)-like, squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), pre-acrosomal protein binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), kinase anchor protein 4 (AKAP-4), synovial sarcoma,X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitin 1 (RU1), renal ubiquitin 2 (RU2), legumin, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, mutant heat shock protein 70-2 (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), mucin-like hormone receptor-like 2 with EGF-like module (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0355] In some embodiments, the lentiviral vector described herein comprises more than one transgene, 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.

[0356] In some embodiments, the dual CAR lentiviral vector described herein encodes two different CARs, for example, CD19CAR and CD22CAR. In some embodiments, the two CARs are part of a single open reading frame and are separated by a protease cleavage site, for example, a self-cleavage site, for example, a P2A site. In some embodiments, the open reading frame encodes a first leader sequence, a first scFv (for example, it binds CD22), optionally a first hinge domain, a first transmembrane domain, a first costimulatory domain (for example, 4-1BB), a first primary signaling domain (for example, CD3-ζ), a protease cleavage site (for example, P2A), a second leader sequence, a second scFv (for example, it binds CD19), optionally a second hinge domain, a second transmembrane domain, a second costimulatory domain (for example, 4-1BB) and a second primary signaling domain (for example, 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.

[0357] Additional CARs that can be encoded by the transgenes described herein are provided, for example, in the section herein entitled "CAR Targets."

[0358] In certain embodiments, lentiviral vectors as described herein encode siRNA or shRNA of nucleic acid in targeting immune effector cells.For example, siRNA or shRNA can target nucleic acid and / or T cell encoding TCR and / or HLA (for example, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (for example, 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). Expression systems for siRNA and shRNA and exemplary shRNAs are described, for example, in paragraphs 649 and 650 of International Application WO2015 / 142675, filed on March 13, 2015, which is incorporated by reference in its entirety. These nucleic acids can also be targeted, for example, using the CRISPER system, zinc finger nucleases, or TALENs. The immune effector cells can be autologous or allogeneic to the subject to be treated.

[0359] In some embodiments, the lentiviral vector described herein comprises or encodes one or more inhibitors of methylcytosine dioxygenase genes (e.g., Tet1, Tet2, or Tet3). It is also contemplated that such compositions and methods are used to increase the functional activity of engineered cells (e.g., genetically modified antigen-specific T cells, e.g., CAR T cells). The destruction of a single allele of the Tet gene (e.g., Tet1, Tet2, or Tet3) can result in a decrease in the total level of 5-hydroxymethylcytosine, accompanied by enhanced proliferation, regulation of effector cytokine production, and degranulation, thereby increasing the proliferation and / or function of CAR T cells. In some embodiments, the expression and / or function of Tet2 in the cell has been reduced or eliminated.

[0360] In some embodiments, the inhibitor of Tet1, Tet2 and / or Tet3 is a siRNA or shRNA specific for 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, for example, a target sequence of the shRNA listed in Table 4 of WO2017 / 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 targeting one or more sites within a gene encoding Tet1, Tet2 and / or Tet3 or its regulatory element, such as Tet2 or its regulatory element; (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 large range of nuclease systems.

[0361] In some embodiments, the lentiviral vectors described herein comprise a transgene, e.g., a transgene encoding a chimeric antigen receptor (CAR), and further comprise a siRNA or shRNA targeting a nucleic acid in an immune effector cell.

[0362] Characteristics of Lentiviral Vectors

[0363] In some embodiments, the lentiviral vector is characterized by a hydrodynamic radius of 90-200 nm as measured by dynamic light scattering (DLS).For example, the lentiviral vector can maintain a hydrodynamic radius of 90-200 nm over a temperature range of 25°C to 37°C.

[0364] 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% over a temperature range of 25°C to 37°C.

[0365] In some embodiments, the lentiviral vector maintains a concentration of about 70% to about 100% after 3, 6, or 9 freeze / thaw cycles relative to the concentration of the lentiviral vector in the aqueous composition before the freeze / thaw cycle, wherein each freeze / thaw cycle comprises freezing the aqueous composition and then allowing the aqueous composition to thaw at room temperature.

[0366] 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, compared to a lentivirus not produced, purified or stored by a method or formulation described herein, e.g., when tested at an MOI of 1.

[0367] Lentiviral vector packaging system

[0368] The packaging system can be used to package a nucleic acid, e.g., an RNA encoding a transgene, into a lentiviral vector. Thus, the systems and methods described herein can include, e.g., 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 that can be used to produce lentiviral vectors are known in the art. See, e.g., Zufferey et al., 1997, Nat. Biotechnol. [Nature Biotechnology] 15: 871-875 and Dull et al., 1998, J. Virol. [Journal of Virology] 72 (11): 8463-8471. Different functions suitable for producing lentiviral vectors can be provided to host cells in a lentiviral packaging system comprising one or more nucleic acids (e.g., plasmids), for example, at least one, two, three or four plasmids, wherein one plasmid encodes a retroviral envelope protein (Env plasmid), one plasmid encodes one or more retroviral packaging proteins, for example, Gag and Pol proteins (packaging plasmid or Gag-Pol plasmid), 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 host cells, wherein 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, wherein one plasmid encodes an expression vector, one plasmid encodes Tat (e.g., pcDNATat), one plasmid encodes Rev protein (e.g., pHCMV-Rev), one plasmid encodes gagpol (e.g., pHCMV-gagpol), and one plasmid encodes VSV-G (e.g., pVSVG), for example, as described in Rout-Pitt et al., J Biol. Methods [Biological Methods] 5 (2): 1-9, 2018). In some embodiments, the plasmid may comprise a dual gene expression cassette, for example, a bicistronic cassette, for example, a bicistronic construct encoding two transgenics of interest. In some embodiments, the first transgenic of interest encodes a first CAR, for example, a CD19 CAR, and the second transgenic of interest encodes a second CAR, for example, a CD22 CAR. In some embodiments, the retroviral packaging protein is derived from a lentivirus, for example, a lentiviral packaging protein, for example, a lentiviral gag and pol protein. In some embodiments, the lentiviral gag protein is a wild-type lentiviral gag protein, and in other embodiments, it has one or more sequence modifications relative to the wild-type sequence.In some embodiments, the lentiviral pol protein is a wild-type lentiviral pol protein, and in other embodiments, it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the rev protein is a wild-type rev protein, and in other embodiments, it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral vector can be a pseudotype vector comprising a modified envelope protein, e.g., an envelope protein or a chimeric envelope protein derived from a different virus, e.g., an Env plasmid can encode a VSV-G Env protein, e.g., a wild-type VSV-G protein or a modified variant.

[0369] In some embodiments, a lentiviral vector is generated using a packaging system comprising the pMDLgpRRE, pRSV-Rev, and pMD.G plasmids (Dull et al., supra), but a kanamycin resistance marker is used, e.g., a marker that confers kanamycin and neomycin resistance, e.g., neomycin phosphotransferase II, rather than the ampicillin gene.

[0370] In some embodiments, the system described herein comprises a transfer vector comprising a kanamycin resistance marker, e.g., a marker conferring resistance to both kanamycin and neomycin, e.g., neomycin phosphotransferase II, e.g., replacing the ampicillin gene. In some embodiments, the transfer vector comprises a sequence from, e.g., a pELPS construct disclosed in WO 2017087861A or Milone et al., Mol. Ther. [Molecular Therapy] 17 (8): 1453-1464, 2009, each of which is incorporated herein by reference in its entirety. In some embodiments, the therapeutic protein is encoded on a self-inactivating transfer vector, and the vector comprises 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 transfer vector lacks one or more of the following, for example, all: a promoter active in bacteria (e.g., lacking all T7 promoters, T3 promoters, and lac promoters), an M13 primer binding site (e.g., lacking both an M13 forward primer binding site and an M13 reverse primer binding site), a phage origin (e.g., f1ori), and a fluorescent protein encoding gene (e.g., GFP, e.g., EGFP). In some embodiments, the transfer vector lacks a CAP binding site and a lac operator. In some embodiments, the transfer vector comprises a pELPS construct as disclosed in WO 2017087861, except that the transfer vector lacks a T7 promoter, an M13 forward primer binding site, a f1 ori, a CAP binding site, an IPTG inducible promoter, a lac operator, an M13 reverse primer binding site, a T3 promoter, and EGFP, wherein optionally the transfer vector encodes a therapeutic protein, e.g., a CAR. In one embodiment, the transfer vector has one or more of the following properties: (a) is more stable than an otherwise similar control transfer vector, (b) results in 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, a f1 ori, a CAP binding site, an IPTG inducible promoter, a lac operator, an M13 reverse primer binding site, and a T3 promoter.

[0371] In some embodiments, the gene expression cassette encodes a protein, e.g., a chimeric antigen receptor (CAR). In some embodiments, the gene expression cassette encodes two proteins, e.g., a first CAR and a second CAR. Exemplary transgenes suitable for gene expression cassettes are described in the present disclosure.

[0372] Transfection

[0373] In some embodiments, different functions for producing lentiviral vectors are provided by transfection (e.g., transient or stable transfection) to multiple host cells of a lentiviral packaging system suitable for producing lentiviral vectors, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells (e.g., multiple Expi293F cells grown in suspension under serum-free conditions). 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%, 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 skilled 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 is provided per million cells for transfection. In some embodiments, a transfection reagent is used to transfect a host cell, e.g., a mammalian cell, e.g., a HEK293 cell, e.g., an Expi293F cell. In some embodiments, a transfection reagent is used. Transfection reagents are well known in the art and can be obtained from commercial suppliers. Examples of transfection reagents include, but are not limited to, Lipofectamine TM (Invitrogen), Polifectamine, LentiTran (Origene), (Polyplus), -AAV (Polypro Corporation) and (Promega). In some embodiments, the transfection reagent is, for example, - 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. - AAV is used at a ratio of about 1:0.5, 1:0.75, 1:1, 1:1.5 or 1:2, or any range therebetween, for transfection.

[0374] In some embodiments, the transfection reagent comprises a synthetic transfection reagent. Synthetic transfection reagents include those that are chemical-based and / or do not contain animal components. In some embodiments, the transfection reagent comprises -AAV transfection reagent. - AAV can be obtained, for example, from Biolipo (850 bd Sébastien Brant, 67400 Illkirch, FRANCE; 1251 Ave of the Americas; 3rd Fl, New York; NY 10020 USA). -AAV is a synthetic, chemical-based, animal-free transfection reagent.

[0375] In some embodiments, at the time of transfection, the density of cells (e.g., Expi293F cells) is about 0.5 x 10 6 cells / mL–1x10 7 cells / mL, 1x10 6 cells / mL–6x10 6 cells / mL, 1x10 6 cells / mL–5x10 6 cells / mL, 1.50x10 6 cells / mL-2.50x10 6 cells / mL, 2.0x10 6 cells / mL–3.0x10 6 cells / mL, 2.0x10 6 cells / mL–2.5x10 6 In some embodiments, at the time of transfection, the cell population has a viability of at least about 80%, 90%, or 95%.

[0376] In some embodiments, the PP / IP (physical particle / infectious particle) ratio after transfection is less than 500, 700, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000.

[0377] Host cells

[0378] Any host cell suitable for expressing a viral vector, e.g., a lentiviral vector, e.g., a lentiviral vector disclosed herein, can be used to practice the methods 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. Many mammalian cell lines are suitable host cells for viral recombinant expression. 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 myeloma (e.g., SP2 / 0 and NS0), and C2C12 cells, as well as transformed primate cell lines, hybridomas, normal diploid cells, and cell lines derived from primary tissues and primary explants cultured in vitro. In some embodiments, the host cell is a HEK293 cell, including cells derived from a HEK293 cell, e.g., a 293F cell, e.g., an Expi293F cell. In some embodiments, at least 80%, at least 85%, at least 90%, at least 90%, at least 95% of the host cells in culture express a large T antigen, e.g., a polyoma virus 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%, at least 95% of the host cells in culture do not express a large T cell antigen. In some embodiments, the host cell is suitable for growth in suspension.

[0379] Cultivation process

[0380] The cell lines described herein can be cultured under conditions that allow the production of lentiviral vector particles with high titers. Eukaryotic cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells can be cultured as: non-anchorage-dependent cells that grow freely in suspension throughout bulk culture; or anchorage-dependent cells that need to attach to a solid substrate for propagation (e.g., as a monolayer).

[0381] In certain embodiments, microcarrier systems can be used to adapt to cell growth. In certain embodiments, microcarrier systems can include suspension cultures, for example, large-scale suspension cultures. Suspension cultures can be operated in open or closed systems, for example, in batches or in batches of fed-batch closed systems. In certain embodiments, nutrients are not added, and waste products are not removed during incubation. In certain embodiments, although cells, products, byproducts and waste products including toxic metabolites are not removed, nutrients are continuously supplied to the system to extend the growth cycle. In certain embodiments, the culture system can be open, for example, a continuous system, for example, a perfusion system or a chemostat system. In certain embodiments, the system can include one or more cell retention devices. The cell retention device can include, for example, microcarriers, fine mesh rotary filters, hollow fibers, flat membrane filters, sedimentation tubes, ultrasonic cell retention devices, etc. In certain embodiments, the cell concentration in the bioreactor is higher than the cell concentration present in the supernatant harvested from the bioreactor. In certain embodiments, the cell concentration in the bioreactor is substantially the same as the cell concentration in the supernatant harvested from the bioreactor.

[0382] In a continuous fermentation process, a defined medium is usually added continuously to a bioreactor while an equal amount of culture volume is removed to recover the product. Continuous cultures typically maintain cells in a logarithmic growth phase at a constant cell density. Continuous or semi-continuous culture methods allow for the regulation of one factor or any number of factors that affect cell growth or end product concentration. For example, a method may limit the carbon source and allow all other parameters to regulate metabolism. In some systems, many factors that affect growth may be constantly changing while the cell concentration measured by the turbidity of the culture medium remains constant. Continuous systems typically maintain steady-state growth, and therefore the cell growth rate is typically balanced with the cell loss caused by the withdrawal of the culture medium from the culture. Methods for regulating nutrients and growth factors for continuous culture processes are known, and a variety of methods are known in the art.

[0383] In some embodiments, a culture of suspension cells comprises only cells that are in suspension. In some embodiments, a culture of suspension cells may comprise a small number (eg, less than 1%) of cells that are adhered (eg, transiently) to a surface.

[0384] "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., untransformed cells), and any other cell populations maintained in vitro.

[0385] In some embodiments, the systems or methods described herein utilize packaging cells or packaging cell lines to produce viral vectors. The cell line can be stably transfected with elements (e.g., retroviral packaging proteins and retroviral envelope proteins) for producing lentiviral vectors. Typically, such packaging cells contain one or more expression cassettes capable of expressing viral proteins (e.g., gag, pol, and env), but the expression cassette does not contain a packaging signal. The packaging cell can be a cell cultured in vitro. The packaging cell line can be used to produce a production cell line for lentiviral particles, for example, by providing at least one plasmid containing at least one transgenic (TOI) expression cassette of interest. In some embodiments, the production cell transiently expresses a plasmid (e.g., a transfer plasmid) encoding a therapeutic effector and containing enough LTR sequences to allow RNA containing one or more LTRs to be packaged into a viral vector. In some embodiments, the production cell line stably expresses an expression cassette encoding a therapeutic effector and containing enough LTR sequences to allow RNA containing one or more LTRs to be packaged into a viral vector.

[0386] Culture medium

[0387] Any suitable medium (e.g., one that supports growth and maintenance of cells under the conditions disclosed herein) can be used to culture eukaryotic cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells. The terms "cell culture medium" and "culture medium" (or simply "culture medium") refer to a nutrient solution used to grow eukaryotic cells (e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells), typically providing 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 culture medium; (2) an energy source, typically in the form of a carbohydrate such as glucose; (3) all essential amino acids, and typically a basic set of twenty amino acids; (4) vitamins and / or other organic compounds that are required at low concentrations; and (5) trace elements, where trace elements are defined as inorganic compounds that are typically required at very low concentrations (typically in the micromolar range). The composition of such culture media is known in the art (e.g., see 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 hereby incorporated 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) hydrolysates of plants, yeasts, and / or tissues, including protein hydrolysates thereof.

[0388] In some embodiments, the culture medium may include serum, for example, fetal bovine serum (FBS). In some embodiments, the culture medium does not contain serum. In some embodiments, the culture medium is chemically defined, for example, the culture medium lacks components of animal origin. As used herein, "animal-derived" components are any components produced in intact animals (such as proteins separated and purified from serum), or components produced in intact animals (such as amino acids prepared by hydrolyzing plant materials using enzymes separated and purified from animals). In contrast, the following proteins are not "animal-derived" components, which have animal protein sequences (i.e., have animal genomic origins), but are produced in vitro in cell cultures using a culture medium lacking components produced in intact animals or separated and purified from intact animals (such as in recombinant yeast or bacterial cells or established continuous eukaryotic cell lines, whether recombinant or not).

[0389] Chemically defined medium is a medium in which all components have known chemical structures. Chemically defined medium is available from commercial suppliers, such as, for example, Sigma, ThermoFisher, Invitrogen, JRH Biosciences, and Gibco. In some embodiments, the medium is FreeStyle TM 293 expression medium. In some embodiments, concentrated serum may be used, for example, a medium containing a higher concentration of nutrients than is normally required and normally provided to a growing culture. In some embodiments, the medium may contain one or more amino acids derived from any source or method known in the art.

