Mesoporous silica particle compositions for viral delivery

Abstract

To provide compositions for the delivery of viral vectors.SOLUTION: The present invention provides compositions including mesoporous silica particles that may be surface-modified, and viral vectors. In some embodiments, the viral vectors are used to transduce T cells to express a chimeric antigen receptor (CAR), in order to treat a subject having a disease, e.g., a disease associated with expression of a tumor antigen.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Patent Application No. 62 / 810,260, filed February 25, 2019, the contents of which are incorporated by reference herein in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on February 20, 2020, is named N2067-7161WO_SL.txt and is 232,920 bytes in size.

[0003] The present invention relates generally to the use of mesoporous silica compositions for the delivery of viral vectors or drug substances. In some embodiments, the viral vector comprises a nucleotide sequence that is engineered to express a chimeric antigen receptor (CAR) to treat a subject having a disease, such as a disease associated with the expression of a tumor antigen. [Background technology]

[0004] T cell adoptive transfer protocols show potential in many therapeutic applications, such as cancer, where CAR T cell therapy has recently been approved for the treatment of B cell malignancies. There is a need to locally deliver viral vectors or drug substances and find efficient manufacturing processes. Summary of the Invention [Means for solving the problem]

[0005] Contemplated herein is a composition comprising a first population of mesoporous silica particles and a viral vector. In some embodiments, the viral vector is conjugated to the first population of mesoporous silica particles. In some embodiments, the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles. In some embodiments, the first population of mesoporous silica particles is surface modified. In some embodiments, the surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, optionally C1-C 20 Alkyl or (-O(CH2-CH2-) 1~25A linker is used. In some embodiments, the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary or quaternary amine. In some embodiments, the surface modification of the first population of mesoporous silica particles is a polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da or about 10,000 Da as measured by gel permeation chromatography (GPC). In some embodiments, the viral vector is a retrovirus, adenovirus, adeno-associated virus, herpes virus or lentivirus. In some embodiments, the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. In some embodiments, the nucleotide sequence encodes a chimeric antigen receptor (CAR), an engineered TCR, one or more cytokines, one or more chemokines, shRNA to block inhibitory molecules, or the nucleotide sequence comprises an mRNA to induce expression of a protein. In some embodiments, the nucleotide sequence encodes a polypeptide engineered to target a tumor antigen. In some embodiments, the polypeptide encodes a polypeptide encoding a tumor antigen, such as TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, 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, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, 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, mutThe composition targets a tumor antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and any combination thereof. In some embodiments, the protein is a CAR comprising an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a signaling domain. In some embodiments, the signaling domain is a CD3 zeta signaling domain. In some embodiments, the composition further comprises a T cell stimulatory compound or a tumor antigen. In some embodiments, the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles, and the T cell stimulatory compound is selected from the group consisting of IL-2, IL-15, anti-CD2 mAb, anti-CD3 mAb, anti-CD28 mAb, neoantigen peptides, peptides from common antigens such as TRP2, gp100, tumor cell lysate, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3TCR or a combination thereof. In some embodiments, the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles. In some embodiments, the composition comprises a second population of mesoporous silica particles, the T cell stimulatory compound or tumor antigen is conjugated to the second population of mesoporous silica particles or to a lipid envelope on the surface of the second population of mesoporous silica particles. In some embodiments, the composition further comprises a cytokine. In some embodiments, the cytokine is conjugated or adsorbed to the first or second population of mesoporous silica particles. In some embodiments, the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21 or transforming growth factor beta (TGF-β) or an agonist, mimetic, variant, functional fragment, or combination thereof. In some embodiments, the mesoporous silica particles comprise pores with a diameter of 2 to 50 nm. In some embodiments, the mesoporous silica particles have a pore size of at least about 100 mm. 2 / g. In some embodiments, the composition is suitable for injection. In some embodiments, the mesoporous silica particles are in the form of mesoporous silica rods.

[0006] Also contemplated herein is a method comprising contacting a T lymphocyte with a composition comprising a first population of mesoporous silica particles and a viral vector, wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. In some embodiments, the contacting occurs in vitro. In some embodiments, the T lymphocyte is activated before or after contacting with the first population of mesoporous silica particles. In some embodiments, the viral vector is conjugated to the first population of mesoporous silica particles. In some embodiments, the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles. In some embodiments, the first population of mesoporous silica particles is surface modified. In some embodiments, the surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, optionally C1-C 20 Alkyl or (-O(CH2-CH2-) 1~25A linker is used. In some embodiments, the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary or quaternary amine. In some embodiments, the first population of mesoporous silica particles is surface modified with polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da or about 10,000 Da as measured by gel permeation chromatography (GPC). In some embodiments, the viral vector is a lentivirus, retrovirus or adenovirus. In some embodiments, the nucleotide sequence encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR is engineered to target a tumor antigen. In some embodiments, the T lymphocytes are activated by contacting the T lymphocytes with a T cell stimulating compound or a tumor antigen. In some embodiments, the T cell stimulating compound or the tumor antigen is conjugated or adsorbed to a first population of mesoporous silica particles or a second population of mesoporous silica particles. In some embodiments, the T cell stimulating compound or the tumor antigen is conjugated or adsorbed to a first population of mesoporous silica particles. In some embodiments, the T cell stimulating compound or the tumor antigen is directly conjugated to a second population of mesoporous silica particles or a lipid envelope on the surface of the second population of mesoporous silica particles. In some embodiments, the method further comprises contacting the T lymphocytes with a cytokine. In some embodiments, the cytokine is in the medium or is conjugated or adsorbed to the first or second population of mesoporous silica particles. In some embodiments, the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, or transforming growth factor beta (TGF-β), or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof.In some embodiments, the mesoporous silica particles are in the form of mesoporous silica rods.