[0390] In some embodiments, enzymes can be added to the culture medium, e.g., nucleases, e.g., endonucleases, e.g., recombinant endonucleases, e.g., 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 In some embodiments, between 2 U / mL and 10 U / mL, between 10 U / mL and 20 U / mL, between 20 U / mL and 30 U / mL, between 30 U / mL and 40 U / mL, between 40 U / mL and 50 U / mL, or between 50 U / mL and 60 U / mL is added. In some embodiments, the addition of the cytokine is performed after about 5-40, 10-40, 10-30, 20-30, or about 20 hours or about 24 hours after transfection of the host cell, e.g., Expi293F cell. In some embodiments, benzonatase is added at a concentration of 3-7U / mL (e.g., about 5U / mL) 20-30 hours (e.g., about 24 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 3-7U / mL (e.g., about 5U / mL) 1-5 hours (e.g., about 3 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 3-7U / mL (e.g., about 5U / mL) 4-8 hours (e.g., about 6 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 12-18U / mL (e.g., about 15U / mL) 1-5 hours (e.g., about 3 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 12-18U / mL (e.g., about 15U / mL) 4-8 hours (e.g., about 6 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 12-18U / mL (e.g., about 15U / mL) 20-30 hours (e.g., about 24 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 20-30U / mL (e.g., about 25U / mL) 1-5 hours (e.g., about 3 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 20-30U / mL (e.g., about 25U / mL) 4-8 hours (e.g., about 6 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 20-30U / mL (e.g., about 25U / mL) 20-30 hours (e.g., about 24 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 40-60U / mL (e.g., about 50U / mL) 1-5 hours (e.g., about 3 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 40-60U / mL (e.g., about 50U / mL) 4-8 hours (e.g., about 6 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 40-60U / mL (e.g., about 50U / mL) 20-30 hours (e.g., about 24 hours) after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 5U / mL about 3 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 15U / mL about 3 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 25U / mL about 3 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 50U / mL about 3 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 5U / mL about 6 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 15U / mL about 6 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 25 U / mL about 6 hours after transfection of the host cells.In some embodiments, benzonatase is added at a concentration of 50 U / mL about 6 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 5 U / mL about 24 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 15 U / mL about 24 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 25 U / mL about 24 hours after transfection of the host cells. In some embodiments, benzonatase is added at a concentration of 50 U / mL about 24 hours after transfection of the host cells. In some embodiments, salt, e.g., MgCl. 2 Add to In some embodiments, the methods disclosed herein may include adding in the production and / or purification process

[0391] In some embodiments, the compound can be added to the culture medium to affect culture growth, for example, inhibit proliferation, induce differentiation, and induce or inhibit gene expression. In some embodiments, the compound is sodium butyrate. In some embodiments, the cell culture medium described herein comprises sodium butyrate.

[0392] Culture and transfection conditions

[0393] Culture conditions can include any culture conditions suitable for maintaining cells (e.g., in a static or proliferative state). 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), stirring level (e.g., revolutions per minute), airflow rate (e.g., air, oxygen, nitrogen), redox potential, cell density (e.g., optical density), cell viability, etc. Changes in culture conditions can include changes, modifications, or conversions in one or more culture parameters. For example, the culture conditions can be changed by increasing or decreasing temperature, increasing or decreasing pH (e.g., adding or removing acid, alkali, 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 stirring and / or adding or removing nutrients (e.g., one or more sugars or sugar sources, biomass, vitamins, etc.), increasing or decreasing the ratio of culture and flask volume, or a combination of the foregoing. In some embodiments, changes in culture conditions are introduced at a certain time during the culture, e.g., before transfection, e.g., increasing or decreasing pH. In some embodiments, the pH is modified, 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.

[0394] Culture volume and culture unit

[0395] The method disclosed herein can be carried out in small cell culture, for example, in laboratory scale, or in large-scale culture, for example, in industrial scale. The method can be carried out in an appropriate culture unit, for example, a culture bottle or a bioreactor. The bioreactor can be of any size as long as it can be used to culture cells, for example, mammalian cells. In some embodiments, the method disclosed herein is highly scalable, for example, a plurality of mammalian cells in a scaled culture (for example, at least 1L, at least 2L, at least 5L, at least 10L, at least 15L, at least 20L), the number of transduction units per ml of culture produced is not less than 30%, 40%, 50%, 60%, 70% or 80% of the number of transduction units per ml of culture in otherwise similar small-scale cultures (for example, 100ml, 200ml, 300ml, 400ml, 500ml). In some embodiments, scale culture (i.e., culture volume greater than 50L) and may be particularly suitable for expansion from small laboratory scale culture (for example, 10L) to production scale culture (for example, 50L and larger) with minimal modification of culture conditions. The internal conditions of the culture unit, including but not limited to pH, pO 2 The production culture unit refers to the final culture unit for producing polypeptides, viruses and / or any other purpose products. The volume of the large-scale production culture unit is generally greater than about 50 liters, and can be about 100, about 200, about 300, about 500, about 800, about 1000, about 2500, about 5000, about 8000, about 10,000, about 12,0000L or more or any intermediate volume. Suitable culture unit or production culture unit can be composed of (i.e., composed of) any material suitable for keeping the cell culture suspended in the culture medium under the culture conditions considered herein and helping mammalian cells such as HEK293 cells, such as Expi293F cell growth and viability. Examples of suitable materials include but are not limited to glass, plastics and / or metals. In certain embodiments, one or more materials do not interfere, or do not significantly or substantially interfere with the expression and / or stability of the desired product such as a lentiviral vector.

[0396] In some embodiments, the cell culture process is carried out in more than one different culture unit, such as using one or more seed culture units, followed by a production culture unit. In some embodiments, the process involves transferring the propagated seed culture from one or more seed culture units to a large production unit. In some embodiments, the expansion of cells to the production culture unit and the production phase can be completed in one physical culture unit, for example, the cells can be expanded to the final production scale and the process can be switched to production conditions. The spent culture medium is harvested at the end of the culture period for downstream processing of lentivirus or lentiviral vectors. In some embodiments, the harvest can be collected 24 hours, 48 ​​hours, 72 hours, 96 hours, or 120 hours after transfection.

[0397] 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 contains the lentivirus at a concentration of, for example, about 5×10 6 Transduction units / mL (TU / mL) to about 6x10 9 TU / mL (e.g., 5x10 6 TU / mL, 5.5x10 6 TU / mL, 6x10 6 TU / mL, 6.5x10 6 TU / mL, 7x10 6 TU / mL, 7.5x10 6 TU / mL, 8x10 6 TU / mL, 8.5x10 6 TU / mL, 9x10 6 TU / mL, 9.5x10 6 TU / mL, 1x10 7 TU / mL, 1.5x10 7 TU / mL, 2x10 7 TU / mL, 2.5x10 7 TU / mL, 3x10 7 TU / mL, 3.5x10 7 TU / mL, 4x10 7 TU / mL, 4.5x10 7 TU / mL, 5x10 7 TU / mL, 5.5x10 7 TU / mL, 6x10 7 TU / mL, 6.5x10 7 TU / mL, 7x10 7 TU / mL, 9x10 7 TU / mL, 1x108 TU / mL, 2.5x10 8 TU / mL, 5x10 8 TU / mL, 8x10 8 TU / mL, 1x10 9 TU / mL, 1.5x10 9 TU / mL, 2x10 9 TU / mL, 2.5x10 9 TU / mL, 3x10 9 TU / mL, 4x10 9 TU / mL, 5x10 9 TU / mL or 6x10 9 TU / mL). In some embodiments, the viral harvest collected at the end of the culture period contains lentivirus at a concentration of at least 5×10 6 TU / mL, 5.5x10 6 TU / mL, 6x10 6 TU / mL, 6.5x10 6 TU / mL, 7x10 6 TU / mL, 7.5x10 6 TU / mL, 8x10 6 TU / mL, 8.5x10 6 TU / mL, 9x10 6 TU / mL, 9.5x10 6 TU / mL, 1x10 7 TU / mL, 1.5x10 7 TU / mL, 2x10 7 TU / mL, 2.5x10 7 TU / mL, 3x10 7 TU / mL, 3.5x10 7 TU / mL, 4x10 7 TU / mL, 4.5x10 7 TU / mL, 5x10 7 TU / mL, 5.5x10 7 TU / mL, 6x10 7 TU / mL, 6.5x10 7 TU / mL, 7x10 7 TU / mL, 9x10 7 TU / mL, 1x10 8 TU / mL, 2.5x10 8 TU / mL, 5x10 8 TU / mL, 8x108 TU / mL, 1x10 9 TU / mL, 1.5x10 9 TU / mL, 2x10 9 TU / mL, 2.5x10 9 TU / mL, 3x10 9 TU / mL, 4x10 9 TU / mL, 5x10 9 TU / mL or 6x10 9 TU / mL. In some embodiments, the viral harvest collected at the end of the culture period contains lentivirus at a concentration of 5x10 6 TU / mL-6x10 6 TU / mL, 6x10 6 TU / mL-7x10 6 TU / mL, 7x10 6 TU / mL-8x10 6 TU / mL, 8x10 6 TU / mL-9x10 6 TU / mL, 9x10 6 TU / mL-1x10 7 TU / mL, 1x10 7 TU / mL-2x10 7 TU / mL, 2x10 7 TU / mL-3x10 7 TU / mL, 3x10 7 TU / mL-4x10 7 TU / mL, 4x10 7 TU / mL-5x10 7 TU / mL, 5x10 7 TU / mL-6x10 7 TU / mL, 6x10 7 TU / mL-7x10 7 TU / mL, 7x10 7 TU / mL-9x10 7 TU / mL, 9x10 7 TU / mL-1x10 8 TU / mL, 1x10 8 TU / mL-2.5x10 8 TU / mL, 2.5x10 8 TU / mL-5x10 8 TU / mL, 5x10 8 TU / mL-8x108 TU / mL, 8x10 8 TU / mL-1x10 9 TU / mL, 1x10 9 TU / mL-1.5x10 9 TU / mL, 1.5x10 9 TU / mL-2x10 9 TU / mL, 2x10 9 TU / mL-2.5x10 9 TU / mL, 2.5x10 9 TU / mL-3x10 9 TU / mL, 3x10 9 TU / mL-4x10 9 TU / mL, 4x10 9 -5x10 9 TU / mL, or 5x10 9 -6x10 9 TU / mL.

[0398] Purification methods include filtration and chromatography

[0399] In some aspects, the present disclosure provides a process for purifying a lentiviral vector with improved efficiency, for example, so as to recover a higher amount of lentiviral vector. In some embodiments, at least one step in the purification process includes adding a reagent (e.g., an amino acid or a salt thereof, for example, arginine or a salt thereof, for example, arginine-HCl) to a purified intermediate composition (an intermediate composition comprising a buffer before purification is completed) before further purification (e.g., centrifugation, filtration, or chromatography) to improve the purification process. In some embodiments, filtration may refer to, but is not limited to, flow filtration, depth filtration, tangential flow filtration. In some embodiments, chromatography may include, but is not limited to, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography.

[0400] In some embodiments, the 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.

[0401] In some embodiments of the methods described herein, the solution or suspension is subjected to a semipermeable membrane (filtration) that retains larger particles, such as viral particles, while allowing solvents and small solute molecules to pass through. In some embodiments, the methods described herein use filters to remove and exchange salts, sugars, and non-aqueous solvents, separate free species from bound species, remove low molecular weight materials, and / or cause rapid changes in ions and / or pH environments. The filtration step can be used to increase the concentration of the carrier in the solution or suspension. In some embodiments, the filtration step is used to increase the concentration of lentiviral particles at harvest. In some embodiments, the methods described herein utilize processes, techniques, or combinations of techniques, comprising sequential or simultaneous filtration steps (e.g., one or more of microfiltration, ultrafiltration, nanofiltration, and diafiltration). In some embodiments, filtration is performed using a flat membrane or hollow fiber. In some embodiments, filtration is performed using an average transmembrane pressure of about 0.1-0.5 bar (e.g., about 0.1, 0.2, 0.3, 0.4, or 0.5 bar). In some embodiments, 4-100 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 are filtered. In certain embodiments of the present disclosure, the filtration step is used to exchange various buffers associated with the present disclosure, optionally combined with chromatography or other purification steps, and optionally remove impurities from the virus yield.

[0402] Filtration techniques, such as those described above and known in the art, can be used to produce lentiviral preparations that are substantially free of 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 cells that produce the lentiviral vector, such as DNA, RNA, or other polynucleotides not included in the lentiviral transgene).

[0403] In some embodiments, a composition substantially free of a particular component contains less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.01%, less than 0.001% of the component in the composition by weight. In some embodiments, in a composition substantially free of a particular component, the component is undetectable, for example according to conventional methods known in the art.

[0404] Buffers, e.g., for purification

[0405] Various buffers, for example, aqueous compositions comprising buffers for viral vector purification are known in the art and may include, but are not limited to, sulfonic acid buffers, for example, 1,4-piperazinediethanesulfonic acid (PIPES)-based buffers (PIPES buffers), polyol buffers, tris buffers, phosphate buffers, acetate buffers, citrate buffers. In some embodiments, the buffer used in connection with the purification process disclosed herein is a sulfonic acid buffer, for example, a PIPES buffer. In some embodiments, the PIPES buffer may comprise a buffer, for example, 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, for example, about 20 mM PIPES.

[0406] In some embodiments, the purification buffer may further comprise a salt, such as sodium chloride (NaCl), magnesium chloride (MgCl 2 ) or calcium chloride (CaCl 2 ) or any combination thereof. The salt can be present in the aqueous lentiviral formulation, for example, 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, mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM, 375mM, 400mM, 450mM, 475mM, 500mM, 525mM, 575mM, 600mM, 625 mM, 650mM, 675mM, 700mM, 725mM, 750mM, 775mM, 800mM, 825mM, 850mM, 875mM, 900mM, 925mM, 950mM, 957mM or 1M). In some embodiments, the concentration of the salt is about 25mM to about 250mM, about 50mM to about 75mM, about 50mM to about 200mM, or about 100mM to about 150mM (e.g., 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 100mM, 125mM, or 150mM). In some embodiments, the concentration of the salt can be 50mM or 75mM, as desired.

[0407] In some embodiments, the purification buffer may further include carbohydrates, e.g., non-reducing carbohydrates, e.g., sucrose or trehalose. In some embodiments, carbohydrates such as sucrose are present at a concentration of about 30mM to about 300mM, about 40mM to about 275mM, about 50mM to about 250mM, about 60mM to about 240mM, about 70mM to about 220mM, about 30mM to 150mM, or about 150-300mM. In some embodiments, the purification buffer (e.g., PIPES buffer) includes sucrose at a concentration of about 50mM to about 80mM (e.g., about 73mM). In some embodiments, the purification buffer (e.g., PIPES buffer) includes sucrose at a concentration of about 200mM to 250mM (e.g., about 220mM).

[0408] In some embodiments, 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% / volume (w / v) of the aqueous lentiviral formulation by weight during manufacture. For example, a carbohydrate (such as a non-reducing carbohydrate as described herein) can be present in an aqueous lentiviral formulation 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 during manufacture. In some embodiments, a carbohydrate (such as a non-reducing carbohydrate described herein) may be present in the aqueous lentiviral formulation during manufacturing 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, a carbohydrate (such as a non-reducing carbohydrate described herein) may be present in the aqueous lentiviral formulation during manufacturing at a concentration of 1% w / v-2% w / v, 2% w / v-3% w / v, 3% w / v-4% w / v, 4% w / v-5% w / v, 5% w / v-6% w / v, 6% w / v-7% w / v, 7% w / v-8% w / v, 8% w / v-9% w / v, 9% w / v-10% w / v.

[0409] In some embodiments, the buffer comprises arginine or a salt thereof, e.g., arginine-HCl. In some embodiments, the agent, e.g., arginine or a salt thereof, e.g., 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 (e.g., about 300) mM. In some embodiments, at least one buffer, e.g., a PIPES buffer for virus purification (e.g., lentivirus purification using a process disclosed herein) comprises arginine, e.g., arginine-HCl. In some embodiments, the pH of the buffer used in the purification process 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.

[0410] In some embodiments, the PIPES buffer can be used as one or more of an exchange buffer and / or a filtration buffer. In some embodiments, the concentration ratio of PIPES, NaCl and sucrose is different in the PIPES filtration buffer and the PIPES exchange buffer. In some embodiments, the concentration ratio of PIPES, NaCl and sucrose is the same in the PIPES filtration buffer and the PIPES exchange buffer. In some embodiments, the concentration ratio of PIPES, NaCl and sucrose is the same in the PIPES exchange buffer and the PIPES filtration buffer.

[0411] Arginine spike

[0412] In some embodiments, arginine, such as arginine-HCl, is added to the cell culture harvest during purification. In some embodiments, arginine, such as arginine-HCl, is added to a purified intermediate composition comprising a buffer such as a PIPES buffer or a PIPES buffer during purification. In some embodiments, arginine, such as arginine-HCl, is added to a PIPES buffer not comprising arginine. In some embodiments, arginine, such as arginine-HCl, is added to a PIPES buffer comprising arginine. In some embodiments, an agent, such as arginine or a salt thereof, such as arginine monochloride (arginine-HCl) is added at a concentration of about 25-50mM (e.g., about 50mM), 50-100mM (e.g., about 75mM), 100-200mM (e.g., about 150mM) or 200-400 (e.g., about 300) mM.

[0413] In some embodiments, the vector recovery rate (e.g., the amount of transduction units of the lentivirus) is increased by about 10%-300%, about 20%-180%, about 30%-160%, about 50%-150%, about 75%-125%, or about 100% in a purification process comprising a purification step (the purification step comprising adding arginine to the purified intermediate composition) relative to a purification process that does not include a purification step (the purification step comprises adding arginine to the purified intermediate composition). In some embodiments, the addition of arginine reduces the process time of purification. In some embodiments, when purification includes adding, for example, arginine or a salt thereof (e.g., arginine-HCl) to the purified intermediate composition, the process 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 an otherwise similar purification process that does not include adding arginine to the purified intermediate composition. In some embodiments, the purified intermediate composition after adding arginine or a salt thereof (e.g., arginine-HCl) and subsequent purification steps shows a total particle concentration of less than 400,000, less than 300,000, less than 200,000, or less than 100,000 per ml, as measured by microfluidic imaging. Without wishing to be bound by theory, in some embodiments, microfluidic imaging does not substantially detect individual lentiviral particles (e.g., infectious viral particles), but rather detects larger particles comprising aggregates, e.g., aggregates of non-functional viruses. In some embodiments, the purified intermediate composition after adding arginine or a salt thereof (e.g., arginine-HCl) and subsequent purification steps shows a particle concentration of less than about 5,000, about 4,500, about 4,000, about 3,500, about 3,000, or about 2,500 per ml of ≥10 μm, as measured by microfluidic imaging. In some embodiments, the purified intermediate composition after addition of arginine or a salt thereof (e.g., arginine-HCl) and subsequent purification steps exhibits a concentration of less than about 500, about 400, about 300, or about 200 μM per ml ≥ 25 μM as measured by microfluidic imaging. In some embodiments, reducing aggregates reduces clogging of the filter membrane at a given time point. In some embodiments, arginine stabilizes the lentiviral particles.

[0414] In some embodiments, the purified lentiviral composition comprises a concentration of, for example, about 1×10 7 Transduction units / mL (TU / mL) to about 7x10 7 TU / mL (e.g., 1x10 7 TU / mL, 1.5x10 7 TU / mL, 2x10 7 TU / mL, 2.5x10 7 TU / mL, 3x107 TU / mL, 3.5x10 7 TU / mL, 4x10 7 TU / mL, 4.5x10 7 TU / mL, 5x10 7 TU / mL, 5.5x10 7 TU / mL, 6x10 7 TU / mL, 6.5x10 7 TU / mL or 7x10 7 TU / mL) of lentiviral vector.

[0415] Aqueous compositions, e.g., for lentiviral formulation and storage

[0416] In some embodiments, the present disclosure provides a formulation, 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 a storage buffer, e.g., a HEPES buffer, e.g., a HEPES buffer comprising free positively charged amino acids (e.g., arginine (e.g., L-arginine), lysine, or histidine) and free non-polar amino acids (e.g., proline (e.g., L-proline), methionine, or tryptophan)). In some embodiments, a lentiviral formulation comprising a formulation buffer or a storage buffer (e.g., a HEPES buffer, e.g., a HEPES buffer comprising free positively charged amino acids (e.g., arginine (e.g., L-arginine), lysine, or histidine) and free non-polar amino acids (e.g., proline (e.g., L-proline), methionine, or tryptophan)) exhibits improved biological properties relative to a lentiviral formulation containing a conventional lentiviral formulation buffer (e.g., PIPES). These improved biological characteristics include increased resistance to aggregation within a range of temperatures herein. In some embodiments, HEPES buffer shows improved transduction capacity at physiological temperature and shows greater resistance to loss of infectivity during multiple freeze / thaw cycles. Other buffers that can be used in conjunction with the lentiviral preparations disclosed herein include histidine buffer, phosphate buffer, sodium citrate buffer, MES buffer, MOPS buffer, and PIPES buffer. The lentiviral preparations disclosed herein may optionally include salts, such as sodium chloride, and may optionally contain carbohydrates, such as non-reducing carbohydrates.

[0417] In some embodiments, the HEPES formulation buffer and / or storage buffer can comprise a buffer, for example, HEPES at a concentration of about 10 mM to about 200 mM, about 10 mM to about 150, about 10 mM to about 100 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, or about 15 mM to about 25 mM, for example, about 20 mM.

[0418] The lentiviral vector preparation disclosed herein may include amino acids or salts thereof; such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine or salts thereof. It should be understood that unless the context clearly specifies otherwise (e.g., compared to referring to a polypeptide molecule consisting of a series of amino acids), "amino acid" is synonymous with "free amino acid" in this context. Therefore, in some embodiments, the HEPES formulation buffer and / or storage buffer may include one or both of free positively charged amino acids (e.g., arginine, lysine or histidine) or salts thereof and free non-polar amino acids (e.g., proline, methionine or tryptophan) or salts thereof.

[0419] In some embodiments, the HEPES formulation buffer and / or storage buffer comprises free arginine (e.g., L-arginine) or a salt thereof. In some embodiments, the concentration of free arginine or a salt thereof is at least 25, 50, 75, 100, or 150 mM. In some embodiments, the concentration of free arginine or a salt thereof is about 150 mM.

[0420] In some embodiments, the HEPES formulation buffer and / or storage buffer comprises free proline (e.g., L-proline) or a salt thereof. In some embodiments, the concentration of free proline or a salt thereof is 25-200, 50-200, 100-200, 125-175, or 150 mM. In some embodiments, the concentration of free proline or a salt thereof is about 150 mM.

[0421] In some embodiments, the HEPES formulation buffer and / or storage buffer comprises both free arginine and free proline or salts thereof (eg, arginine-HCl and proline-HCl).

[0422] In some embodiments, the HEPES formulation buffer and / or storage buffer comprises a total amount of less than 20, less than 10, less than 5, less than 2, or less than 1 mM of inorganic salts. In some embodiments, the HEPES formulation buffer and / or storage buffer comprises a total amount of inorganic salts between about 0.1 mM to about 20 mM. In some embodiments, the HEPES formulation buffer and / or storage buffer is substantially free of (e.g., free of) inorganic salts. In some embodiments, the HEPES formulation buffer and / or storage buffer is free of inorganic salts. In some embodiments, the HEPES formulation buffer and / or storage buffer is substantially free of (e.g., free of) NaCl and MgCl 2 In some embodiments, the HEPES formulation buffer and / or storage buffer does not contain NaCl and MgCl 2 One or both of the following.

[0423] The lentiviral vector preparation of the present disclosure may further contain carbohydrates, such as non-reducing carbohydrates as described herein, for example, cryoprotectants. Exemplary non-reducing carbohydrates include sucrose and trehalose, etc. When included in a lentiviral vector preparation, carbohydrates (e.g., non-reducing carbohydrates, e.g., sucrose) can be present at a concentration of, for example, about 25-200mM, about 50-200mM, about 100-200mM, about 125-175mM, or about 150mM. For example, carbohydrates (non-reducing carbohydrates as described herein, e.g., sucrose) can be present in aqueous lentiviral preparations at a concentration of about 150mM.

[0424] In some embodiments, HEPES preparation buffer and / or storage buffer further include stabilizer. In some embodiments, HEPES preparation buffer and / or storage buffer further include human serum albumin (HSA). In some embodiments, HSA is human-derived HSA (e.g., HSA separated from human serum). Human-derived HSA can be obtained by, for example, separating HSA from human serum according to conventional methods known in the art. In some embodiments, HSA is recombinant HSA (rHSA). In some embodiments, HSA is present in 0.5%-3%, 0.5%-2%, 0.5%-1%, 1%-2%, 1.5%-2.5% or 2% w / v. In some embodiments, HSA is present in about 2% w / v. In other embodiments, HEPES preparation buffer and / or storage buffer are substantially free of (e.g., free of) HSA. In other embodiments, HEPES preparation buffer and / or storage buffer are free of HSA.

[0425] The lentiviral vector preparations described herein can exhibit, for example, a pH of about 6.0 to about 7.5, for example, 6.0-7.5, 6.0-7.0, 6.0-6.5, 6.5-7.0, 6.2-6.8, 6.4-6.6, or 6.5. In some embodiments, the pH of the lentiviral vector preparation is 6.5.

[0426] In some embodiments, HEPES formulation buffer and / or storage buffer are substantially free of (e.g., free of) one, two or three of PEG lipids, F108 and cholesterol. In some embodiments, HEPES formulation buffer and / or storage buffer are free of one, two or three of PEG lipids, F108 and cholesterol.

[0427] In some embodiments, the HEPES formulation buffer and / or storage buffer comprises L-arginine, L-proline, sucrose, and is substantially free of inorganic salts. In some embodiments, the HEPES formulation buffer and / or storage buffer comprises L-arginine, L-proline, sucrose, and is substantially free of inorganic salts.

[0428] In some embodiments, the HEPES formulation buffer and / or storage buffer comprises L-arginine at a concentration of 100-200 mM, L-proline at a concentration of 25-200 mM, and sucrose at a concentration of 25-200 mM.

[0429] In some embodiments, the HEPES formulation buffer and / or storage buffer comprises 20 mM HEPES, 150 mM L-arginine, 150 mM L-proline, and 150 mM sucrose. In some embodiments, the HEPES formulation buffer and / or storage buffer further comprises 2% w / v HSA.

[0430] In some embodiments, the HEPES formulation buffer and the HEPES storage buffer comprise the same composition. In some embodiments, the HEPES formulation buffer and the HEPES storage buffer comprise different compositions.

[0431] The lentiviral vector can be present in the lentiviral preparation of the present disclosure at a certain concentration range. For example, the lentiviral vector can be present at, for example, about 1×10 7 Transduction units / mL (TU / mL) to about 1x10 9 TU / mL (e.g., 1x10 7 TU / mL, 2x10 7 TU / mL, 3x10 7 TU / mL, 4x10 7 TU / mL, 5x10 7 TU / mL, 6x107 TU / mL, 7x10 7 TU / mL, 8x10 7 TU / mL, 9x10 7 TU / mL, 1x10 8 TU / mL, 1.5x10 8 TU / mL, 2x10 8 TU / mL, 2.5x10 8 TU / mL, 3x10 8 TU / mL, 3.5x10 8 TU / mL, 4x10 8 TU / mL, 4.5x10 8 TU / mL, 5x10 8 TU / mL, 5.5x10 8 TU / mL, 6x10 8 TU / mL, 6.5x10 8 TU / mL, 7x10 8 TU / mL, 7.5x10 8 TU / mL, 8x10 8 TU / mL, 8.5x10 8 TU / mL, 9x10 8 TU / mL, 9.5x10 8 TU / mL, 1x10 9 TU / mL, 2x10 9 TU / mL, 3x10 9 TU / mL, 4x10 9 TU / mL, 5x10 9 TU / mL or 6x10 9 TU / mL) is present in the lentiviral preparation. If desired, the lentiviral preparation may contain a concentration of about 3x10 8 TU / mL to about 5x10 8 TU / mL (e.g., 3x10 8 TU / mL, 3.5x10 8 TU / mL, 4x10 8 TU / mL, 4.5x10 8 TU / mL or 5x10 8 TU / mL) of lentiviral vector.

[0432] In some embodiments, the aqueous composition comprises cholesterol. In some embodiments, the aqueous composition comprises MgCl 2In some embodiments, the aqueous composition comprises lysine. In some embodiments, the aqueous composition comprises lactose. In some embodiments, the aqueous composition comprises sorbitol. In some embodiments, the aqueous composition comprises glycerol. In some embodiments, the aqueous composition comprises PEG lipids. In some embodiments, the aqueous composition comprises F108. In some embodiments, the aqueous composition comprises glutamic acid.

[0433] In some embodiments, an aqueous composition (e.g., an aqueous composition comprising a lentiviral vector described herein) can be stored at a low temperature, e.g., at 10°C, at 6°C, at 4°C, at 0°C, at -10°C, at -20°C, at -30°C, at -40°C, at -50°C, at -60°C, at -70°C, at -80°C, or at -90°C for a period of time, e.g., for about 20 minutes, 40 minutes, 60 minutes, 1.5 hours, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days, 25 days, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, or longer. In some embodiments, the aqueous composition is stored at less than 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, the purified lentiviral sample stored in HEPES storage buffer is stored at -80°C under frozen conditions immediately after purification. In some embodiments, the lentiviral preparation thus stored can be thawed and refrozen (e.g., freeze-thaw cycles) before use. In some embodiments, the lentiviral preparations prepared and stored as disclosed herein can undergo at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 freeze-thaw cycles without any significant loss of stability and / or infectivity. In some embodiments, the formulation exhibits 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 in stability and / or infectivity compared to a lentiviral formulation that has never undergone a freeze-thaw cycle.

[0434] In some embodiments, the lentiviral formulations disclosed herein can be stored under refrigeration at 4° C. for a period of time, for example, for about 20 minutes, 40 minutes, 60 minutes, 1.5 hours, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days, 25 days, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months or more. In some embodiments, the lentiviral preparations disclosed herein can be stored under -80°C freezing conditions for a period of time, for example, for about 20 minutes, 40 minutes, 60 minutes, 1.5 hours, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days, 25 days, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months or longer. In some embodiments, compared to lentivirus that has never been frozen, the lentiviral preparations disclosed herein (e.g., stored under freezing conditions) show 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. In some embodiments, after undergoing more than one (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) freeze-thaw cycles, the infectivity loss of the lentiviral preparation is no more than 0.5%, no more than 1%, no more than 2%, no more than 5%, no more than 7%, 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%, no more than 80%. In some embodiments, the lentiviral preparations disclosed herein (e.g., stored under frozen conditions) 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% stable compared to lentivirus that has never been frozen. In some embodiments, the lentiviral preparation is used after freezing 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, at least 7 days to improve vector integration.

[0435] The present disclosure further includes dried or lyophilized compositions prepared by drying or lyophilizing aqueous compositions described herein, as well as aqueous compositions prepared by reconstituting such dried or lyophilized compositions in a buffer described herein (or another standard vehicle for administration).

[0436] While the HEPES buffers disclosed in this section are suitable for lentiviral formulation and storage, they can also be used in other steps of the lentiviral manufacturing and purification process, such as during filtration (e.g., ultrafiltration prior to sterile filtration).

[0437] The present disclosure provides, inter alia, a formulation comprising a lentiviral vector described herein and an aqueous composition. As used herein, "formulation" is synonymous with "composition". In some embodiments, the composition is a pharmaceutical composition.

[0438] CAR Target

[0439] Described herein are viral vectors for transducing immune effector cells (e.g., T cells, NK cells), which are engineered to contain one or more chimeric antigen receptors (CARs) that direct immune effector cells to undesirable cells (e.g., cancer cells). This is achieved by the antigen binding domains on CAR, which are specific to cancer-associated antigens. Two types of cancer-associated antigens (tumor antigens) that CAR can target are: (1) cancer-associated antigens expressed on the surface of cancer cells; and (2) cancer-associated antigens in cells themselves, however, fragments of this antigen (peptide) are presented on the surface of cancer cells by MHC (major histocompatibility complex).

[0440] In some embodiments, the tumor antigen is selected from one or more of the following: CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, 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 member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD21 3A2); mesothelin; interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testosterone 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); prostatic enzyme; prostatic acid phosphatase (PAP); mutated elongation factor 2 (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (pro, megalin factor) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); by breakpoint Oncogene fusion protein composed of clustering region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-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 beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related protein (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycosylceramide (GloboH); breast differentiation antigen (NY-BR-1); urine protein 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVC R1); adrenergic receptor beta 3 (ADRB3); pan-nexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma 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 mutants; prostate-specific protein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1); 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 myelocytoma 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 (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3);Paired box protein Pax-5 (PAX5); pre-acrosomal protein binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); legumin; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (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); mucin-like hormone receptor-like 2 with EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). ;

[0441] CAR described herein may include an antigen binding domain (e.g., an antibody or antibody fragment, TCR or TCR fragment) bound to a tumor-supporting antigen (e.g., an antigen supporting a tumor as described herein). In some embodiments, the tumor-supporting antigen is an antigen present on a stromal cell or a bone marrow-derived containment cell (MDSC). 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, cells expressing CAR destroy tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival.

[0442] In an embodiment, the stromal cell antigen is selected from one or more of the following: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) and tenascin. In one embodiment, the FAP-specific antibody is sirolimus, competing with sirolimus for binding, or having the same CDR as sirolimus. In an embodiment, the MDSC antigen is selected from one or more of the following: CD33, CD11b, C14, CD15 and CD66b. Therefore, in some embodiments, the tumor-supporting antigen is selected from one or more of the following: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) or tenascin, CD33, CD11b, C14, CD15, and CD66b.

[0443] CD19

[0444] A non-limiting exemplary tumor antigen is CD19. CARs that bind CD19 are known in the art. For example, those disclosed in WO2012 / 079000 and WO2014 / 153270 can be used according to the present disclosure. Any CD19 CAR known 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 is described in the following: U.S. Pat. No. 8,399,645; U.S. Pat. No. 7,446,190; Xu et al., Leuk Lymphoma. 2013 54(2):255-260 (2012); Cruz et al., Blood 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) [American Society for Gene and Cell Therapy (ASGCT) 16th Annual Meeting] (May 15-18, Salt Lake City) 2013, Abstract 10.

[0445] Non-limiting exemplary CD19 CARs include the CD19 CARs described herein, or the anti-CD19 CARs 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, NC T02546739, NCT02656147, NCT02772198, NCT00709033, NCT02081937, NCT00924326, NCT02735083, NCT02794246, NCT02746952, NCT0159369 6. NCT02134262, NCT01853631, NCT02443831, NCT02277522, NCT02348216, NCT02614066, NCT02030834, NCT02624258, NCT02625480, NCT02 030847, NCT02644655, NCT02349698, NCT02813837, NCT02050347, NCT01683279, NCT02529813, NCT02537977, NCT02799550, NCT02672501, N CT02819583, NCT02028455, NCT01840566, NCT01318317, NCT01864889, NCT02706405, NCT01475058, NCT01430390, NCT02146924, NCT02051 257. NCT02431988, NCT01815749, NCT02153580, NCT01865617, NCT02208362, NCT02685670, NCT02535364, NCT02631044, NCT02728882, NCT0 2735291, 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.