[0007] Also contemplated herein is a method of genetically transducing T lymphocytes with a recombinant polynucleotide in vivo, comprising administering a composition comprising a first population of mesoporous silica particles and a viral vector to a subject having one or more T lymphocytes, wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed, and wherein upon contact of the composition with one or more T lymphocytes, the T lymphocytes are genetically transduced by the recombinant polynucleotide. In some embodiments, the viral vector is conjugated to the first population of mesoporous silica particles. In some embodiments, the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles. In some embodiments, the first population of mesoporous silica particles is surface modified. In some embodiments, the surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, and optionally C1-C 20 Alkyl or (-O(CH2-CH2-) 1~25A linker is used. In some embodiments, the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary or quaternary amine. In some embodiments, the first population of mesoporous silica particles is surface modified with polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da or about 10,000 Da as measured by gel permeation chromatography (GPC). In some embodiments, the viral vector is a lentivirus, retrovirus or adenovirus. In some embodiments, the nucleotide sequence encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR is engineered to target a tumor antigen. In some embodiments, the composition further comprises a T cell stimulating compound or a tumor antigen conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles. In some embodiments, the T cell stimulating compound or the tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles. In some embodiments, the composition comprises a second population of mesoporous silica particles, and the T cell stimulating compound or the tumor antigen is conjugated directly to the second population of mesoporous silica particles or to the lipid envelope on the surface of the second population of mesoporous silica particles. In some embodiments, the first or second population of mesoporous silica particles further comprises a cytokine conjugated or adsorbed to the first or second population of mesoporous silica particles. In some embodiments, the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, or transforming growth factor beta (TGF-β), or an agonist, mimetic, variant, functional fragment, or combination thereof. In some embodiments, the subject's T lymphocytes are expanded in vivo.In some embodiments, the mesoporous silica particles are in the form of mesoporous silica rods.

[0008] Also contemplated herein is a method of expanding a T lymphocyte population in vitro, comprising: (a) contacting the T lymphocyte population with a composition comprising a first population of mesoporous silica particles and a viral vector to provide a transduced T lymphocyte population; and (b) contacting the transduced T lymphocyte population with a T cell stimulatory compound or a tumor antigen and optionally a cytokine, wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. In some embodiments, the viral vector is conjugated to the first population of mesoporous silica particles. In some embodiments, the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles. In some embodiments, the first population of mesoporous silica particles is surface modified. In some embodiments, the surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, and optionally C1-C 20 Alkyl or (-O(CH2-CH2-) 1~25A linker is used. In some embodiments, the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary or quaternary amine. In some embodiments, the first population of mesoporous silica particles is surface modified with polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da or about 10,000 Da as measured by gel permeation chromatography (GPC). In some embodiments, the viral vector is a lentivirus, retrovirus or adenovirus. In some embodiments, the nucleotide sequence encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR is engineered to target a tumor antigen. In some embodiments, the T cell stimulating compound or tumor antigen is conjugated or adsorbed to a first population of mesoporous silica particles or a second population of mesoporous silica particles, and the T cell stimulating compound or tumor antigen is IL-2, IL-15, anti-CD2 mAb, anti-CD3 mAb, anti-CD28 mAb, neo-antigen peptide, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or a combination thereof. In some embodiments, the T cell stimulating compound or tumor antigen is conjugated or adsorbed to a first population of mesoporous silica particles. In some embodiments comprising the second population of mesoporous silica particles, the T cell stimulatory compound or the tumor antigen is conjugated to the second population of mesoporous silica particles or to a lipid envelope on the surface of the second population of mesoporous silica particles.In some embodiments, the method further comprises (c) contacting the T lymphocytes with a cytokine, the cytokine being IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21 or transforming growth factor beta (TGF-β) or an agonist, mimetic, variant, functional fragment or combination thereof. In some embodiments, the mesoporous silica particles are in the form of mesoporous silica rods.

[0009] Also contemplated herein is a method of treating a subject having a disease, injury or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a composition comprising a first population of mesoporous silica particles and a viral vector, the viral vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR) engineered to target the tumor antigen, thereby treating the subject. In some embodiments, the viral vector is conjugated to the first population of mesoporous silica particles. In some embodiments, the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles. In some embodiments, the first population of mesoporous silica particles is surface modified. In some embodiments, the surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, optionally C1-C 20 Alkyl or (-O(CH2-CH2-) 1~25A linker is used. In some embodiments, the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary or quaternary amine. In some embodiments, the first population of mesoporous silica particles is surface modified with polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da or about 10,000 Da as measured by gel permeation chromatography (GPC). In some embodiments, the viral vector is a lentivirus, a retrovirus or an adenovirus. In some embodiments, the composition further comprises a T cell stimulatory compound or a tumor antigen conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles, hi some embodiments, the T cell stimulatory compound or the tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles. In some embodiments, where the composition comprises a second population of mesoporous silica particles, the T cell stimulatory compound or tumor antigen is conjugated directly to the second population of mesoporous silica particles or to the lipid envelope on the surface of the second population of mesoporous silica particles, and the T cell stimulatory compound or tumor antigen is IL-2, IL-15, anti-CD2 mAb, anti-CD3 mAb, anti-CD28 mAb, neo-antigen peptide, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or combinations thereof. In some embodiments, the first or second population of mesoporous silica particles further comprises a cytokine conjugated or adsorbed to the first or second population of mesoporous silica particles.In some embodiments, the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21 or transforming growth factor beta (TGF-β) or an agonist, mimetic, variant, functional fragment or combination thereof. In some embodiments, the mesoporous silica particles are in the form of mesoporous silica rods.

[0010] Also contemplated herein is a method of delivering a viral vector to a desired site of action in a subject, comprising administering to the subject a composition comprising a first population of mesoporous silica particles and a viral vector. In some embodiments, the viral vector is conjugated to the first population of mesoporous silica particles. In some embodiments, the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles. In some embodiments, the first population of mesoporous silica particles is surface modified. In some embodiments, the surface modification of the first population of mesoporous silica particles is C 1~20 Alkylamines, C 1~20 Carboxylic acid, C 1~20 Azide and substituted or unsubstituted C 1~20 In some embodiments, the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary, or quaternary amine. In some embodiments, the viral vector is a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, or a lentivirus. In some embodiments, the first population of mesoporous silica particles comprises pores with diameters of 2 to 50 nm. In some embodiments, the first population of mesoporous silica particles comprises pores with diameters of at least about 100 nm. 2 / g. In some embodiments, the mesoporous silica particles are in the form of mesoporous silica rods.

[0011] Also contemplated herein is a method of expanding a chimeric antigen receptor (CAR) T (CAR-T) cell population, comprising contacting the CAR-T cell population with mesoporous silica particles conjugated to a targeting moiety, where the targeting moiety is complementary to the CAR. In some embodiments, the CAR is a protein engineered to target a tumor antigen.In some embodiments, the tumor antigen is TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, 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, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, 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 Selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1 and any combination thereof.In some embodiments, the mesoporous silica particles are in the form of mesoporous silica rods.