[0446] In some embodiments, the antigen binding domain binds CD19 and has the same or similar binding specificity as the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. [Molecular Immunology] 34 (16-17): 1157-1165 (1997). In some embodiments, the antigen binding domain binds CD19 and includes a scFv fragment described in Nicholson et al. Mol. Immun. [Molecular Immunology] 34 (16-17): 1157-1165 (1997).

[0447] In some embodiments, the antigen binding domain (e.g., a humanized antigen binding domain) binds CD19 and comprises a sequence from Table 3 of WO2014 / 153270 (incorporated herein by reference). WO2014 / 153270 also describes methods for determining the binding and efficacy of various CAR constructs.

[0448] For clinical settings, humanization of mouse CD19 antibodies may be desirable, where mouse-specific residues can induce human-anti-mouse antigen (HAMA) responses in patients receiving CART19 therapy (i.e., T cell therapy transduced with CAR19 constructs). The generation, characterization, and efficacy of humanized CD19 CAR sequences are described in International Application WO2014 / 153270, which is incorporated herein by reference in its entirety, including Examples 1-5 (pages 115-159).

[0449] In certain embodiments, antigen binding domains include the parent mouse scFv sequence of the CAR19 construct provided in WO2012 / 079000 (incorporated herein by reference). In certain embodiments, antigen binding domains bind CD19 and include the scFv described in WO2012 / 079000.

[0450] In some embodiments, the CD19 CAR comprises a fusion polypeptide sequence provided as SEQ ID NO: 12 in WO2012 / 079000, which provides a murine scFv fragment that specifically binds to human CD19.

[0451] In some embodiments, the CD19 CAR comprises the amino acid sequence provided as SEQ ID NO: 12 in WO2012 / 079000.

[0452] In some embodiments, the CD19 CAR comprises the amino acid sequence:

[0453] diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggs gggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgq gtsvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggc elrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr(SEQ ID NO:757), or a sequence substantially homologous thereto.

[0454] In some embodiments, the CD19 CAR comprises the amino acid sequence:

[0455] eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfc qqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwg settyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss(SEQ ID NO:758)

[0456] In some embodiments, the CD19 CAR is a humanized CD19 CAR comprising the amino acid sequence:

[0457] eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggs ggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgq gtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggc elrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr(SEQ ID NO:759)

[0458] In some embodiments, the CD19 CAR comprises a sequence, e.g., a CDR, VH, VL, scFv, or complete CAR sequence disclosed in Table 1 below, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the CD19 CAR comprises a sequence, e.g., a CDR, VH, VL, scFv, or complete CAR sequence disclosed in Table 1 below, or a sequence having at least one amino acid substitution (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 substitutions) relative thereto.

[0459] Table 1. Amino acid and nucleic acid sequences of exemplary anti-CD19 molecules

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468] Exemplary CD19 CAR A

[0469] In some embodiments, CD19 CAR is a binding domain of a single-chain variable fragment (scFv) derived from an FMC63 monoclonal antibody, an IgG4 hinge region, a CD28 transmembrane domain, a 4-1BB (CD137) co-stimulatory domain, and a CD3ζ activation domain. In some embodiments, CD19 CAR is encoded by a nucleotide sequence of Table 25 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity thereto. In some embodiments, CD19 CAR is encoded by a nucleotide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleotide sequence of Table 25. In some embodiments, CD19 CAR comprises a polypeptide encoded by a nucleotide sequence of Table 25 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity thereto. In some embodiments, CD19 CAR comprises a polypeptide encoded by a nucleotide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleic acid sequence of Table 25. In some embodiments, CD19 CAR comprises a polypeptide sequence of Table 25 or a polypeptide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity thereto. In some embodiments, CD19 CAR comprises a polypeptide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 amino acid substitutions relative to the amino acid sequence of Table 25. In some embodiments, CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence of Table 25. In some embodiments, CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence according to Kabat's Table 25. In some embodiments, CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence according to Josiah's Table 25. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence of Table 25. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 according to a sequence of Table 25 of Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 according to a sequence of Table 25 of Josiah. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 of a sequence of Table 25. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Table 25 of Kabat. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Table 25 of Josiah.

[0470] Table 25: Amino acid and nucleic acid sequences of exemplary CD19 CARs

[0471]

[0472]

[0473]

[0474]

[0475] Exemplary CD19 CAR B

[0476] In some embodiments, CD19 CAR comprises a mouse anti-CD19 single chain variable fragment (scFv) connected to CD28 and CD3-ζ costimulatory domains. In certain embodiments, anti-CD19 single chain variable fragments include FMC63 antibodies (e.g., Nicholson et al., Molecular Immunology [Molecular Immunology], 34 (16-17): 1157-1165, antibodies described in 1997; The entire contents of these documents are incorporated herein by reference). In some embodiments, CD19 CAR is encoded by a nucleotide sequence of Table 26 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity thereto. In some embodiments, CD19 CAR is encoded by a nucleotide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleotide sequence of Table 26. 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% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleic acid sequence of Table 26. 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% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 amino acid substitutions relative to the amino acid sequence of Table 25. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence of Table 26. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 according to a sequence of Table 26 of Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 according to a sequence of Table 26 of Josiah. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 according to a sequence of Table 26 of Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 according to a sequence of Table 26 of Josiah. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 according to a sequence of Table 26 of Kabat. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Table 26 of Josiah. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Table 26 of Kabat. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Table 26 of Josiah.

[0477] Table 26: Amino acid and nucleic acid sequences of exemplary CD19 CARs

[0478]

[0479]

[0480]

[0481] Exemplary CD19 CAR C

[0482] In some embodiments, CD19 CAR comprises a mouse anti-CD19 single-chain variable fragment (scFv) connected to a CD28 and CD3-ζ costimulatory domain. In some embodiments, 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% sequence identity thereto. In some embodiments, CD19 CAR is encoded by a nucleotide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleotide sequence of Table 27. In some embodiments, CD19 CAR comprises a polypeptide encoded by a nucleotide sequence of Table 27 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity thereto. In some embodiments, CD19 CAR comprises a polypeptide encoded by a nucleotide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleic acid sequence of Table 27. 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% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the amino acid sequence of Table 27. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence of Table 27. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence according to Table 27 of Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence according to Table 27 of Josiah. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence of Table 27. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence according to Table 27 of Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 according to a sequence of Josiah's Table 27. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Table 27. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Kabat's Table 27. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Josiah's Table 27.

[0483] Table 27: Amino acid and nucleic acid sequences of exemplary CD19 CARs

[0484]

[0485]

[0486]

[0487]

[0488]

[0489] Exemplary CD19 CAR F

[0490] In some embodiments, the CD19 CAR is encoded by a nucleotide sequence of Table 34 or a nucleotide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity thereto. In some embodiments, the CD19 CAR is encoded by a nucleotide sequence with no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleotide sequence of Table 34. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence of Table 34 or a nucleotide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide encoded by a nucleotide sequence with no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleic acid sequence of Table 34. In some embodiments, the CD19 CAR comprises a polypeptide sequence of Table 34 or a polypeptide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity thereto. In some embodiments, the CD19 CAR comprises a polypeptide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 amino acid substitutions relative to the amino acid sequence of Table 34. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence of Table 34. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 according to a sequence of Table 34 of Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 according to a sequence of Table 34 of Josiah. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 of a sequence of Table 34. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 according to a sequence of Table 34 of Kabat. In some embodiments, the CD19 CAR comprises a heavy chain CDR1-3 according to a sequence of Table 34 of Josiah. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 of a sequence of Table 34. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Table 34 of Kabat. In some embodiments, the CD19 CAR comprises a light chain CDR1-3 according to a sequence of Table 34 of Josiah.

[0491] Table 34: Amino acid and nucleic acid sequences of exemplary CD19 CARs

[0492]

[0493]

[0494] Exemplary CD19-CD20 CAR G

[0495] In some embodiments, CD19 CAR is a bispecific CAR. In certain embodiments, CD19 bispecific CAR includes a light chain variable domain targeting CD19 and a heavy chain variable domain targeting different targets (e.g., CD20). In some embodiments, bispecific CAR is anti-CD19 and anti-CD20 CAR. In some embodiments, bispecific CAR is encoded by a nucleotide sequence of Table 35 or a nucleotide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity. In some embodiments, bispecific CAR is encoded by a nucleotide sequence with no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleotide sequence of Table 35. In some embodiments, bispecific CAR includes a polypeptide encoded by a nucleotide sequence of Table 35 or a nucleotide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity relative to it. In some embodiments, the bispecific CAR comprises a polypeptide encoded by a nucleotide sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleic acid sequence of Table 35. 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% sequence identity relative thereto. In some embodiments, the bispecific CAR comprises a polypeptide sequence having or no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 amino acid substitutions relative to the amino acid sequence of Table 35. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence of Table 35. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 according to a sequence of Table 35 of Kabat. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 according to a sequence of Table 35 of Josiah. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 of a sequence of Table 35. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 according to a sequence of Table 35 of Kabat. In some embodiments, the bispecific CAR comprises a heavy chain CDR1-3 according to a sequence of Table 35 of Josiah. In some embodiments, the bispecific CAR comprises a light chain CDR1-3 of a sequence of Table 35. In some embodiments, the bispecific CAR comprises a light chain CDR1-3 according to a sequence of Table 35 of Kabat. In some embodiments, the bispecific CAR comprises a light chain CDR1-3 according to a sequence of Table 35 of Josiah.

[0496] Table 35: Amino acid and nucleic acid sequences of exemplary CD19 CARs

[0497]

[0498]

[0499]

[0500] BCMA

[0501] A non-limiting exemplary tumor antigen is BCMA. CARs that bind BCMA are known in the art. For example, those disclosed in WO2016 / 014565 or WO2019 / 241426 can be used according to 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 according to 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 ​​disclosed in WO2016 / 014565 , 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-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.

[0502] In some embodiments, the BCMACAR comprises 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_ one or more CDRs, VHs, VLs, scFvs, 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%-99%) identical thereto.

[0503] Exemplary antigen binding domains that bind BCMA are disclosed in WO2012 / 0163805, WO 2017 / 021450, WO2017 / 011804, WO 2017 / 025038, WO 2016 / 090327, WO 2016 / 130598, WO 2016 / 210293, WO2016 / 090320, WO 2016 / 014789, WO 2016 / 094304, WO 2016 / 154055, WO 2015 / 166073, WO2015 / 188119, WO 2015 / 158671, US 9,243,058, US 8,920,776, US 9,273,141, US 7,083,785、U.S. 9,034,324, US2007 / 0049735, US2015 / 0284467, US2015 / 0051266, US2015 / 0344844, US2016 / 013 1655、US2016 / 0297884、US2016 / 0297885、US2017 / 0051308、US2017 / 0051252、WO2016 / 020332、WO 2016 / 087531、WO 2016 / 079177、WO 2015 / 172800、WO 2017 / 008169、US 9,340,621, US2013 / 0273055, US2016 / 0176973, US2015 / 0368351, US2017 / 0051068, US2016 / 0368988, and US2015 / 0232557, which are incorporated herein by reference in their entirety. In some embodiments, one or more BCMA antigen binding domains disclosed therein are antigen binding domains.

[0504] In some embodiments, the antigen binding domain comprises a human antibody or a 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 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 herein (e.g., in Tables 2-14). In some embodiments, the human anti-BCMA binding domain comprises a human VL herein (e.g., in Tables 2, 6, and 10) and / or a human VH herein (e.g., in Tables 2, 6, and 10). In some embodiments, the antigen binding domain is a scFv comprising a VL and a VH of an amino acid sequence of 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) but not more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions) of an amino acid sequence provided in Tables 2, 6 and 10, or a sequence with 95%-99% identity with 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) but not more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions) of an amino acid sequence provided in Tables 2, 6 and 10, or a sequence with 95%-99% identity with an amino acid sequence of Tables 2, 6 and 10.

[0505] In certain embodiments, the antigen binding domains described herein include:

[0506] (1) One, two or three light chain (LC) CDRs selected from the following:

[0507] (i) LC CDR1 of SEQ ID NO:54, LC CDR2 of SEQ ID NO:55 and LC CDR3 of SEQ ID NO:56; and / or

[0508] (2) one, two or three heavy chain (HC) CDRs selected from one of the following:

[0509] (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.

[0510] In certain embodiments, the antigen binding domains described herein include:

[0511] (1) one, two or three light chain (LC) CDRs selected from one of the following:

[0512] (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

[0513] (2) one, two or three heavy chain (HC) CDRs selected from one of the following:

[0514] (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.

[0515] In certain embodiments, the antigen binding domains described herein include:

[0516] (1) one, two or three light chain (LC) CDRs selected from one of the following:

[0517] (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

[0518] (2) one, two or three heavy chain (HC) CDRs selected from one of the following:

[0519] (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.

[0520] In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, 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 CDR1, HC CDR2, HC CDR3, LC CDR1, 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 CDR1, HC CDR2, HC CDR3, LC CDR1, 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 CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55, and 56, respectively.

[0521] In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, 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 CDR1, HC CDR2, HC CDR3, LC CDR1, 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 CDR1, HC CDR2, HC CDR3, LC CDR1, 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 CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 47, 48, 76, 57, 58, and 59, respectively.

[0522] In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, 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 CDR1, HC CDR2, HC CDR3, LC CDR1, 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 CDR1, HC CDR2, HC CDR3, LC CDR1, 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 CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 49, 50, 77, 60, 58, and 56, respectively.

[0523] In some embodiments, BCMACAR comprises a sequence, e.g., a CDR, VH, VL, scFv, or complete CAR sequence disclosed in Tables 2-14, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0524] Table 2. Amino acid and nucleic acid sequences of exemplary PALLAS-derived anti-BCMA molecules

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536] Table 3. Kabat CDRs of exemplary PALLAS-derived anti-BCMA molecules

[0537]

[0538]

[0539] Table 4. Josiah CDRs of Exemplary PALLAS-Derived Anti-BCMA Molecules

[0540]

[0541]

[0542] Table 5. IMGT CDRs of exemplary PALLAS-derived anti-BCMA molecules

[0543]

[0544]

[0545] Table 6. Amino acid and nucleic acid sequences of exemplary B cell-derived anti-BCMA molecules

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559] Table 7. Kabat CDRs of Exemplary B Cell-Derived Anti-BCMA Molecules

[0560]

[0561] Table 8. Josiah CDRs of Exemplary B Cell-Derived Anti-BCMA Molecules

[0562]

[0563]

[0564] Table 9. IMGT CDRs of exemplary B cell-derived anti-BCMA molecules

[0565]

[0566] Table 14. Amino acid and nucleic acid sequences of exemplary anti-BCMA molecules based on PI61

[0567]

[0568]

[0569]

[0570]

[0571] Table 10. Amino acid and nucleic acid sequences of exemplary hybridoma-derived anti-BCMA molecules

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580] Table 11. Kabat CDRs of exemplary hybridoma-derived anti-BCMA molecules

[0581]

[0582]

[0583] Table 12. Josiah CDRs of Exemplary Hybridoma-Derived Anti-BCMA Molecules

[0584]

[0585]

[0586] Table 13. IMGT CDRs of exemplary hybridoma-derived anti-BCMA molecules

[0587]

[0588] In certain embodiments, BCMACAR is produced using VH and VL sequences from WO 2012 / 0163805 (the content of the document is hereby incorporated by reference in its entirety). In certain embodiments, BCMACAR can be produced using CDR, VH, VL, scFv or complete CAR sequences from WO2019 / 241426 (the content of the document is hereby incorporated by reference in its entirety).

[0589] Exemplary BCMACAR D

[0590] In some embodiments, BCMACAR comprises a mouse extracellular single-chain variable fragment (scFv) specific for recognizing B cell maturation antigen (BCMA), followed by a human CD8α hinge and transmembrane domain fused in series with the T cell cytoplasmic signaling domain of CD137 (4-1BB) and CD3ζ chains. The combination of BCMACAR D with target cells expressing BCMA leads to signal transduction initiated by CD3ζ and 4-1BB domains and subsequent CAR positive T cell activation. Antigen-specific activation of BCMACAR D leads to cell lysis and killing of CAR positive T cells, cytokine secretion, and subsequent expression of BCMA cells. In some embodiments, BCMACAR is encoded by the nucleotide sequence of Table 28 or a nucleotide sequence with respect to which there is at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity. In some embodiments, BCMACAR is encoded by a nucleotide sequence with no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitution relative to the nucleotide sequence of Table 28. In some embodiments, BCMACAR comprises a polypeptide encoded by a nucleotide sequence of table 28 or a nucleotide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity relative to it. In some embodiments, BCMACAR comprises a polypeptide encoded by a nucleotide sequence with no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitutions relative to the nucleotide sequence of table 28. In some embodiments, BCMACAR comprises a polypeptide sequence of table 28 or a polypeptide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity relative to it. In some embodiments, BCMACAR comprises a polypeptide sequence with no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 amino acid substitutions relative to the amino acid sequence of table 28. In some embodiments, BCMACAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence of table 28. In some embodiments, BCMACAR comprises a heavy chain CDR1-3 and a light chain CDR1-3 of a sequence according to Kabat's table 28. In some embodiments, BCMACAR comprises heavy chain CDR1-3 and light chain CDR1-3 according to the sequence of Table 28 of Josiah. In some embodiments, BCMACAR comprises heavy chain CDR1-3 of the sequence of Table 28. In some embodiments, BCMACAR comprises heavy chain CDR1-3 of the sequence of Table 28 of Kabat. In some embodiments, BCMACAR comprises heavy chain CDR1-3 of the sequence of Table 28 of Josiah. In some embodiments, BCMACAR comprises light chain CDR1-3 of the sequence of Table 28 of Kabat. In some embodiments, BCMACAR comprises light chain CDR1-3 of the sequence of Table 28 of Kabat.In some embodiments, the BCMACAR comprises a light chain CDR1-3 according to the sequence of Table 28 of Josiah.

[0591] Table 28. Amino acid sequences of exemplary BCMACARs

[0592]

[0593]

[0594] Exemplary BCMACAR E

[0595] In some embodiments, BCMACAR comprises two single domain antibodies connected to 4-1BB costimulatory domain and CD3-ζ signaling domain. In some embodiments, chimeric antigen receptor as described herein comprises a polypeptide comprising: (a) an extracellular antigen binding domain comprising a first anti-BCMA single domain antibody (sdAb) and a second anti-BCMA sdAb. In some embodiments, the first and second anti-BCMA antibodies are each independently a VhH domain. In certain embodiments, the first anti-BCMA sdAb comprises CDR1, CDR2 and CDR3 as listed in the VhH domain, and the domain comprises SEQ ID NO: 377 amino acid sequence or a peptide sequence with respect to it having at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity. In some embodiments, the first anti-BCMA sdAb comprises CDR1, CDR2, and CDR3 as set forth in a VhH domain comprising an amino acid sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 substitutions relative to the amino acid sequence of SEQ ID NO: 377. In certain embodiments, the second anti-BCMA sdAb comprises CDR1, CDR2, and CDR3 as set forth in a VhH domain comprising an amino acid sequence of SEQ ID NO: 381, or a peptide sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity thereto. In some embodiments, the second anti-BCMA sdAb comprises CDR1, CDR2, and CDR3 as set forth in a VhH domain comprising an amino acid sequence having no more than 20, 15, 10, 8, 5, 4, 3, 2, or 1 substitutions relative to the amino acid sequence of SEQ ID NO: 381. In certain embodiments, BCMACAR is any BCMACAR described in U.S. Patent No. 11,186,647, the entire contents of which are incorporated herein by reference. In some embodiments, CD19 CAR is encoded by a nucleotide sequence of Table 29 or a nucleotide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity relative to it. In some embodiments, CD19 CAR is encoded by a nucleotide sequence with no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitution relative to the nucleotide sequence of Table 29. In some embodiments, CD19 CAR includes a polypeptide encoded by a nucleotide sequence of Table 29 or a nucleotide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity relative to it. In some embodiments, CD19 CAR includes a polypeptide encoded by a nucleotide sequence with no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 substitution relative to the nucleotide sequence of Table 29.In some embodiments, CD19 CAR comprises a polypeptide sequence of Table 29 or a polypeptide sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity thereto. In some embodiments, CD19 CAR comprises a polypeptide sequence with no more than 20, 15, 10, 8, 5, 4, 3, 2 or 1 amino acid substitutions relative to the amino acid sequence of Table 29. In some embodiments, BCMACAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence of Table 29. In some embodiments, BCMACAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence of Table 29 according to Kabat. In some embodiments, BCMACAR comprises heavy chain CDR1-3 and light chain CDR1-3 of the sequence of Table 29 according to Josiah. In some embodiments, BCMACAR comprises heavy chain CDR1-3 of the sequence of Table 29. In some embodiments, BCMACAR comprises heavy chain CDR1-3 of the sequence of Table 29 according to Kabat. In some embodiments, BCMACAR comprises a heavy chain CDR1-3 according to a sequence of Josiah's Table 29. In some embodiments, BCMACAR comprises a light chain CDR1-3 according to a sequence of Table 29. In some embodiments, BCMACAR comprises a light chain CDR1-3 according to a sequence of Kabat's Table 29. In some embodiments, BCMACAR comprises a light chain CDR1-3 according to a sequence of Josiah's Table 29.

[0596] Table 29. Amino acid and nucleic acid sequences of exemplary BCMACAR D

[0597]

[0598]

[0599] Other exemplary targets

[0600] Additional non-limiting exemplary tumor antigens include CD20, CD22, EGFR, CD123, and CLL-1.

[0601] CARs that bind to CD20 are known in the art. For example, those disclosed in WO2018 / 067992 or WO2016 / 164731, which are incorporated herein by reference, can be used according to the present disclosure. Any known CD20 CAR in the art, for example, the CD20 antigen binding domain of any known CD20 CAR can be used according to the present disclosure. Exemplary sequences or CD20 CAR sequences that bind to CD20 are disclosed in, for example, Tables 1-5 of WO2018 / 067992 (incorporated herein by reference). In some embodiments, CD20 CAR is included in the CDR, variable region, scFv or full-length sequence of CD20 CAR disclosed in WO2018 / 067992 or WO2016 / 164731 (both incorporated herein by reference). In some embodiments, the CD20 CAR comprises a sequence, e.g., a CDR, VH, VL, scFv, or complete CAR sequence disclosed in Table 23 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0602] Exemplary antigen binding domains that bind to CD20 are described in WO2016 / 164731 and WO2018 / 067992 (incorporated herein by reference). In some embodiments, an antigen binding domain of one or more CD20 antigen binding domains disclosed therein.

[0603] Exemplary antigen binding domains that bind CD22 are described in WO2016 / 164731 and WO2018 / 067992 (incorporated herein by reference).

[0604] In some embodiments, the antigen binding domain comprises 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 LC CDR1, LC CDR2, and LC CDR3. In embodiments, the antigen binding domain comprises LC CDR1, LC CDR2, and LC CDR3 amino acid sequences listed in Table 16.

[0605] In some embodiments, the antigen binding domain comprises one, two or all of the 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 the HC CDR1, HC CDR2, and HC CDR3 of any heavy chain binding domain amino acid sequence listed in Table 15.

[0606] Exemplary antigen binding domains that bind EGFRvIII are described in WO2014 / 130657.

[0607] Exemplary antigen binding domains that bind CD123 are described in WO 2014 / 130635 and WO 2016 / 028896 (incorporated herein by reference).

[0608] In some embodiments, the antigen binding domain comprises a sequence from Table 1-2 of WO2014 / 130635 (incorporated herein by reference).

[0609] In some embodiments, the antigen binding domain comprises a sequence from Tables 2, 6, and 9 of WO2016 / 028896 (incorporated herein by reference).

[0610] Exemplary antigen binding domains that bind CLL-1 are disclosed in WO2016 / 014535 (incorporated herein by reference).

[0611] 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, from an antibody described herein (e.g., an antibody described in WO2015 / 142675, US-2015-0283178-A1, US-2016-0046724-A1, US2014 / 0322212A1, US2016 / 0068601A1, US2016 / 0051651A1, US2016 / 0096892A1, US2014 / 0322275A1, or WO2015 / 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. CDR3, these light chain CDRs are from antibodies described herein (e.g., antibodies described in WO2015 / 142675, US-2015-0283178-A1, US-2016-0046724-A1, US2014 / 0322212A1, US2016 / 0068601A1, US2016 / 0051651A1, US2016 / 0096892A1, US2014 / 0322275A1 or WO2015 / 090230, which are incorporated herein by reference). In some embodiments, the antigen binding domain comprises a heavy chain variable region and / or a variable light chain region of the antibodies listed above.

[0612] In an embodiment, the antigen binding domain is an antigen binding domain described in WO 2015 / 142675, US-2015-0283178-A1, US-2016-0046724-A1, US2014 / 0322212 A1, US2016 / 0068601 A1, US2016 / 0051651 A1, US 2016 / 0096892 A1, US2014 / 0322275 A1, or WO 2015 / 090230 (incorporated herein by reference).

[0613] Exemplary target antigens that can be targeted using CAR-expressing cells include, but are not limited to, CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFRα-4, among others, described in, e.g., 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).

[0614] In some embodiments, any CAR described herein (e.g., any one of CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFRα-4) antigen binding domains include one, two, three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3 from the antibodies listed above, and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3 from the antigen binding domains listed above. In some embodiments, the antigen binding domain includes a heavy chain variable region and / or a variable light chain region of an antibody listed or described above.

[0615] 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) from the antibodies listed above, and / or one, two, three (e.g., all three) light chain CDRs (LC CDR1, LC CDR2, and LC CDR3) from the antibodies 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.

[0616] Table 23. Amino acid sequences of exemplary anti-CD20 molecules

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

[0644]

[0645]

[0646]

[0647] CARs that bind to CD22 are known in the art. For example, those disclosed in WO2018 / 067992 or WO2016 / 164731 can be used according to the present disclosure. Any CD22 CAR known in the art, for example, the CD22 antigen binding domain of any known CD22 CAR can be used according to the present disclosure.

[0648] Exemplary CD22 binding sequences or CD22 CAR sequences are disclosed in, e.g., Tables 6A, 6B, 7A, 7B, 7C, 8A, 8B, 9A, 9B, 10A and 10B of WO 2016164731 and Tables 6-10 of WO 2018067992. In some embodiments, the CD22 CAR sequence comprises a CDR, variable region, scFv or full-length sequence of a CD22 CAR disclosed in WO 2018067992 or WO 2016164731.

[0649] In an embodiment, the CAR comprises an antigen binding domain that binds 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.

[0650] The sequence of human CD22 CAR is provided below. In some embodiments, the human CD22 CAR is CAR22-65.

[0651] Human CD22 CAR scFv sequence

[0652] EVQLQQSGPGLVKPSQTLSLTCAISGDSMLSNSDTWNWIRQSPSRGLEWLGRTYHRSTWYDDYASSVRGRVSINVDTSKNQYSLQLNAVTPEDTGVYYCARVRLQDGNSWSDAFDVWGQGTMVTVSSG GGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGTGTQLTVL(SEQ ID NO:753)

[0653] Human CD22 CAR heavy chain variable region

[0654] EVQLQQSGPGLVKPSQTLSLTCAISGDSMLSNSDTWNWIRQSPSRGLEWLGRTYHRSTWYDDYASSVRGRVSINVDTSKNQYSLQLNAVTTPEDTGVYYCARVRLQDGNSWSDAFDVWGQGTMVTVSS(SEQ ID NO:754)

[0655] Human CD22 CAR light chain variable region

[0656] QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGTGTQLTVL(SEQID NO 755)

[0657] In some embodiments, the CD22 CAR comprises a sequence, e.g., a CDR, VH, VL, scFv, or complete CAR sequence disclosed in Tables 15-16 and 24 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0658] Table 15. Heavy chain variable domain CDRs of CD22 CAR (CAR22-65)

[0659]

[0660] Table 16. Light chain variable domain CDRs of CD22 CAR (CAR22-65). The LC CDR sequences in the table have the same sequence under Kabat or combined definition.

[0661]

[0662] Table 24. Amino acid sequences of exemplary anti-CD22 molecules

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678] CAR with EGFR is known in the art.For example, those disclosed in WO2014 / 130657, which is incorporated herein by reference, can be used according to this disclosure. Any known EGFR CAR in the art can be used according to this disclosure, for example, the EGFR antigen binding domains of any known EGFR CAR. Exemplary EGFRvIIICAR can include WO2014 / 130657 (incorporated herein by reference), for example, CDR, variable region, scFv or full-length CAR sequences disclosed in Table 2 of WO2014 / 130657.

[0679] CAR with CD123 is known in the art. For example, those disclosed in WO2014 / 130635 or WO2016 / 028896 can be used according to this disclosure. Any known CD123 CAR in the art, for example, the CD123 antigen binding domains of any known CD123 CAR can be used according to this disclosure. For example, CAR1 to CAR8 disclosed in WO2014 / 130635; or CAR123-1 to CAR123-4 and hzCAR123-1 to hzCAR123-32 disclosed in WO2016 / 028896. The amino acid sequence and nucleotide sequence encoding CD123CAR molecules and antigen binding domains (for example, including one, two, three VH CDRs according to Kabat or Josiah; and one, two, three VL CDRs) are specified in WO 2014 / 130635 and WO2016 / 028896.

[0680] CARs that bind CLL-1 are known in the art. For example, those disclosed in US2016 / 0051651A1 (incorporated herein by reference). Any known CLL-1CAR in the art, for example, the CLL-1 antigen binding domain of any known CLL-1CAR can be used according to this disclosure.

[0681] In some embodiments, CAR comprises a CLL-1CAR or antigen binding domain according to Table 2 of WO2016 / 014535 (incorporated herein by reference). The amino acid sequence and nucleotide sequence encoding the CLL-1CAR molecule and antigen binding domain (e.g., comprising one, two, three VH CDRs according to Kabat or Josiah; and one, two, three VL CDRs) are specified in WO2016 / 014535.

[0682] CARs that bind to CD33 are known in the art. For example, those disclosed in US2016 / 0096892A1 and WO2016 / 014576, which are incorporated herein by reference, can be used according to the present disclosure. Any CD33 CAR known in the art can be used according to the present disclosure, for example, the CD33 antigen binding domain of any known CD33 CAR. For example, CAR33-1 to CAR33-9 disclosed in WO2016 / 014576 can be used according to the present disclosure.

[0683] In some embodiments, the CAR comprises a CD33 CAR or antigen binding domain according to Table 2 or 9 of WO2016 / 014576 (incorporated herein by reference). The amino acid sequences and nucleotide sequences encoding the CD33 CAR molecules and antigen binding domains (e.g., comprising one, two, three VH CDRs according to Kabat or Josiah; and one, two, three VLCDRs) are specified in WO2016 / 014576.

[0684] In one embodiment, the antigen binding domain against CD33 is an antigen binding portion (e.g., CDRs) of an antibody described, e.g., in 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 [progress in hematology] 2012:683065 (2012); and Pizzitola et al., Leukemia [leukemia] doi:10.1038 / Lue.2014.62 (2014). In one embodiment, the antigen binding domain against CD33 is an antigen binding portion (e.g., CDR) of an antibody, antigen binding fragment, or CAR described in WO / 2016 / 014576.

[0685] In one embodiment, an antigen binding domain against GD2 is an antigen binding portion (e.g., CDRs) of an antibody described, e.g., in Mujoo et al., Cancer Res 47(4):1098-1104 (1987); Cheung et al., Cancer Res 45(6):2642-2649 (1985), Cheung et al., J Clin Oncol 5(9):1430-1440 (1987), Cheung et al., J Clin Oncol 16(9):3053-3060 (1998), Handgretinger et al., Cancer Immunol Immunother 35(3):199-204 (1992). In some embodiments, the antigen binding domain against GD2 is an antigen binding portion of an antibody selected from: mAb 14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9, see, e.g., WO 2012033885, WO 2013040371, WO 2013192294, WO 2013061273, WO 2013123061, WO 2013074916, and WO 201385552. In some embodiments, the antigen binding domain against GD2 is an antigen binding portion of an antibody described in U.S. Publication No.: 20100150910 or PCT Publication No.: WO2011160119.

[0686] In one embodiment, the antigen binding domain against a Tn antigen is an antigen binding portion (e.g., CDR) of an antibody described in, for example, US 8,440,798; Brooks et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America] 107(22): 10056-10061 (2010); and Stone et al., OncoImmunology [Tumor Immunology] 1(6): 863-873 (2012).

[0687] In one embodiment, the antigen binding domain against PSMA is an antigen binding portion (e.g., CDRs) of an antibody described, for example, in: Parker et al., Protein Expr Purif 89(2):136-145 (2013), US20110268656 (J591 ScFv); Frigerio et al., European J Cancer 49(9):2223-2232 (2013) (scFv D2B); WO 2006125481 (mAbs 3 / A12, 3 / E7 and 3 / F11) and single-chain antibody fragments (scFv A5 and D7).

[0688] In one embodiment, an antigen binding domain against ROR1 is an antigen binding portion (e.g., CDR) of an antibody described in, e.g., Hudecek et al., Clin Cancer Res 19(12):3153-3164 (2013); WO 2011159847; and US20130101607.

[0689] In one embodiment, the antigen binding domain against FLT3 is an antigen binding portion (e.g., CDR) of the antibodies described in, e.g., WO 2011076922, US5777084, EP 0754230, US20090297529, and several commercial catalog antibodies (R&D, ebiosciences, Abcam).

[0690] In one embodiment, an antigen binding domain against TAG72 is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Hombach et al., Gastroenterology 113(4): 1163-1170 (1997); and Abeam ab691.

[0691] In one embodiment, an antigen binding domain against FAP is an antigen binding portion (e.g., CDRs) of an antibody described, e.g., in Ostermann et al., Clinical Cancer Research 14:4584-4592 (2008) (FAP5), U.S. Patent Publication No. 2009 / 0304718; sirolimusumab (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).

[0692] In one embodiment, the antigen binding domain against CD38 is an antigen binding portion (e.g., CDRs) of daratumumab (see, e.g., Groen et al., Blood 116(21):1261-1262 (2010); MOR202 (see, e.g., US 8,263,746); or an antibody described in US 8,362,211.

[0693] In one embodiment, an antigen binding domain against CD44v6 is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Casucci et al., Blood 122(20):3461-3472 (2013).

[0694] In one embodiment, an antigen binding domain against CEA is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Chmielewski et al., Gastoenterology 143(4): 1095-1107 (2012).

[0695] In one embodiment, the antigen binding domain against EPCAM is an antigen binding portion (e.g., CDR) of an antibody selected from: MT110, EpCAM-CD3 bispecific Ab (see, e.g., clinicaltrials.gov / ct2 / show / NCT00635596); edrecolomab; 3622W94; ING-1; and adelimumab (MT201).

[0696] In one embodiment, an antigen binding domain against PRSS21 is an antigen binding portion (eg, CDRs) of an antibody described in U.S. Pat. No. 8,080,650.

[0697] In one embodiment, an antigen binding domain against B7H3 is an antigen binding portion (eg, CDRs) of the antibody MGA271 (Macrogenics).