[0012] Also contemplated herein are compositions comprising mesoporous silica particles conjugated to polyethyleneimine. In some embodiments, the mesoporous silica particles are in the form of mesoporous silica rods. In some embodiments, the composition further comprises an active agent. In some embodiments, the active agent is absorbed or adsorbed to the mesoporous silica particles.

[0013] Also contemplated herein is a method of delivering an active agent to a desired site of action in a subject, comprising administering to the subject a composition comprising mesoporous silica particles conjugated to polyethyleneimine and further comprising an active agent. In some embodiments, the active agent is absorbed or adsorbed to the mesoporous silica particles. In some embodiments, the composition provides sustained delivery of the active agent to the subject.

[0014] Also contemplated herein is a method of treating a subject having a disease, disorder, or condition, comprising administering to the subject a composition comprising mesoporous silica particles conjugated to polyethyleneimine and further comprising an active agent. In some embodiments, the active agent is absorbed or adsorbed to the mesoporous silica particles. In some embodiments, the disease, disorder, or condition is associated with a tumor antigen.

[0015] Also contemplated herein is a composition comprising mesoporous silica particles conjugated to polyethyleneimine and further comprising an active agent for use in a method of treating a subject having a disease, disorder or condition. In some embodiments, the active agent is absorbed or adsorbed to the mesoporous silica particles. In some embodiments, the disease, disorder or condition is associated with a tumor antigen. Also contemplated herein is a composition comprising cells produced as described herein for use in a method of treating a subject having a disease, disorder or condition. In some embodiments, the disease, disorder or condition is associated with a tumor antigen.

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0017] [Figure 1] A series of surface modifications of mesoporous silica particles are presented. [Diagram 2] Results from staining of the viral envelope protein (VSV-G) on the MSR surface after adsorption of VSV-G pseudotyped lentivirus onto the MSR are shown. A control MSR is shown in the upper panel, and rods incubated with virus are shown in the lower panel. [Diagram 3] FIG. 1 is a schematic diagram of virus adsorption to MSR and transduction of T cells. [Figure 4] Results of GFP expression by T cells incubated with free lentivirus or MSR-bound lentivirus are provided. Dilutions from a starting concentration of 40 μg / ml of virus-coated MSR are as indicated. The "1× lenti" condition corresponds to the amount of virus incubated in the MSR condition. The "2× lenti" condition corresponds to twice the amount used to coat the MSR condition. [Diagram 5]A schematic diagram of the overall strategy for ligand presentation on the MSR surface is provided. Liposomes are incubated with the MSR to form a supported lipid bilayer. Ligands can be attached to the MSR-lipid bilayer using streptavidin-biotin interactions. [Figure 6] Photographs of an MSR coated with POPC liposomes containing 1 mol % PE-carboxyfluorescein. Bright field (left), fluorescent (center) and overlay (right) images are shown. [Figure 7] 1 shows the peptide sequence of the EGFRvIII CAR binding peptide (LEEKKGNYVVTDH (SEQ ID NO: 674)). [Figure 8] Figure 1 shows cytokine production of EGFRvIII CART by peptide immobilization on MSR. The results provide interferon gamma and interleukin-2 production of EGFRvIII CART stimulated by lipid-coated MSR (1% PE-biotin in lipid coating) presenting EGFRvIII-CAR binding peptide compared to control conditions. [Figure 9] Figure 1 shows the proliferation of EGFRvIII CART by peptide immobilization on MSR. A lipid-coated MSR composition of 0.01% PE-biotin was used for peptide immobilization, and the MSR concentration was 30 μg / ml in the well. Cell counts were performed on day 7 of culture under the indicated conditions. [Figure 10A-10B] Figure 10 shows the expansion and final cell composition of EGFRvIII CART by peptide immobilization on MSR. The starting MSR concentration was 50 μg / ml and the dilution of MSR from this starting concentration is as indicated on the axis. Figure 10A: Percentage of CD4 and CD8 T cells at the end of the culture period with the indicated materials. Figure 10B: FACS analysis of CD8+ and CD4+ CAR T cells diluting CFSE during a 3 day culture period using MSR containing various amounts of EGFRvIII CAR binding peptide with or without anti-CD28 on the MSR surface. [Figure 11A-11B]Figure 11 shows the expansion and final cell composition of BCMA CARTs by BCMA protein immobilization on MSR. The starting MSR concentration was 50 μg / ml and the dilution of MSR from this starting concentration is as indicated on the axis. Figure 11A: Percentage of CD4 and CD8 T cells at the end of the culture period with the indicated materials. Figure 11B: FACS analysis of CD8+ and CD4+ CAR T cells diluting CFSE during a 3 day culture period using MSR containing various amounts of EGFRvIII CAR binding peptide with or without anti-CD28 on the MSR surface. [Figure 12] 1 shows a schematic of the co-stimulation and transduction of unstimulated human T cells using MSRs according to some embodiments. Two populations of MSRs are generated - 1) MSRs presenting agonistic CD3 / CD28 antibodies to stimulate T cells, 2) positively charged PEI-MSRs conjugated with lentivirus to facilitate virus delivery to T cells. The two types of MSRs can be mixed in different ratios to tune the amount of stimulus and virus to which T cells are exposed. [Figure 13] Transduction efficiency of T cells exposed to stimulatory (anti-CD3 / CD28 antibody immobilized MSR) and PEI-MSR incubated with virus is shown. T cells were incubated with different amounts of stimulatory rod (Stim1.00 represents 70 μg / ml MSR) and exposed to GFP lentivirus at different multiplicities of infection (MOI), bound to PEI-MSR or in the form of free virus. The highest concentration of MSR in the viral conditions was 22 μg / ml. [Figure 14] Figure 1 shows the transduction efficiency of T cells exposed to stimulatory (anti-CD3 / CD28 antibody immobilized) MSR and PEI-MSR incubated with virus. The plot shows the transduction efficiency as a function of the concentration of stimulatory MSR at different total amounts of virus. The MSR concentration in stimulatory MSR condition 1.0 is 70 μg / ml. The MSR concentration in PEI MSR condition 1 is 22 μg / ml. Transduction was evaluated 3 days after the start of culture. [Figure 15]Results from a comparison of virus delivery strategies for transduction efficiency are provided. In "PEI" and "free" conditions, T cells were stimulated with "high" levels of CD3 / CD28 antibodies bound to MSR (MSR concentration 70 μg / ml) and a given virus was either bound to PEI-MSR or delivered free in the medium, respectively (virus concentration 1.0 contains 22 μg / ml MSR, MOI approx. 6.7). In "PEI+CD3 / CD28", virus and CD3 / CD28 agonist antibodies were bound to PEI-MSR (concentration 1.0 is 22 μg / ml MSR). Transduction was assessed 3 days after initiation of culture. [Figure 16] Results from a comparison of various delivery strategies for transduction in PBMC populations are provided. The conditions in Figure 15 were added to PBMCs. The percentage of transduced cells in each cell type was quantified. Transduction was assessed 3 days after initiation of culture. [Figure 17] The different transduced fractions of PBMCs due to various viral delivery strategies are provided. The top panel provides the total cell composition present in the PBMC population under the conditions of Figure 15. The bottom panel provides the composition of the transduced cell fraction present after viral delivery using the conditions of Figure 15. Transduction was assessed 3 days after initiation of culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical referent of the article. By way of example, "an element" means one element or more than one element.