[0698] In one embodiment, the antigen binding domain against KIT is the antigen binding portion (e.g., CDRs) of the antibodies described in, e.g., US 7915391, US20120288506, and several commercial catalog antibodies.

[0699] In one embodiment, the antigen binding domain against IL-13Ra2 is an antigen binding portion (e.g., CDRs) of an antibody described in, e.g., WO 2008 / 146911, WO 2004087758, several commercial catalog antibodies, and the antibody in WO 2004087758.

[0700] In one embodiment, the antigen binding domain against CD30 is an antigen binding portion (eg, CDRs) of an antibody described in, for example, US 7090843 Bl and EP0805871.

[0701] In one embodiment, the antigen binding domain against GD3 is an antigen binding portion (eg, CDR) of an antibody described in, for example, US 7253263; US 8,207,308; US20120276046; EP1013761; WO2005035577; and US 6437098.

[0702] In one embodiment, the antigen binding domain against CD171 is an antigen binding portion (eg, CDR) of an antibody described in, e.g., Hong et al., J Immunother 37(2):93-104 (2014).

[0703] In one embodiment, the antigen binding domain against IL-11Ra is an antigen binding portion (e.g., CDR) of an antibody available from Abcam (Catalog No. ab55262) ​​or Novus Biologicals (Catalog No. 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).

[0704] In one embodiment, an antigen binding domain against PSCA is an antigen binding portion (e.g., CDRs) of an antibody described, e.g., in 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 Publication No. 20090311181.

[0705] In one embodiment, an antigen binding domain against VEGFR2 is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Chinnasamy et al., J Clin Invest 120(11):3953-3968 (2010).

[0706] In one embodiment, an antigen binding domain against Lewis Y is an antigen binding portion (e.g., CDRs) of an antibody described, for example, in Kelly et al., Cancer Biother Radiopharm 23(4):411-423 (2008) (hu3S193 Ab (scFvs)); Dolezal et al., Protein Engineering 16(1):47-56 (2003) (NC10 scFv).

[0707] In one embodiment, an antigen binding domain against CD24 is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Maliar et al., Gastroenterology 143(5): 1375-1384 (2012).

[0708] In one embodiment, the antigen binding domain against PDGFR-β is an antigen binding portion (e.g., CDRs) of the antibody Abeam ab32570.

[0709] In one embodiment, the antigen binding domain against SSEA-4 is the antigen binding portion (eg, CDRs) of the antibody MC813 (Cell Signaling) or other commercially available antibodies.

[0710] In one embodiment, the 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 WO2016 / 164731.

[0711] In one embodiment, the antigen binding domain against folate receptor alpha is the antibody IMGN853 or an antigen binding portion (e.g., CDRs) of an antibody described in: US20120009181; US ​​4851332, LK26: US5952484.

[0712] In one embodiment, the antigen binding domain against ERBB2 (Her2 / neu) is an antigen binding portion (e.g., CDRs) of the antibody trastuzumab or pertuzumab.

[0713] In one embodiment, an antigen binding domain against MUCl is an antigen binding portion (eg, CDRs) of antibody SAR566658.

[0714] In one embodiment, the antigen binding domain against EGFR is an antigen binding portion (e.g., CDR) of the antibody cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.

[0715] In one embodiment, an antigen binding domain against NCAM is an antigen binding portion (eg, CDRs) of the following antibody: Antibody clone 2-2B: MAB5324 (EMD Millipore).

[0716] In one embodiment, an antigen binding domain against ephrin B2 is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Abengozar et al., Blood 119(19):4565-4576 (2012).

[0717] In one embodiment, the antigen binding domain against IGF-I receptor is an antigen binding portion (eg, CDRs) of an antibody described in, for example, US 8344112 B2; EP 2322550 A1; WO 2006 / 138315, or PCT / US2006 / 022995.

[0718] In one embodiment, an antigen binding domain against CAIX is an antigen binding portion (e.g., CDRs) of antibody clone 303123 (R&D Systems).

[0719] In one embodiment, the antigen binding domain against LMP2 is an antigen binding portion (eg, CDRs) of an antibody described in, for example, US 7,410,640 or US20050129701.

[0720] In one embodiment, the antigen binding domain against gp100 is an antigen binding portion (e.g., CDR) of the antibody HMB45, NKIβB, or an antibody described in WO2013165940 or US20130295007

[0721] In one embodiment, an antigen binding domain against tyrosinase is an antigen binding portion (e.g., CDRs) of an antibody described in, e.g., US 5843674; or US19950504048.

[0722] In one embodiment, an antigen binding domain against EphA2 is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Yu et al., Mol Ther 22(1): 102-111 (2014).

[0723] In one embodiment, the antigen binding domain against GD3 is an antigen binding portion (e.g., CDRs) of an antibody described in, e.g., US 7253263; US 8,207,308; US20120276046; EP1013761 A3; 20120276046; WO 2005035577; or US 6437098.

[0724] In one embodiment, the antigen binding domain against Fucosyl GM1 is an antigen binding portion (eg, CDRs) of an antibody described in, for example, US20100297138; or WO 2007 / 067992.

[0725] In one embodiment, the antigen binding domain against sLe is an antigen binding portion (e.g., CDRs) of antibody G193 (for lewis Y), see Scott AM et al., Cancer Res 60:3254-61 (2000), also as described in Neeson et al., J Immunol 2013 May 190 (Conference Abstract Supplement) 177.10.

[0726] In one embodiment, the antigen binding domain against GM3 is an antigen binding portion (e.g., CDRs) of antibody CA 2523449 (mAb 14F7).

[0727] In one embodiment, the antigen binding domain against HMWMAA is an antigen binding portion (e.g., CDRs) of an antibody described, for example, in: Kmiecik et al., Oncoimmunology 3(1):e27185 (2014) (PMID:24575382) (mAb9.2.27); US6528481; WO 2010033866; or US20140004124.

[0728] In one embodiment, the antigen binding domain against o-acetyl-GD2 is an antigen binding portion (eg, CDRs) of antibody 8B6.

[0729] In one embodiment, an antigen binding domain against TEM1 / CD248 is an antigen binding portion (e.g., CDRs) of an antibody described, for example, in: Marty et al., Cancer Lett 235(2):298-308 (2006); Zhao et al., J Immunol Methods 363(2):221-232 (2011).

[0730] In one embodiment, the 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.

[0731] In one embodiment, an antigen binding domain against TSHR is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., US 8,603,466; US 8,501,415; or US 8,309,693.

[0732] In one embodiment, the antigen binding domain against GPRC5D is an antigen binding portion (eg, CDR) of the following antibodies: antibody FAB6300A (R&D Systems); or LS-A4180 (Lifespan Biosciences).

[0733] In one embodiment, the antigen binding domain against CD97 is an antigen binding portion (e.g., CDRs) of an antibody described, for example, in: US 6,846,911; de Groot et al., J Immunol 183(6):4127-4134 (2009); or the antibody from R&D: MAB3734.

[0734] In one embodiment, an antigen binding domain against ALK is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Mino-Kenudson et al., Clin Cancer Res 16(5): 1561-1571 (2010).

[0735] In one embodiment, the antigen binding domain against polysialic acid is an antigen binding portion (eg, CDR) of an antibody described in, e.g., Nagae et al., J Biol Chem 288(47):33784-33796 (2013).

[0736] In one embodiment, an antigen binding domain against PLAC1 is an antigen binding portion (eg, CDRs) of an antibody described in, eg, Ghods et al., Biotechnol Appl Biochem 2013 doi: 10.1002 / bab.1177.

[0737] In one embodiment, the antigen binding domain against GloboH is an antigen binding portion of the following antibody: antibody VK9; or an antibody described, for example, in the following: Kudryashov V et al., Glycoconj J. [Journal of Glycoconjugates] 15(3):243-9 (1998), Lou et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 111(7):2482-2487 (2014); MBr1: Bremer EG et al. J Biol Chem [Journal of Biological Chemistry] 259:14773–14777 (1984).

[0738] In one embodiment, an antigen binding domain against NY-BR-1 is an antigen binding portion (e.g., CDRs) of an antibody described in, e.g., Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007).

[0739] In one embodiment, an antigen binding domain against WT-1 is an antigen binding portion (e.g., CDR) of an antibody described in, e.g., Dao et al., Sci Transl Med 5(176):176ra33 (2013); or WO 2012 / 135854.

[0740] In one embodiment, the antigen binding domain against MAGE-A1 is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Willemsen et al., J Immunol 174(12):7853-7858 (2005) (TCR-like scFv).

[0741] In one embodiment, an antigen binding domain against sperm protein 17 is an antigen binding portion (e.g., CDR) of an antibody described in, e.g., Song et al., Target Oncol. Aug 14, 2013 (PMID: 23943313); Song et al., Med Oncol. 29(4): 2923-2931 (2012).

[0742] In one embodiment, an antigen binding domain against Tie 2 is an antigen binding portion (e.g., CDRs) of antibody AB33 (Cell Signaling Technology).

[0743] In one embodiment, an antigen binding domain against MAD-CT-2 is an antigen binding portion (eg, CDRs) of an antibody described, e.g., in: PMID: 2450952; US7635753.

[0744] 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).

[0745] In one embodiment, the antigen binding domain against MelanA / MART1 is an antigen binding portion (eg, CDRs) of an antibody described in: EP 2514766 A2; or US 7,749,719.

[0746] In one embodiment, an antigen binding domain against a sarcoma translocation breakpoint is an antigen binding portion (eg, CDR) of an antibody described in, e.g., Luo et al., EMBO Mol. Med. 4(6):453-461 (2012).

[0747] In one embodiment, an antigen binding domain against TRP-2 is an antigen binding portion (eg, CDRs) of an antibody described in, e.g., Wang et al., J Exp Med. 184(6):2207-16 (1996).

[0748] In one embodiment, an antigen binding domain against CYP1B1 is an antigen binding portion (eg, CDRs) of an antibody described in, eg, Maecker et al., Blood 102(9):3287-3294 (2003).

[0749] In one embodiment, the antigen binding domain against RAGE-1 is an antigen binding portion (eg, CDRs) of the antibody MAB5328 (EMD Millipore).

[0750] In one embodiment, the antigen binding domain against human telomerase reverse transcriptase is the antigen binding portion (eg, CDR) of the following antibody: Antibody catalog number: LS-B95-100 (Lasbon Biotech)

[0751] In one embodiment, the antigen binding domain against intestinal carboxylesterase is an antigen binding portion (eg, CDR) of the following antibody: Antibody 4F12: Catalog No.: LS-B6190-50 (Lasbon Biotech).

[0752] In one embodiment, the antigen binding domain against mut hsp70-2 is an antigen binding portion (eg, CDR) of the following antibody: antibody (Lasbon Biotech: monoclonal: catalog number: LS-C133261-100 (Lasbon Biotech).

[0753] In one embodiment, the antigen binding domain against CD79a is the antigen binding portion (e.g., CDR) of the following antibodies: antibody anti-CD79a antibody [HM47 / A9] (ab3121) available from Abcam; antibody CD79A antibody No. 3351 available from Cell Signaling Technology; or antibody HPA017748-anti-CD79A antibody generated in rabbit and available from Sigma Aldrich.

[0754] In one embodiment, the antigen binding domain against CD79b is an antigen binding portion (e.g., CDRs) of the antibody polatuzumab vedotin (anti-CD79b) (described in 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 the bispecific antibody anti-CD79b / CD3 (described in "4507 Pre-Clinical Characterization Preclinical characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b / CD3 As a Potential Therapy for B Cell Malignancies [4507 Preclinical characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b / CD3 As a Potential Therapy for B Cell Malignancies]" Abstracts of 56 th ASH Annual Meeting and Exposition, San Francisco, California, December 6-9, 2014.

[0755] In one embodiment, the antigen binding domain against CD72 is an antigen binding portion (e.g., CDRs) of antibody J3-109 (described in Myers and Uckun, "An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia." Leuk Lymphoma. 1995 Jun;18(1-2):119-22) or 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. 2009 Mar 15 69;2358.

[0756] In one embodiment, the antigen binding domain against LAIR1 is an antigen binding portion (eg, CDR) of the following antibodies: antibody ANT-301LAIR1 antibody available from ProSpec; or anti-human CD305 (LAIR1) antibody available from BioLegend.

[0757] In one embodiment, the antigen binding domain against FCAR is the antigen binding portion (e.g., CDR) of the antibody CD89 / FCAR antibody (Catalog No. 10414-H08H) available from Sino Biological.

[0758] In one embodiment, the antigen binding domain against LILRA2 is an antigen binding portion (e.g., CDRs) of the antibody LILRA2 monoclonal antibody (M17) (clone 3C7) available from Abnova, or the mouse anti-LILRA2 antibody (monoclonal (2D7)) available from Lasbon Biotech.

[0759] In one embodiment, the antigen binding domain against CD300LF is the antigen binding portion (e.g., CDR) of the antibody mouse anti-CMRF35-like molecule 1 antibody (monoclonal [UP-D2]) available from Biogen Biotech; or the rat anti-CMRF35-like molecule 1 antibody (monoclonal [234903]) available from R&D Systems.

[0760] In one embodiment, the antigen binding domain against CLEC12A is an antigen binding portion (e.g., CDR) of the following antibodies: Antibody Bispecific T cell engager (BiTE) scFv-antibody and ADC (described in Noordhuis et al., "Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1xCD3 BiTE Antibody" 53 rd ASH Annual Meeting and Exposition [53rd ASH Annual Meeting and Exposition, December 10-13, 2011), and MCLA-117 (Merus).

[0761] In one embodiment, an antigen binding domain against BST2 (also known as CD317) is an antigen binding portion (e.g., CDRs) of the antibody mouse anti-CD317 antibody (monoclonal [3H4]) available from Antibodies-Online or mouse anti-CD317 antibody (monoclonal [696739]) available from R&D Systems.

[0762] In one embodiment, the antigen binding domain against EMR2 (also known as CD312) is an antigen binding portion (e.g., CDRs) of the antibody mouse anti-CD312 antibody (monoclonal [LS-B8033]) available from Lasbon Biotech, Inc. or mouse anti-CD312 antibody (monoclonal [494025]) available from R&D Systems, Inc.

[0763] In one embodiment, an antigen binding domain against LY75 is an antigen binding portion (e.g., CDRs) of the antibody mouse anti-LY75 antibody (monoclonal [HD30]), available from EMD Millipore, or mouse anti-LY75 antibody (monoclonal [A15797]), available from Life Technologies.

[0764] In one embodiment, the antigen binding domain against GPC3 is an antigen binding portion (e.g., CDRs) of antibody hGC33 (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 antibodies 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).

[0765] In one embodiment, the antigen binding domain against FCRL5 is an antigen binding portion (e.g., CDRs) of an anti-FcRL5 antibody described in Elkins et al., "FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma," Mol Cancer Ther. 2012 Oct; 11(10):2222-32. In one embodiment, an antigen binding domain against FCRL5 is an antigen binding portion (e.g., CDRs) of an 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.

[0766] In one embodiment, the antigen binding domain against IGLL1 is an antigen binding portion (e.g., CDR) of the following antibodies: antibody mouse anti-immunoglobulin λ-like polypeptide 1, monoclonal [AT1G4] available from Lifespan Biosciences; mouse anti-immunoglobulin λ-like polypeptide 1 antibody, monoclonal [HSL11] available from BioLegend.

[0767] 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 the antibodies listed above, and / or one, two, three (e.g., all three) light chain CDRs (LC CDR1, LC CDR2, and LC CDR3) from the antibodies listed above. In one embodiment, the antigen binding domain comprises a heavy chain variable region and / or a variable light chain region of an antibody listed above.

[0768] On the other hand, the antigen binding domain comprises a humanized antibody or antibody fragment. In some aspects, the non-human antibody is humanized, wherein the specific sequence or region of the antibody is modified to increase the similarity with the antibody or its fragment produced naturally in humans. On the one hand, the antigen binding domain is humanized.

[0769] CARs that bind to mesothelin are known in the art. For example, those that bind to human mesothelin disclosed in WO 2015090230 and WO 2017112741 (incorporated herein by reference, e.g., Tables 2, 3, 4, and 5 of WO 2017112741). Any mesothelin CAR known in the art, e.g., the mesothelin antigen binding domain of any known mesothelin CAR can be used according to the present disclosure.

[0770] CARs incorporating GFRα-4 are known in the art. For example, those disclosed in WO2016 / 025880 can be used according to the present disclosure. Any known GFRα-4CAR in the art, for example, the GFRα-4 antigen binding domain of any known GFRα-4CAR can be used according to the present disclosure. The amino acid sequence and nucleotide sequence encoding the GFRα-4CAR molecule and antigen binding domain (for example, comprising one, two, three VH CDRs according to Kabat or Josiah; and one, two, three VLCDRs) are specified in WO2016 / 025880.

[0771] Antigen binding domain structure

[0772] In some embodiments, the antigen binding domain of the encoded CAR molecule comprises an antibody, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single domain antibody (SDAB), a VH or VL domain, a camelid VHH domain, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. [European Journal of Immunology] 17, 105 (1987)).

[0773] In some cases, scFvs can be prepared according to 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 generated by linking the VH and VL regions together using a flexible polypeptide linker. ScFv molecules contain a linker (e.g., a Ser-Gly linker) with an optimized length and / or amino acid composition. The linker length can greatly affect how the variable regions of the scFv fold and interact. In fact, if a short polypeptide linker (e.g., between 5-10 amino acids) is used, intrachain folding can be prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size, 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 PCT Publication Nos. WO 2006 / 020258 and WO 2007 / 024715 (incorporated herein by reference).

[0774] The scFv may comprise a linker of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more amino acid residues between its VL region and VH region. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequence comprises multiple sets of glycine and serine repeats, such as (Gly 4Ser) n, wherein n is a positive integer equal to or greater than 1 (SEQ ID NO: 22). In some embodiments, the linker can be (Gly 4 Ser) 4 (SEQ ID NO: 29) or (Gly 4 Ser) 3 (SEQ ID NO:30). Variations in linker length can retain or enhance activity, resulting in superior efficacy in activity studies.

[0775] On the other hand, the antigen binding domain is a T cell receptor ("TCR") or a fragment thereof, e.g., a single-chain TCR (scTCR). Methods for preparing such TCRs are known in the art. See, e.g., Willemsen RA et al., Gene Therapy [gene therapy] 7: 1369–1377 (2000); Zhang T et al., Cancer Gene Ther [cancer gene therapy] 11: 487–496 (2004); Aggen et al., Gene Ther. [gene therapy] 19 (4): 365-74 (2012) (the reference is incorporated herein in its entirety). For example, scTCRs can be engineered to contain Vα and Vβ genes from T cell clones connected by a linker (e.g., a flexible peptide). This approach is very useful for cancer-related targets that are themselves intracellular, however, fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC.

[0776] In certain embodiments, the encoded antigen binding domain has 10 -4 M to 10 -8 The binding affinity KD of M.

[0777] In some embodiments, the encoded CAR molecule comprises an antigen binding domain having a binding affinity KD of 10 for a target antigen. -4 M to 10 -8 M, for example, 10 -5 M to 10 -7 M, for example, 10 -6 M or 10 -7M. In some embodiments, the binding affinity of the antigen binding domain is at least five times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, or at least 1,000 times lower than the binding affinity of a reference antibody (e.g., an antibody described herein). In some embodiments, the binding affinity of the encoded antigen binding domain is at least 5 times lower than the binding affinity of a reference antibody (e.g., an antibody from which the antigen binding domain is derived). In some aspects, such antibody fragments are functional in that they provide a biological response that can include, but are not limited to, activation of an immune response, inhibition of signal transduction originating from its target antigen, inhibition of kinase activity, and the like, as understood by the skilled artisan.

[0778] In some aspects, the antigen binding domain of the CAR is a scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived.

[0779] In some aspects, the antigen binding domains (e.g., scFv) of CAR described herein are encoded by nucleic acid molecules, and the sequence of the nucleic acid molecules has been codon optimized to be expressed in mammalian cells. In some aspects, the entire CAR construct is encoded by nucleic acid molecules, and the entire sequence of the nucleic acid molecules has been codon optimized to be expressed in mammalian cells. Codon optimization refers to the following discovery: the frequency of occurrence of synonymous codons (i.e., codons encoding the same amino acids) in coding DNA is biased in different species. This codon degeneracy allows the same polypeptide to be encoded by various nucleotide sequences. A variety of codon optimization methods are known in the art, and include, for example, methods disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

[0780] As will be appreciated by those skilled in the art, the percentage (%) of amino acid sequence identity for nucleic acids (e.g., DNA or RNA), peptides, polypeptides can be calculated as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to the nucleotides or amino acid residues in a particular nucleotide, peptide, or polypeptide sequence, after the sequences are aligned and gaps are introduced (if necessary) to achieve the maximum percentage of sequence identity and any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining the percentage of nucleic acid amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN. TM (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required to achieve maximum comparison over the full length of the compared sequences. Unless otherwise indicated herein, BLAST is used to calculate percent identity in this article.

[0781] Specific antigen-antibody pairs are known in the art. Non-limiting exemplary embodiments of antigen-antibody pairs and components thereof are provided herein above in the section entitled Targets and below.

[0782] Bispecific CAR

[0783] In certain embodiments, the antigen binding domain is a bispecific or multispecific molecule (e.g., a multispecific antibody molecule). In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. The bispecific antibody molecule is characterized by having a first immunoglobulin variable domain sequence with binding specificity to a first epitope and a second immunoglobulin variable domain sequence with binding specificity to a second epitope. In some embodiments, the first and second epitopes are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In some embodiments, the first epitope and the second epitope overlap. In some embodiments, the first epitope and the second epitope do not overlap. In some embodiments, the first epitope and the second epitope are on different antigens, for example, on different proteins (or different subunits of a multimeric protein). In some embodiments, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity to the first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity to the second epitope. In some embodiments, the bispecific antibody molecule comprises a half antibody with binding specificity to the first epitope and a half antibody with binding specificity to the second epitope. In some embodiments, the bispecific antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope, and a half antibody or fragment thereof having binding specificity for a second epitope. In some embodiments, the bispecific antibody molecule comprises a scFv or fragment thereof having binding specificity for a first epitope, and a scFv or fragment thereof having binding specificity for a second epitope.

[0784] In some embodiments, the antibody molecule is a multispecific (e.g., bispecific or trispecific) antibody molecule. Such molecules include bispecific fusion proteins, for example, an expression construct containing two scFvs (with a hydrophilic helical peptide linker between them) and a complete constant region, as described, for example, in US5637481; a mini antibody construct with connected VL and VH chains (which are further connected to the antibody hinge region and CH3 region with a peptide spacer), which can dimerize to form a bispecific / multivalent molecule, as described, for example, in US5837821; a string of VH domains (or VL domains in family members), which are connected to the cross-linkable groups at the C-terminus by peptide bonds, which are further associated with the VL domains to form a series of FV (or scFv), as described, for example, in US 5864019; and single-chain binding polypeptides having both VH and VL domains connected by a peptide linker are combined into multivalent structures by non-covalent or chemical cross-linking to form, for example, homobivalent, heterobivalent, trivalent and tetravalent structures using scFV or diabody type formats, as described, for example, in US5869620. The contents of the above-referenced applications are incorporated herein by reference in their entirety.

[0785] In each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, VH may be upstream or downstream of VL. In some embodiments, an upstream antibody or antibody fragment (e.g., scFv) has its VH (VH1) arranged upstream of its VL (VL1), and a downstream antibody or antibody fragment (e.g., scFv) has its VL (VL2) arranged upstream of its VH (VH2), such that the entire bispecific antibody molecule has an arrangement of VH1-VL1-VL2-VH2. In other embodiments, an upstream antibody or antibody fragment (e.g., scFv) has its VL (VL1) arranged upstream of its VH (VH1), and a downstream antibody or antibody fragment (e.g., scFv) has its VH (VH2) arranged upstream of its VL (VL2), such that the entire bispecific antibody molecule has an arrangement of VL1-VH1-VH2-VL2. Optionally, if the construct is arranged as VH1-VL1-VL2-VH2, the linker is disposed between two antibodies or antibody fragments (e.g., scFv), such as VL1 and VL2, and if the construct is arranged as VL1-VH1-VH2-VL2, the linker is disposed between VH1 and VH2. The linker may be a linker as described herein, for example, (Gly 4 -Ser) nJoint, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 691). In general, the joint between the two scFvs should be long enough to avoid mismatching between the domains of the two scFvs. Optionally, the joint is arranged between the VL and VH of the first scFv. Optionally, the joint is arranged between the VL and VH of the second scFv. In a construct with multiple joints, any two or more of the joints may be the same or different. Therefore, in some embodiments, the bispecific CAR comprises VL, VH and optionally one or more joints in an arrangement as described herein.

[0786] Transmembrane domain

[0787] About transmembrane domain, in various embodiments, chimeric molecules described herein (for example, CAR) can be designed to include transmembrane domains of extracellular domains attached to chimeric molecules. Transmembrane domains may include one or more other amino acids adjacent to transmembrane region, for example, one or more amino acids related to the extracellular region of a protein derived from a transmembrane source (for example, 1,2,3,4,5,6,7,8,9,10 to 15 amino acids in the extracellular region) and / or one or more other amino acids related to the intracellular region of a protein derived from a transmembrane protein (for example, 1,2,3,4,5,6,7,8,9,10 to 15 amino acids in the intracellular region). In some aspects, a transmembrane domain is associated with one of the other domains of a chimeric protein (for example, CAR), for example, in some embodiments, a transmembrane domain may be derived from the same protein from which a signaling domain, a costimulatory domain or a hinge domain is derived. In another aspect, a transmembrane domain is not derived from the same protein from which any other domain of a chimeric protein (for example, CAR) is derived. In some cases, the transmembrane domain can be selected or modified by amino acid replacement to avoid such a domain from binding to the transmembrane domain of the same or different surface membrane proteins, for example, to minimize the interaction with other members of the receptor complex. In some aspects, the transmembrane domain can homodimerize with another CAR on the cell surface of the cell expressing CAR. In different aspects, the amino acid sequence of the transmembrane domain can be modified or replaced to minimize the interaction with the binding domain of the natural binding partner present in the same cell expressing CAR.

[0788] The transmembrane domain can be derived from a natural source or from a recombinant source. In the case where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In some aspects, whenever CAR is bound to a target, the transmembrane domain is able to conduct signals to one or more intracellular domains. In some embodiments, the transmembrane domain may include at least one or more transmembrane regions of, for example, α, β or ζ chains of a T cell receptor, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain may include at least one or more of the following transmembrane regions: 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, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, L y9(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.

[0789] In some cases, the transmembrane domain can be attached to the extracellular region of the CAR (e.g., the antigen binding domain of the CAR) by 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, 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 an amino acid sequence of SEQ ID NO: 4 (e.g., consisting thereof). In some aspects, the transmembrane domain comprises a transmembrane domain of SEQ ID NO: 12 (e.g., consisting thereof).

[0790] In some embodiments, the encoded transmembrane domain comprises an 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 of 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.

[0791] In other embodiments, the nucleic acid molecule encoding CAR comprises a nucleotide sequence of a CD8 transmembrane domain, e.g., a sequence comprising SEQ ID NO: 13, or a sequence having 95%-99% identity thereof.

[0792] 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 with 95%-99% identity to SEQ ID NO: 4 or 6. In other embodiments, the nucleic acid sequence encoding the hinge region comprises a sequence of SEQ ID NO: 5 or SEQ ID NO: 7 corresponding to a CD8 hinge or an IgG4 hinge, respectively, or a sequence with 95%-99% identity to SEQ ID NO: 5 or 7.

[0793] In some aspects, the hinge or spacer comprises an IgG4 hinge. For example, in some embodiments, the hinge or spacer comprises a hinge of the amino acid sequence ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO: 6).In some embodiments, the hinge or spacer comprises a hinge encoded by the nucleotide sequence of GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG(SEQ ID NO:7).

[0794] In some aspects, the hinge or spacer comprises an IgD hinge. For example, in some embodiments, the hinge or spacer comprises a hinge of the amino acid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO: 8).In some embodiments, the hinge or spacer comprises a hinge encoded by the nucleotide sequence of (SEQ ID NO: 9).

[0795] In some aspects, the transmembrane domain can be recombinant, in which case it will comprise primarily hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.

[0796] Optionally, a short oligopeptide or polypeptide linker of between 2 and 10 amino acids in length can form a bond between the transmembrane domain and the cytoplasmic region of the CAR. Glycine-serine doublets provide particularly suitable linkers. For example, in some aspects, the linker comprises an amino acid sequence of GGGGSGGGGS (SEQ ID NO: 10). In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 11). In some embodiments, the linker comprises an amino acid sequence of GGGGS (SEQ ID NO: 877). In some embodiments, the linker is encoded by a nucleotide sequence of SEQ ID NO: 876.

[0797] In some aspects, the hinge or spacer comprises a KIR2DS2 hinge.

[0798] Signaling domain

[0799] In some embodiments of the CAR described herein having an intracellular signaling domain, such a domain may contain, 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.

[0800] The intracellular signaling sequences in the cytoplasmic part of CAR can be connected to each other in a random or specified order. Optionally, a short oligopeptide or polypeptide linker, for example, a length between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) can form a key between 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.

[0801] In some aspects, the intracellular signaling domain is designed to include two or more (e.g., 2, 3, 4, 5, or more) costimulatory signaling domains. In some embodiments, 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 includes two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0802] Primary signaling domain

[0803] The primary signaling domain regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary intracellular signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. In CAR, such a domain serves the same purpose.

[0804] Examples of primary intracellular signaling domains containing ITAMs include those of CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10 and DAP12. In some embodiments, the CAR comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-ζ.

[0805] In some embodiments, the encoded primary signaling domain comprises a functional signaling domain of CD3 zeta. The encoded CD3 zeta primary signaling domain may comprise an amino acid sequence having at least one, two or three modifications, but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20, or a sequence having 95%-99% identity to an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, the encoded primary signaling domain comprises a sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In other embodiments, the nucleic acid sequence encoding the primary signaling domain comprises a sequence of SEQ ID NO: 19 or SEQ ID NO: 21, or a sequence having 95%-99% identity thereto.

[0806] co-stimulatory signaling domain

[0807] In some embodiments, the encoded intracellular signaling domain comprises a co-stimulatory signaling domain. For example, the intracellular signaling domain can comprise a primary signaling domain and a co-stimulatory signaling domain. In some embodiments, the encoded co-stimulatory signaling domain comprises a functional signaling domain of a protein selected from one or more of the following: 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, 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, IT GAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TR ANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD22 9), 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.

[0808] 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 of an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16, or a sequence with 95%-99% identity to an amino acid sequence of 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 thereof.

[0809] 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, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

[0810] In some embodiments, the nucleic acid sequence encoding the intracellular signaling domain comprises a sequence of SEQ ID NO: 15 or SEQ ID NO: 17, or a sequence with 95%-99% identity thereof; and a sequence of SEQ ID NO: 19 or SEQ ID NO: 21, or a sequence with 95%-99% identity thereof.

[0811] 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.

[0812] In some aspects, the intracellular signaling domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28. In some aspects, the intracellular signaling domain is designed to include the signaling domain of CD3-ζ and the signaling domain of 4-1BB. In some aspects, the signaling domain of 4-1BB is the signaling domain of SEQ ID NO: 14. In some aspects, the signaling domain of CD3-ζ is the signaling domain of SEQ ID NO: 18.

[0813] In some aspects, the intracellular signaling domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD27. In some aspects, the signaling domain of CD27 includes the amino acid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 16). In some aspects, the signaling domain of CD27 is encoded by the nucleic acid sequence of AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 17).

[0814] Inhibitory domain

[0815] 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 inhibitor KIR (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 having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology or differing by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues with a naturally occurring inhKIR.

[0816] 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 a sequence that has 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.

[0817] In some embodiments, the vector is an in vitro transcribed vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. In some embodiments, the nucleic acid sequence in the vector further comprises a poly (A) tail, e.g., a poly A tail. In some embodiments, the nucleic acid sequence in the vector further comprises a 3'UTR, e.g., a 3'UTR described herein, e.g., comprising at least one repeat of a 3'UTR derived from human β-globulin. In some embodiments, the nucleic acid sequence in the vector further comprises a promoter, e.g., a T2A promoter.

[0818] Promoter

[0819] In some embodiments, the vector further comprises a promoter. In some embodiments, the promoter is selected from EF-1 promoter, CMV IE gene promoter, EF-1α promoter, ubiquitin C promoter or phosphoglycerate kinase (PGK) promoter. In some embodiments, the promoter is EF-1 promoter. In some embodiments, the EF-1 promoter comprises a sequence of SEQ ID NO: 1.

[0820] In some embodiments, immune effector cells (e.g., T cells) can be expressed using a variety of techniques known to those of skill in the art (e.g., Ficoll TM In some aspects, the cells of the circulating blood from an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In some aspects, the cells collected by apheresis can be washed to remove the plasma portion, and the cells are optionally suspended in a buffer or culture medium for subsequent processing steps. In certain embodiments, cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution lacks calcium and may lack magnesium, or may lack many (if not all) divalent cations.

[0821] Table 17: Sequences of various components of CAR (aa-amino acid, na-nucleic acid encoding the corresponding protein)

[0822]

[0823]

[0824]

[0825]

[0826]

[0827]

[0828] Manufacturing method

[0829] The lentiviral vectors described herein can be used, for example, in the in vitro manufacture of CAR-T cells (e.g., using the methods described herein).

[0830] The CART disclosed herein can be manufactured in vitro by any method known in the art. For example, the method described in WO2012 / 079000 or WO2020 / 047452 (both incorporated herein by reference) can be used. The CART disclosed herein can also be manufactured in vivo by any method known in the art. For example, the method disclosed in WO2020 / 176397 (incorporated herein by reference). In vivo CART production is also described in, for example, WO / 2022 / 040586 (incorporated herein by reference in its entirety).

[0831] The immune effector cell (e.g., a T cell or a NK cell) can express one CAR or two or more CARs.

[0832] In some embodiments, the method disclosed herein can manufacture immune effector cells engineered to express one or more CARs in less than 24 hours. Without wishing to be bound by theory, the method provided herein retains the undifferentiated phenotype of T cells, such as initial T cells, during the manufacturing process. These cells expressing CAR with undifferentiated phenotype can continue longer and / or better expand in vivo after infusion. In some embodiments, compared with CART cells produced by traditional manufacturing processes (e.g., as measured using scRNA-seq), the CART cells produced by the manufacturing method provided herein include a higher percentage of stem cell memory T cells.

[0833] In some embodiments, the CART cells produced by the manufacturing methods provided herein include a higher percentage of effector T cells than CART cells produced by traditional manufacturing processes, for example, measured using scRNA-seq. In some embodiments, compared with CART cells produced by traditional manufacturing processes (e.g., as measured using scRNA-seq), CART cells produced by the manufacturing methods provided herein better maintain the rigidity of T cells. In some embodiments, CART cells produced by the manufacturing methods provided herein show lower hypoxia levels than CART cells produced by traditional manufacturing processes, for example, measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein show lower levels of autophagy than CART cells produced by traditional manufacturing processes, for example, measured using scRNA-seq. In some embodiments, immune effector cells are engineered to include nucleic acid molecules encoding one or more CARs disclosed herein.