[0020] The term "about," when referring to a measurable value, such as an amount, temporal duration, and the like, means that variations of ±20%, or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value are encompassed, as such variations are appropriate to the practice of the methods of the present disclosure.

[0021] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains of a CAR polypeptide construct are on the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the domains of a CAR polypeptide construct are not contiguous with each other, e.g., on different polypeptide chains, e.g., as provided in a RCAR as described herein.

[0022] In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule is selected from 41BB (i.e., CD137), CD27, ICOS, and / or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and cell membrane localization of the CAR.

[0023] A CAR that comprises an antigen binding domain (e.g., an scFv, single domain antibody, or TCR (e.g., a TCR alpha binding domain or a TCR beta binding domain)) that targets a particular tumor marker X (where X can be a tumor marker as described herein) is also referred to as an XCAR. For example, a CAR that comprises an antigen binding domain that targets BCMA is referred to as a BCMA CAR. CARs can be expressed in any cell, such as an immune effector cell (e.g., a T cell or an NK cell) as described herein.

[0024] The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell to regulate cellular activity through a defined signaling pathway, either by generating second messengers or by functioning as an effector in response to such messengers.

[0025] The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multichain or single chain or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules.

[0026] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies (VL or VH) such as sdAb, camelid VHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0027] The portion of the CAR of the invention that comprises an antibody or antibody fragment thereof can exist in various forms, with the antigen-binding domain expressed as part of a contiguous polypeptide chain, including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR composition of the invention comprises an antibody fragment. In further embodiments, the CAR comprises an antibody fragment that comprises an scFv. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (the "Chothia" numbering scheme), or a combination thereof.

[0028] As used herein, the term "binding domain" or "antibody molecule" refers to a protein, such as an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In some embodiments, an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0029] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformation, and which usually determines the class to which the antibody belongs.

[0030] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformation. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0031] The term "recombinant antibody" refers to an antibody made using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody made by synthesis of a DNA molecule encoding an antibody, the DNA molecule expressing the antibody protein, or an amino acid sequence specifying that antibody, where the DNA or amino acid sequence was obtained using well-known recombinant DNA or amino acid sequence techniques available in the art.

[0032] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may include either or both antibody production or activation of specific immunologically competent cells. Those skilled in the art will appreciate that any macromolecule can be an antigen, including virtually any protein or peptide. Furthermore, an antigen can be derived from recombinant or genomic DNA. Those skilled in the art will therefore appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes and that the nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits the desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthesized or obtained from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or bodily fluids along with other biological components.

[0033] The term "anti-cancer effect" refers to a biological effect that may be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" may also be manifested by the ability of peptides, polynucleotides, cells, and antibodies in preventing the development of cancer in the first place. The term "anti-tumor effect" refers to a biological effect that may be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0034] The term "autologous" refers to any material derived from the same individual that is later reintroduced into that individual.

[0035] The term "allogeneic" refers to any material that is derived from a different animal of the same species as the individual into which the material is introduced.Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical.In some embodiments, allogeneic materials from individuals of the same species can be genetically different enough to interact antigenically.

[0036] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0037] The term "cancer" refers to a disease characterized by uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Various examples of cancer are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous and malignant cancers and tumors.

[0038] "Derived from" as 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 method 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. This does not imply or include a limitation on a particular method of making the intracellular signaling domain, such as one must start with the CD3 zeta sequence and delete unnecessary sequences or make mutations to provide the intracellular signaling domain.

[0039] The phrase "diseases associated with expression of tumor antigens described herein" includes diseases associated with expression of tumor antigens described herein or conditions associated with cells expressing tumor antigens described herein or non-cancer related indications associated with cells expressing tumor antigens described herein, including, but not limited to, proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndromes, or preleukemias. In some embodiments, the cancer associated with expression of tumor antigens described herein is a hematological cancer. In some embodiments, the cancer associated with expression of tumor antigens described herein is a solid cancer. Further diseases associated with expression of tumor antigens described herein include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders associated with expression of tumor antigens described herein. Non-cancer related indications associated with expression of tumor antigens described herein include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergy and asthma), and transplantation. In some embodiments, the tumor antigen expressing cells express, or have at any time expressed, mRNA encoding the tumor antigen. In some embodiments, the tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal or reduced levels. In some embodiments, the tumor antigen-expressing cells produce detectable levels of tumor antigen protein temporarily, but subsequently produce substantially no detectable tumor antigen protein.

[0040] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex or signaling through the signaling domain of an appropriate NK receptor or CAR. Stimulation can mediate a change in expression of a molecule.

[0041] The term "stimulatory molecule" refers to a molecule that is expressed by an immune cell (e.g., T cell, NK cell, B cell) and provides a cytoplasmic signaling sequence that stimulatorily regulates immune cell activation for at least some aspects of the immune cell signaling pathway. In some embodiments, the signal is a primary signal, e.g., initiated by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which leads to mediating a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that function in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R16), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In certain CARs of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, such as the primary signaling sequence of CD3-zeta. In certain CARs of the invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 18 or equivalent residues from a non-human species, such as mouse, rodent, monkey, ape, etc. In a specific CAR of the invention, the primary signaling sequence of CD3-zeta is the sequence as provided in SEQ ID NO: 20 or equivalent residues from a non-human species, such as mouse, rodent, monkey, ape, etc.

[0042] The term "antigen-presenting cell" or "APC" refers to a cell of the immune system, such as an accessory cell (e.g., a B cell, a dendritic cell, etc.), that presents foreign antigens complexed with major histocompatibility complexes (MHC) on its surface. T cells can recognize such complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0043] "Intracellular signaling domain," as the term is used herein, refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of a CAR-containing cell, e.g., a CART cell. Examples of immune effector function (e.g., in a CART cell) include cytolytic activity, including secretion of cytokines, and helper activity.

[0044] In some embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain may include a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of 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 costimulatory molecule.

[0045] A primary intracellular signaling domain can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences that contain ITAMs include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 beta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0046] The term "zeta" or alternatively "zeta chain", "CD3 zeta" or "TCR zeta" refers to CD247. Swiss-Prot Accession No. P20963 provides an exemplary human CD3 zeta amino acid sequence. "Zeta stimulatory domain" or alternatively "CD3 zeta stimulatory domain" or "TCR zeta stimulatory domain" refers to the stimulatory domain of CD3 zeta or variants thereof (e.g., molecules having mutations, such as point mutations, fragments, insertions or deletions). In some embodiments, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Accession No. BAG36664.1 or variants thereof (e.g., molecules having mutations, such as point mutations, fragments, insertions or deletions). In some embodiments, "zeta stimulatory domain" or "CD3 zeta stimulatory domain" refers to the sequence provided as SEQ ID NO: 9 or 10 or variants thereof (e.g., molecules having mutations, such as point mutations, fragments, insertions or deletions). Alternatively or additionally, the term "zeta" or alternatively "zeta chain", "CD3-zeta" (or "CD3 zeta, CD3 zeta or CD3z) or "TCR-zeta" is defined as the protein provided as GenBan 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 sufficient to functionally transmit an initial signal required for T cell activation, or a functional derivative thereof. In some embodiments, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Accession No. BAG36664.1, or the equivalent residues from a non-human species that is a functional ortholog thereof, e.g., mouse, rodent, monkey, ape, etc. In some embodiments, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO: 18. In some embodiments, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:20.

[0047] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that contributes to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, These include ligands that specifically bind to 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 CD83.

[0048] The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. The costimulatory molecule can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins) and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3 and a ligand that specifically binds to CD83.

[0049] The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived, or the entire naturally occurring intracellular signaling domain, or a functional fragment or derivative thereof.

[0050] "Immune effector cells," as the term is used herein, refer to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0051] "Immune effector function or immune effector response," as the term is used herein, refers to a function or response, e.g., of an immune effector cell, that enhances or promotes immune attack of a target cell. For example, immune effector function or response refers to a property of a T cell or NK cell that promotes the killing or growth or proliferation inhibition of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0052] The terms "encoding" or "encoding" refer to the inherent property of a specific nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Thus, a gene, cDNA, or RNA codes for a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a transcription template for a gene or cDNA, can be referred to as encoding the protein or other product of that gene's cDNA.

[0053] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that the nucleotide sequence encoding the protein may, in any version, contain one or more introns.

[0054] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition as described herein that is effective to achieve a particular biological result.

[0055] The term "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue or system.

[0056] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0057] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0058] The term "transfer vector" refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid inside a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes self-replicating plasmids or viruses. The term should be construed to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into a cell, such as, for example, polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0059] The term "expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses, "viral vectors") that incorporate the recombinant polynucleotide.

[0060] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and lentiviruses can deliver large amounts of genetic information to the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0061] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, in particular, the self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, LENTIVECTOR® gene delivery technology from Oxford BioMedica, LENTIMAX™ vector system from Lentigen, and the like. Non-clinical types of lentiviral vectors are also available and would be known to those skilled in the art.

[0062] The term "homology" or "identity" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., when a position in each of the two DNA molecules is occupied by adenine, they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in two sequences are homologous (e.g., 5 positions in a polymer 10 subunits long), the two sequences are 50% homologous. If 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0063] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies and antibody fragments thereof are those in which residues from a complementarity determining region (CDR) of the recipient in a human immunoglobulin (recipient antibody or antibody fragment) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, a humanized antibody or antibody fragment thereof will contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin, and all or most of the FR regions being those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0064] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, whose entire molecule is of human origin or consists of an amino acid sequence identical to a human form of an antibody or immunoglobulin.

[0065] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0066] The term "operably linked" or "transcriptional control" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. DNA sequences that are operably linked can be contiguous with each other and in the same reading frame, for example, when necessary to join two protein coding regions.

[0067] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0068] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, of which there are numerous varieties, generally referred to in the art as proteins. "Polypeptides" specifically include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, or combinations thereof.

[0069] The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, necessary to initiate the specific transcription of a polynucleotide sequence.

[0070] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that provides tissue-specific expression of the gene product.

[0071] The term "constitutive promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the production of the gene product in a cell under most or all physiological conditions of the cell.

[0072] The term "inducible promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0073] The term "tissue-specific promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the production of a gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0074] The terms "cancer-associated antigen" or "tumor antigen" refer synonymously to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of cancer cells, either entirely or alternatively as a fragment (e.g., MHC / peptide), and that is useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, the tumor antigen is a marker that is expressed by both normal and cancer cells, such as a lineage marker, e.g., CD19 on B cells. In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed on cancer cells compared to normal cells (e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression or more compared to normal cells). In some embodiments, the tumor antigen is a cell surface molecule that is inappropriately synthesized on cancer cells, such as a molecule that contains a deletion, addition, or mutation compared to the molecule expressed on normal cells. In some embodiments, the tumor antigen will be expressed only on the cell surface of cancer cells, either entirely or alternatively as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. In some embodiments, the CAR of the present invention comprises a CAR that comprises an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Normally, peptides derived from endogenous proteins fit into a pocket of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy.TCR-like antibodies that target peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening a library, such as a human scFv phage display library.

[0075] The terms "tumor-supporting antigen" or "cancer-supporting antigen" refer interchangeably to molecules (typically proteins, carbohydrates, or lipids) expressed on the surface of cells that are not themselves cancerous, but that support cancer cells, for example by promoting proliferation or survival, e.g., resistance to immune cells. Exemplary cells of this type include stromal cells and myeloid-derived suppressor cells (MDSCs). A tumor-supporting antigen itself may not play a role in supporting tumor cells, so long as the antigen is present on a cell that supports cancer cells.