[0834] In some embodiments, the methods disclosed herein do not involve the use of beads, e.g. (e.g., CD3 / CD28 ), and no bead removal step is involved. In some embodiments, the CART cells manufactured by the methods disclosed herein can be administered to a subject with minimal ex vivo expansion, for example, 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. Therefore, the methods described herein provide a rapid manufacturing process for preparing an improved CAR-expressing cell product, which is used to treat a subject's disease.

[0835] In some embodiments, the present disclosure provides methods for preparing a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), the methods comprising: (i) contacting a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukapheresis products) with an agent that stimulates the CD3 / TCR complex and / or with an agent that stimulates co-stimulatory molecules on the surface of the cells; (ii) contacting a population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule; and (iii) harvesting the population of cells (e.g., T cells) for storage (e.g., reconstituting the cell population in a cryopreservation medium) or administration, wherein: (a) step (ii) is performed together with step (i), or is performed no later than 20 hours after the start of step (i) (e.g., no later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), e.g., no later than 18 hours after the start of step (i)). , and step (iii) is performed no later than 26 hours after the start of step (i) (e.g., no later than 22, 23, or 24 hours after the start of step (i), for example, no later than 24 hours after the start of step (i)); (b) step (ii) is performed together with step (i), or no later than 20 hours after the start of step (i) (e.g., no later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example, no later than 18 hours after the start of step (i)), And step (iii) is performed no later than 30 hours after the start of step (ii) (e.g., no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii); or (c) the cell population from step (iii) does not expand, or expands by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, such as no more than 10%, compared to the cell population at the start of step (i), such 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 in step (ii) is 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 in step (ii) is on a non-viral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule in step (ii) is not on any vector. In some embodiments, step (ii) comprises transducing a population of cells (e.g., T cells) with one or more viral vectors comprising a nucleic acid molecule encoding one or more CARs.

[0836] In some embodiments, a population of cells (eg, T cells) is collected from an aliquot sample (eg, a leukocyte aliquot sample) of a subject.

[0837] In some embodiments, an aliquot (e.g., a leukocyte aliquot) is collected from a subject and transported to a cell manufacturing facility as a frozen sample (e.g., a cryopreserved sample). The frozen aliquot is then thawed, and T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the aliquot, e.g., using a cell sorter (e.g., Device). The selected T cells (e.g., CD4+T cells and / or CD8+T cells) are then inoculated using the activation process described herein for CART manufacturing. In some embodiments, the selected T cells (e.g., CD4+T cells and / or CD8+T cells) undergo one or more rounds of freezing-thawing before inoculation for CART manufacturing.

[0838] In some embodiments, an aliquot sample (e.g., a leukocyte aliquot sample) is collected from a subject and transported to a cell manufacturing facility as a fresh product (e.g., a non-frozen sample). T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the aliquot sample, e.g., using a cell sorter (e.g., Device). The selected T cells (e.g., CD4+T cells and / or CD8+T cells) are then inoculated using the activation process described herein for CART manufacturing. In some embodiments, the selected T cells (e.g., CD4+T cells and / or CD8+T cells) undergo one or more rounds of freezing-thawing before inoculation for CART manufacturing.

[0839] In some embodiments, an aliquot (e.g., a leukocyte aliquot) is collected from a subject. T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the aliquot, e.g., using a cell sorter (e.g., Device). The selected T cells (e.g., CD4+T cells and / or CD8+T cells) are then transported to a cell manufacturing facility as a frozen sample (e.g., a frozen sample). The selected T cells (e.g., CD4+T cells and / or CD8+T cells) are then thawed and inoculated for CART manufacturing using the activation process described herein.

[0840] In some embodiments, cells (e.g., T cells) are contacted with anti-CD3 and anti-CD28 antibodies for example 12 hours, followed by transduction with a vector (e.g., lentiviral vector) (e.g., one or more vectors) encoding CAR (e.g., one or more CARs). 24 hours after the start of culture, cells are washed and prepared for storage or administration.

[0841] Without wishing to be bound by theory, brief CD3 and CD28 stimulation can promote the effective transduction of self-renewal T cells. Compared with traditional CART manufacturing methods, the activation process provided herein does not involve extended ex vivo expansion. Similar to the cytokine process, the activation process provided herein also retains undifferentiated T cells during CART manufacturing.

[0842] In some embodiments, the cell population is contacted with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule on the surface of the cell.

[0843] In some embodiments, the agent for stimulating CD3 / TCR complex is the agent for stimulating CD3. In some embodiments, the agent for stimulating costimulatory molecules is the agent for stimulating CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226 or any combination thereof. In some embodiments, the agent for stimulating costimulatory molecules is the agent for stimulating CD28. In some embodiments, the agent for stimulating CD3 / TCR complex is selected from antibody (for example, single domain antibody (for example, heavy chain variable domain antibody), peptide body, Fab fragment or scFv), small molecule or ligand (for example, naturally occurring ligand, recombinant ligand or chimeric ligand). In some embodiments, the agent for stimulating CD3 / TCR complex is antibody. In some embodiments, the agent for stimulating CD3 / TCR complex is anti-CD3 antibody. In some embodiments, the agent that stimulates the co-stimulatory 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 a scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the agent that stimulates the co-stimulatory molecule is an antibody. In some embodiments, the agent that stimulates the co-stimulatory molecule is an anti-CD28 antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex or the agent that stimulates the co-stimulatory molecule does not comprise beads. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates the co-stimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates the co-stimulatory molecule comprise a T cell TransAct TM .

[0844] In certain embodiments, matrix comprises polymer or is made up of it, for example, biodegradable or biocompatible inert material, for example, it is nontoxic to cells.In certain embodiments, matrix is ​​made up of hydrophilic polymer chain, and due to the hydration of chain, it obtains maximum mobility in aqueous solution.In certain embodiments, mobile matrix can be collagen, purified protein, purified peptide, polysaccharide, glycosaminoglycan or extracellular matrix composition.Polysaccharide can include for example cellulose ether, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan gum, guar gum or alginate.Other polymers can include polyester, polyether, polyacrylate, polyacrylamide, polyamine, polyethyleneimine, polyquaternary ammonium salt polymer, polyphosphazene, polyvinyl alcohol, polyvinyl acetate, polyvinyl pyrrolidone, block copolymer or polyurethane.In certain embodiments, mobile matrix is ​​the polymer of dextran.

[0845] In some embodiments, cell colony is contacted with nucleic acid molecules (e.g., one or more nucleic acid molecules) encoding CAR (e.g., one or more CARs). In some embodiments, cell colony is transduced with a DNA molecule (e.g., one or more DNA molecules) encoding CAR (e.g., one or more CARs).

[0846] In some embodiments, in the case of co-transduction of two nucleic acid molecules (e.g., lentiviral vectors), each of which encodes a CAR disclosed herein, each vector containing a nucleic acid molecule encoding a CAR can be added to a reaction mixture (e.g., containing a cell population) at a different multiplicity of infection (MOI).

[0847] Without wishing to be bound by theory, it is believed that in some embodiments, for vectors containing nucleic acid molecules encoding different CAR molecules, using different MOIs may affect the final composition of the cell population. For example, in the case of co-transduction of a lentiviral vector encoding one CAR and encoding another CAR targeting a different target, different MOIs can be used to maximize the percentage of preferred single CART cells and dual CART cells, while resulting in fewer unwanted single CART cells and untransduced cells.

[0848] The precise MOI used for each vector can be adjusted or determined based on many factors (including but not limited to the characteristics of the viral vector batch, the characteristics of the cells to be transduced, and the transduction efficiency). In some embodiments, the cell colony is contacted with one or more nucleic acid molecules encoding one or more CARs, and the cell colony is contacted with the agent stimulating the CD3 / TCR complex and / or the agent stimulating the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell colony is contacted with the agent stimulating the CD3 / TCR complex and / or the agent stimulating the co-stimulatory molecules on the surface of the above-mentioned cells no later than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 hours after the cell colony is contacted with the agent stimulating the CD3 / TCR complex and / or the agent stimulating the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 20 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 19 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 18 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 17 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells.

[0849] In some embodiments, no later than 16 hours after the cell population contacts the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs. In some embodiments, no later than 15 hours after the cell population contacts the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs. In some embodiments, no later than 14 hours after the cell population contacts the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs. In some embodiments, no later than 14 hours after the cell population contacts the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 13 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 12 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 11 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 10 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 9 hours after the cell population is contacted with an agent that stimulates the CD3 / TCR complex and / or a co-stimulatory molecule on the surface of the above-mentioned cell. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 8 hours after the cell population is contacted with an agent that stimulates the CD3 / TCR complex and / or a co-stimulatory molecule on the surface of the above-mentioned cell.In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 7 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-mentioned cells. In some embodiments, the cell population is contacted with one or more nucleic acid molecules encoding one or more CARs no later than 6 hours after the cell population is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the co-stimulatory molecules on the surface of the above-men...

Claims

1. A composition comprising: 1) lentiviral vector; and 2) an aqueous composition, the aqueous composition comprising: a) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES); and b) one or both of free positively charged amino acids and free non-polar amino acids.

2. The composition of claim 1, wherein the aqueous composition is substantially free of inorganic salts.

3. The composition of claim 1, wherein the aqueous composition is substantially free of NaCl and MgCl 2 One or both of the following.

4. The composition of claim 1, wherein the aqueous composition comprises less than 20 mM of inorganic salts in a total amount.

5. A composition as claimed in any one of the preceding claims, wherein the concentration of HEPES is 10-200, 10-150, 10-100, 10-50, 10-40, 10-30, 15-25 or 20 mM.

6. The composition of any of the preceding claims, wherein the concentration of HEPES is about 20 mM.

7. The composition of any one of the preceding claims, comprising the positively charged amino acid, wherein the positively charged amino acid is arginine, lysine or histidine.

8. The composition of claim 7, wherein the positively charged amino acid is arginine.

9. The composition of claim 8, wherein the arginine is L-arginine.

10. The composition of any one of claims 7-9, wherein the concentration of arginine is at least 50, at least 75, at least 100, or at least 150 mM.

11. The composition of any one of claims 7-10, wherein the concentration of arginine is 25-50, 50-75, 75-100, 75-125, 100-200, 125-175 or 150 mM.

12. The composition of any one of claims 7-10, wherein the concentration of arginine is about 150 mM.

13. The composition of any one of the preceding claims, comprising the non-polar amino acid, wherein the non-polar amino acid is proline, methionine or tryptophan.

14. The composition of claim 13, wherein the non-polar amino acid is proline.

15. The composition of claim 14, wherein the proline is L-proline.

16. The composition of any one of claims 13-15, wherein the concentration of proline is 25-200, 50-200, 100-200, 125-175 or 150 mM.

17. The composition of any one of claims 13-15, wherein the concentration of proline is about 150 mM.

18. The composition of any one of the preceding claims, further comprising a cryoprotectant.

19. The composition of claim 18, wherein the cryoprotectant is a carbohydrate.

20. The composition of claim 19, wherein the carbohydrate is a non-reducing carbohydrate.

21. The composition of claim 19, wherein the carbohydrate is sucrose.

22. The composition of any one of embodiments 1-17, further comprising sucrose.

23. The composition of claim 22, wherein the concentration of sucrose is 25-200, 50-200, 100-200, 125-175 or 150 mM.

24. The composition of claim 22, wherein the concentration of sucrose is about 150 mM.

25. The composition of any one of the preceding claims, wherein the aqueous composition further comprises a stabilizer.

26. The composition of any one of claims 1-24, wherein the aqueous composition comprises HSA.

27. The composition of claim 26, wherein the HSA comprises recombinant HSA (rHSA) or human-derived HSA.

28. The composition of any of claims 26 or 27, wherein the HSA is present at 0.5%-3%, 0.5%-2%, 0.5%-1%, 1%-2%, 1.5%-2.5% or 2% w / v.

29. The composition of claim 28, wherein the HSA is present at about 2% w / v.

30. The composition of any one of claims 1-24, wherein the aqueous composition is substantially free of HSA.

31. A composition as claimed in any preceding claim, having a pH of 6.0-7.5, 6.0-7.0, 6.0-6.5, 6.5-7.0, 6.2-6.8, 6.4-6.6 or 6.

5.

32. A composition as claimed in any preceding claim, having a pH of about 6.

5.

33. A composition as described in any of the preceding claims, wherein the aqueous composition is substantially free of one, two or three of PEG lipid, F108 and cholesterol.

34. A composition as claimed in any one of the preceding claims, in: The aqueous composition comprises the positively charged amino acid, wherein the positively charged amino acid comprises L-arginine; The aqueous composition comprises the non-polar amino acid, wherein the non-polar amino acid comprises L-proline; The aqueous composition further comprises sucrose; and The aqueous composition is substantially free of inorganic salts.

35. The composition of claim 34, in: The concentration of the L-arginine is 100-200 mM; The concentration of L-proline is 25-200 mM; and The concentration of sucrose is 25-200 mM.

36. The composition of any of the preceding claims, wherein the aqueous composition has an osmolality of about 400 mOsm / kg to about 700 mOsm / kg.

37. The composition of claim 36, wherein the aqueous composition has an osmolality of about 415 mOsm / kg to about 689 mOsm / kg.

38. A composition as claimed in any one of the preceding claims, comprising at least 1 x 10 6 , at least 1x10 7 , at least 5x 10 7 , at least 1x 10 8 , at least 1x 10 9 , at least 2x 10 9 , at least 3x 10 9 , at least 4x 10 9 , at least 5x 10 9 or at least 6x 10 9 transducing units / ml (TU / mL) of the lentiviral vector.

39. The composition of any of the preceding claims, wherein the lentiviral vector comprises a transgene.

40. The composition of claim 39, wherein the transgene encodes a chimeric antigen receptor (CAR).

41. The composition of claim 39, wherein the CAR comprises an antigen binding domain that binds to a tumor antigen, the antigen binding domain being selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostate enzyme, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-ab l, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumin, HPV E6, HPVE7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1, and any combination thereof.

42. The composition of claim 40 or 41, wherein the CAR comprises an antigen binding domain, a transmembrane domain, one or more primary signaling domains, and / or one or more co-stimulatory signaling domains.

43. The composition of claim 42, wherein the one or more primary signaling domains comprises a CD3-zeta stimulatory domain.

44. The composition of claim 42 or 43, wherein the one or more costimulatory signaling domains comprise an intracellular domain selected from: (a) a co-stimulatory protein selected from the group consisting of: OX40, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, 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(CD22 9), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and (b) a ligand that specifically binds to CD83, wherein the ligand is selected from the group consisting of a 4-1BB (CD137) co-stimulatory domain and a CD28 co-stimulatory domain.

45. A composition as described in any one of claims 42 to 44, in: (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154; (b) the transmembrane domain comprises the transmembrane domain of CD8; (c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 90% sequence identity thereto; or (d) the transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 90% sequence identity thereto.

46. ​​The composition of any of the preceding claims, wherein the composition exhibits a level of subvisible particles greater than or equal to 25 μm of about 50-100 particles / mL or less.

47. The composition of any of the preceding claims, wherein the composition exhibits a level of subvisible particles greater than or equal to 10 μm of about 1100-2000 particles / mL or less.

48. The composition of any of the preceding claims, wherein the lentiviral vector in the composition retains a hydrodynamic diameter between about 85-200, 90-200, or 85-130 nm.

49. A kit comprising: the lentiviral vector according to any one of the preceding claims and an aqueous composition.

50. A method for preparing a cell expressing CAR, the method include: Providing a composition as claimed in any one of the preceding claims; as well as contacting the composition with immune effector cells under conditions that allow transduction of one or more of the immune effector cells, Thereby preparing the CAR-expressing cells.

51. The method of claim 50, which is an in vitro or ex vivo method.

52. The method of claim 50 or 51, wherein the immune effector cell population comprises one or both of T cells and NK cells.

53. The method of any one of claims 50-52, wherein the composition comprises HSA.

54. The method of claim 53, wherein the concentration of HSA is about 0.5% to 3% w / v.

55. The method of claim 53 or 54, wherein the HSA comprises recombinant HSA (rHSA) or human-derived HSA.

56. A method of delivering a transgene to a subject, the method comprising administering to the subject the composition of any one of the preceding claims, wherein the lentiviral vector of the composition comprises the transgene.

57. The method of claim 56, wherein the transgene comprises a CAR.

58. The method of claim 56 or 57, wherein the composition further comprises particles.

59. The method of claim 58, wherein the particles are silica particles.

60. The method of claim 59, wherein the silica particles are mesoporous particles (MSP).

61. The method of claim 60, wherein the mesoporous silica particles are mesoporous silica rods.

62. The method of claim 60 or 61, in: (i) a retroviral vector is non-covalently or covalently associated with the mesoporous silica particles; and / or (ii) a cell activating agent is non-covalently or covalently associated with the mesoporous silica particles.

63. The method of any one of claims 56-61, wherein the composition further comprises a cell activating agent.

64. The method of claim 63, wherein the cell activating agent: (a) comprising an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule and / or a growth factor receptor; (b) is a multispecific binding molecule comprising: (i) an anti-CD3 binding domain, and (ii) a co-stimulatory molecule binding domain; and / or (c) conjugated to or adsorbed onto the particle.

65. The method of claim 64, wherein the co-stimulatory molecule binding domain is an anti-CD2 binding domain or an anti-CD28 binding domain.

66. The method of claim 64 or 65, wherein the particles are mesoporous silica particles.

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