[0076] As used herein, "in vitro transcribed RNA" refers to in vitro synthesized RNA, preferably mRNA. Generally, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector contains the template used to generate the in vitro transcribed RNA.

[0077] As used herein, "poly(A)" is a series of adenosines added to an mRNA by polyadenylation. In a preferred embodiment of a construct for transient expression, the polyA is 50-5000 (SEQ ID NO: 34), preferably more than 64, more preferably more than 100, and most preferably more than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA function such as localization, stability, or translation efficiency.

[0078] As used herein with respect to expression, e.g., expression of a CAR molecule, "transient" refers to expression of an unintegrated transgene for a period of hours, days, or weeks, which is shorter than the period of expression of the gene when integrated into the genome or contained in a stable plasmid replicon in a host cell.

[0079] As used herein, the terms "treat", "treatment" and "treating" refer to a reduction or amelioration of the progression, severity and / or duration of a proliferative disorder or an amelioration of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder resulting from administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the invention). In specific embodiments, the terms "treat", "treatment" and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, that is not necessarily discernible by the patient. In other embodiments, the terms "treat", "treatment" and "treating" refer to either or both of a physical, e.g., by stabilization of a discernible symptom, inhibition of the progression of a proliferative disorder, physiological, e.g., by stabilization of a physical parameter. In other embodiments, the terms "treat", "treatment" and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0080] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in the transmission of a signal from one part of a cell to another part of a cell. The phrase "cell surface receptor" includes molecules and molecular complexes that have the ability to receive a signal and transmit the signal across the membrane of a cell.

[0081] The term "subject" is intended to include living organisms in which an immune response can be generated (eg, mammals, humans).

[0082] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a substantially purified cell population refers to a homogenous cell population. In other instances, the term simply refers to a cell that has been separated from the cells with which it is naturally associated in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0083] The term "therapeutic" as used herein means treatment. A therapeutic effect is achieved by the reduction, suppression, amelioration, or eradication of a disease state.

[0084] The term "prevention" as used herein means the prevention of or prophylactic treatment against a disease or disease state.

[0085] The terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0086] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (e.g., a tumor antigen) protein present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0087] "Refractory" as used herein refers to a disease, e.g., cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. A refractory cancer may also be referred to as a resistant cancer.

[0088] "Relapsed," as used herein, refers to the return of signs and symptoms of a disease (e.g., cancer) or a disease such as cancer, after a period of improvement, e.g., after prior treatment with a therapy, e.g., a cancer therapy.

[0089] The term "system," as used herein, e.g., in connection with gene editing, refers to a group of molecules, e.g., one or more molecules, that act together to produce a desired function.

[0090] "Gene editing system," as the term is used herein, refers to a system, e.g., one or more molecules, that directs and performs modifications, e.g., deletions, of one or more nucleic acids at or near a site of genetic DNA targeted by the system. Gene editing systems are known in the art and are described more fully below.

[0091] A "dominant negative" gene product or protein is one that interferes with the function of a gene product or protein. The affected gene product may be the same as or different from the dominant negative protein. Dominant negative gene products may be in a variety of forms, including full-length proteins or fragments thereof with truncations, point mutations, or fusions of full-length wild-type or mutant proteins or fragments thereof with other proteins. The level of inhibition observed may be very low. For example, this may require a large excess of the dominant negative protein compared to the functional protein or proteins involved in the process to see an effect. Confirmation of the effect under normal biological assay conditions may be difficult.

[0092] The term "ratio" refers to the proportion of a particular molecule relative to the total number of molecules in a population. In an exemplary embodiment, T cells having a particular phenotype (e.g., T SCM A percentage of T cells having a particular phenotype (e.g., CD45RA+CD62L+ cells) refers to the ratio of the number of T cells having that phenotype to the total number of T cells in a population. In an exemplary embodiment, a percentage of T cells having a particular phenotype (e.g., CD45RA+CD62L+ cells) refers to the ratio of the number of T cells having that phenotype to the total number of T cells in a population. It will be understood that such ratios, where indicated, may be measured for a subset of cells. For example, CD4+ T SCM The proportion of cells can be measured relative to the total number of CD4+ T cells.

[0093] The term "population of immune effector cells," as used herein, refers to a composition comprising at least two, e.g., two or more, e.g., more than one, immune effector cells, and no level of purity is indicative of the presence or absence of other cell types. In an exemplary embodiment, the population is substantially free of other cell types. In another exemplary embodiment, the population comprises at least two cells of a particular cell type or having a particular function or property.

[0094] "T SCMThe terms "T cell" and "naive T cell" are used interchangeably and refer to a less differentiated T cell state characterized by surface expression of CD45RA and CD62L (e.g., CD45RA positive and CD62L positive (sometimes written as CD45RA+CD62L+)). In general, T cell differentiation progresses from the most "naive" to the most "exhausted", with T SCM -like (e.g., CD45RA+CD62L+ cells) > T CM (e.g., CD45RA-CD62L+ cells)>T EM (e.g., CD45RA-CD62L- cells)>T EFF Naive T cells may be characterized, for example, by increased self-renewal, anti-tumor efficacy, proliferation and / or survival compared to more exhausted T cell phenotypes. In one exemplary embodiment, naive T cells refer to CD45RA+CD62L+ T cells. In another exemplary embodiment, naive T cells refer to T SCM CD45RA+CD62L+CCR7+CD27+CD95+ T cells.

[0095] "T SCM The term "T cells" refers to T cells with a stem cell memory phenotype, characterized by expression of CD45RA, CD62L, CCR7, CD27, and CD95 on their cell surface (e.g., CD45RA positive, CD62L positive, CCR7 positive, CD27 positive, and CD95 positive (sometimes referred to as CD45RA+CD62L+CCR7+CD27+CD95+)). SCM The cells are an example of naive T cells. The T cells can be CD4+ and / or CD8+ T cells.

[0096] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched (or straight-chain or linear) hydrocarbon moiety containing 1 to 20 carbon atoms. Preferably, an alkyl contains 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms. Representative examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, vert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, and n-heptyl. For example, "C 1~6 The term "alkyl" refers to a hydrocarbon having 1 to 6 carbon atoms, 1~7 The term "alkyl" refers to a hydrocarbon having 1 to 7 carbon atoms.

[0097] As used herein, the term "haloalkyl" refers to an alkyl, as defined herein, substituted by one or more halo groups, as defined herein. Preferably, the haloalkyl can be a monohaloalkyl, dihaloalkyl, or polyhaloalkyl, including perhaloalkyl. A monohaloalkyl can have one iodo, bromo, chloro, or fluoro in the alkyl group. Dihaloalkyl and polyhaloalkyl groups can have two or more of the same halo atoms or a combination of different halo groups in the alkyl. Preferably, a polyhaloalkyl contains up to 12, or 10, or 8, or 6, or 4, or 3, or 2 halo groups. Representative examples of haloalkyl are fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. A perhaloalkyl refers to an alkyl in which all hydrogen atoms are replaced by halo atoms. For example, "Halo-C 1~6 The term "alkyl" refers to a hydrocarbon having 1 to 6 carbon atoms and substituted with one or more halo groups, and is defined as "halo-C 1~7The term "alkyl" refers to a hydrocarbon having 1 to 7 carbon atoms and substituted with one or more halo groups.

[0098] As used herein, "salt" includes pharma- ceutically acceptable acid addition salts which can be formed with inorganic and organic acids, such as acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, and the like. Salts include, but are not limited to, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, naphthate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate and trifluoroacetate.

[0099] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, etc. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0100] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table, in certain embodiments salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper, with particularly suitable salts including the ammonium, potassium, sodium, calcium, and magnesium salts.

[0101] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Particular organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0102] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have all the possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

[0103] Headings, subheadings or numbered or lettered elements, e.g., (a), (b), (i), etc., are provided merely for ease of reading. The use of headings or numbered or lettered elements herein does not require that the steps or elements be performed in alphabetical order or that the steps or elements are necessarily separate from one another.

[0104] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0105] Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims.

[0106] Surface-modified mesoporous silica particles In some embodiments, the present invention provides mesoporous silica particles. Mesoporous silica particles include porous bodies having uniform pores, for example, hexagonal close-packed cylindrical pores. Mesoporous silica particles can be synthesized by using rod-shaped micelles of surfactants as templates, which are formed in water by dissolving and hydrolyzing silica sources, such as alkoxysilanes, sodium silicate solutions, kanemite, and silica microparticles, in water or alcohol in the presence of an acid or base catalyst. See, for example, U.S. Patent Application Publication No. 2015-0072009 and Hoffmann et al., Angewandte Chemie International Edition, 45, 3216-3251, 2006. Many types of surfactants, such as cationic, anionic, and nonionic surfactants, have been investigated as surfactants, and it is generally known that alkyltrimethylammonium salts of cationic surfactants provide mesoporous silica with the largest specific surface area and pore volume. See U.S. Patent Application Publication No. 2013 / 0052117 and Katiyar et al. (Journal of Chromatography 1122(1-2):13-20).

[0107] Mesoporous silica particles can be provided in various forms, such as microspheres, irregularly shaped particles, angular rods, rounded nanorods. Mesoporous silica particles can have various predefined shapes, including, for example, spherical, elliptical, rod-like, or curved cylinders. In certain embodiments, the compositions and methods described herein use mesoporous silica rods (MSR). Methods for assembling mesoporous silica to produce microrods are known in the art. See Wang et al, Journal of Nanoparticle Research, 15:1501, 2013. In some embodiments, mesoporous silica particles are synthesized by reacting tetraethyl orthosilicate with a template made of micellar rods. The result is mesoporous silica spheres or rods filled with ordered pore arrays. The template can then be removed by washing with a solvent adjusted to the appropriate pH. In this example, after removing the surfactant template, the mesoporous silica particles were characterized by uniform and regularly interconnected mesoporosity, e.g., 600 mm. 2 / g~approx. 1200m 2 / g, especially at about 800m 2 / g~about 1000m 2 / g, especially at about 850m 2 / g ~ approx. 950m 2 / g specific surface area. In another embodiment, mesoporous silica particles can be synthesized using sol-gel or spray drying methods. Tetraethyl orthosilicate is used with additional polymer monomers (as templates). In yet another embodiment, one or more tetraalkoxysilanes and one or more (3-cyanopropyl)trialkoxysilanes can be co-condensed to provide mesoporous silicate particles as rods. See US Patent Publication No. 2013-0145488, US Patent Publication No. 2012-0264599 and US Patent Publication No. 2012-0256336, the contents of which are incorporated by reference in their entirety.

[0108] Mesoporous silica particles (MSP) (e.g., MSR) may contain pores that may be ordered or randomly distributed, such as pores 2-100 nm in diameter or 2-50 nm in diameter, e.g., pores 2-5 nm, 10-20 nm, 10-30 nm, 10-40 nm, 20-30 nm, 30-50 nm, 30-40 nm, 40-50 nm. In some embodiments, the microrods contain pores with diameters of approximately 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm or more. The size of the pores may vary depending on the type of application.

[0109] In some embodiments, the length of the MSR is in the micrometer range, ranging from about 5 μm to about 500 μm. In one example, the MSR comprises a length of 5 to 50 μm, such as 10 to 20 μm, 10 to 30 μm, 10 to 40 μm, 20 to 30 μm, 30 to 50 μm, 30 to 40 μm, 40 to 50 μm. In some embodiments, the MSR comprises a length of 50 μm to 250 μm, such as about 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 150 μm, 180 μm, 200 μm, 225 μm or more. In some embodiments, MSRs with higher aspect ratios are used, for example with rods that comprise a length of 50 μm to 200 μm, particularly a length of 80 μm to 120 μm, particularly a length of about 100 μm or more.

[0110] In yet another embodiment, the MSP (e.g., MSR) provides a large surface area for attachment and / or binding to target cells, e.g., T cells. Methods for obtaining high surface area mesoporous silicates are known in the art. See, for example, U.S. Pat. No. 8,883,308 and U.S. Publication No. 2011-0253643, the entire contents of which are incorporated herein by reference. In some embodiments, the high surface area is due to the fibrous morphology of the nanoparticles, which allows for obtaining a high concentration of highly dispersed and easily accessible moieties on the surface. In certain embodiments, the high surface area MSP (e.g., MSR) has a surface area of ​​at least about 100 m 2 / g, at least 150m 2 / g or at least 300m 2 In another embodiment, the high surface area MSP (e.g., MSR) has a surface area of ​​about 100 m 2 / g~about 1000m 2 / g (including all values ​​or subranges therebetween), e.g. 50m 2 / g, 100m 2 / g, 200m 2 / g, 300m 2 / g, 400m 2 / g, 600m 2 / g, 800m 2 / g, 100-500m 2 / g, 100-300m 2 / g, 500-800m 2 / g or 500~1000m 2 / g surface area.

[0111] In some embodiments, the mesoporous silica particles may include a surface modification. As used herein, "surface modification" refers to attaching or appending a functional group to the surface of the MSP (e.g., MSR). In some embodiments, the functional group is adsorbed or covalently attached to the surface covering the pores and / or nanochannels or to the surface of the MSP (e.g., MSR). As used herein, "functional group" defines a chemical moiety linked to the MSR. In some embodiments, the functional group is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, disulfide, polyethyleneimine, hydrophobic moiety, or salt thereof. In some embodiments, the functional group (i.e. -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, disulfide, polyethyleneimine, hydrophobic moiety, or salt thereof) may be separated from the silica surface by a linker. In some embodiments, the functional group is a C1-C 20The functional group is covalently attached to the MSP or MSR surface via an alkyl linker. In other embodiments, the functional group is covalently attached to the MSP or MSR surface via a polyethylene glycol linker. In certain embodiments, the polyethylene glycol linker has the formula (O(CH2-CH2-) 1~25 In certain embodiments, the surface modification has a C1 to C 20 It is alkylperhaloalkyl or C1-C20 alkylperfluoroalkyl.

[0112] The general structure of the surface modification is: [ka] (Wherein, L is a linker, X is a functional group. It could be.

[0113] In some embodiments, L is C1-C 20 or a polyethylene glycol group, and X can be -OH (hydroxyl), a primary, secondary, tertiary or quaternary amine, a carboxylic acid, a phosphonate, a halide, an azide, an alkyne, an epoxide, a sulfhydryl, a disulfide, a polyethyleneimine or a hydrophobic moiety or a salt thereof.

[0114] As used herein, surface modification with phosphonates (also known as phosphonate-modified nanoparticles) refers to a surface modification with at least one phosphonic acid (-P(O)(OH)2) group or phosphinic acid (-P(O)(OH)R, where R is a C1-C 20 (wherein R is an alkyl group). Phosphonic or phosphinic acids can be charged or uncharged depending on the pH. At physiological pH, phosphonic and phosphinic acids are negatively charged or anionic. Phosphonate modifications can be prepared, for example, by treating the surface of the silica body with a phosphonate-containing trialkylsiloxane compound or a phosphonate-containing trihydroxylsilyl compound, such as (trihydroxylsilyl)propylmethylphosphonate.

[0115] In some embodiments, the mesoporous silica particles (e.g., MSR) are surface-modified with primary, secondary, tertiary, or quaternary amines. The secondary, tertiary, and quaternary amines are selected from the group consisting of C1-C 20 and may be charged. In some embodiments, the amine group may be in salt form. In some embodiments, the primary, secondary, tertiary, or quaternary amine may be separated from the MSP surface by a linker. In certain embodiments, the mesoporous silica particles are modified with polyethyleneimine. In certain embodiments, the polyethyleneimine is branched or unbranched. In alternative embodiments, the polyethyleneimine group has an average molecular weight in the range of about 1000 to 100,000 Daltons (Da) as measured by gel permeation chromatography (GPC). In some embodiments, the polyethyleneimine group has an average molecular weight of about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, about 10,000 Da, or about 20,000 Da, as measured by gel permeation chromatography (GPC).

[0116] The structures of various exemplary surface-modified mesoporous silica particles are shown in FIG.

[0117] The MSPs (e.g., MSRs) described herein are prepared by methods known to those of skill in the art, as described herein. In general, MSPs having surface modifications can be prepared by the following methods.

[0118] In general, any reaction capable of reacting with the silyl hydroxide surface of the MSP (e.g., MSR) can be used to covalently modify the surface. For example, the surface of the MSP (e.g., MSR) can be treated with a trialkoxysilyl or trihydroxysilyl compound. In some embodiments, the mesoporous silica particles are suspended in a suitable reaction solvent. In some embodiments, the reaction solvent can be an aqueous solvent or a buffer solution with a pH of 0-14. Further mixtures of the aqueous solution with one or more organic solvents can be used, including, but not limited to, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, toluene, triethylamine, dimethylformamide, dimethylacetamide, dimethylsulfoxide, methanol, ethanol, methylene chloride, or dichloromethane. In some embodiments, the suspended mesoporous silica particles are reacted with a trialkoxysilyl or trihydroxysilyl reagent having a desired functional group as described herein. Amine modifications can be prepared, for example, by treating MSP with an amine-containing trialkoxysilane compound, such as aminopropyltriethoxysilane, 3-(2-aminoethylamino)propyl-trimethoxysilane, or 3-trimethoxysilylpropylethylenediamine. In certain embodiments, the trialkoxysilyl is a trimethoxysilyl or triethoxysilyl group. In alternative embodiments, the trialkoxysilyl reagent is a trialkoxyalkylamine. In some embodiments, the trialkoxyalkylamine comprises a primary, secondary, tertiary, or quaternary amine.

[0119] In certain embodiments, the trialkoxysilyl reagent comprises a polyethyleneimine group. In certain embodiments, the polyethyleneimine is branched or unbranched. In alternative embodiments, the polyethyleneimine group has an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, or about 10,000 Da, as measured by gel permeation chromatography (GPC). In some embodiments, the trialkoxysilyl reagent comprises a C 1~20 In certain embodiments, the trialkoxysilyl reagent comprises an alkyl azide group. 1~20 In another embodiment, the trialkoxysilyl reagent comprises a C 1~20 Contains an alkyl group.

[0120] Sulfhydryl modifications on MSPs (eg, MSRs) can be prepared, for example, by treating the MSPs with a sulfhydryl-containing trialkoxysilane compound, such as 3-mercaptopropyltriethoxysilane.

[0121] Disulfide modifications on MSPs (eg, MSRs) can be prepared, for example, by treating the surface of the nanoparticles with a disulfide-containing trialkoxysilane compound or by treating a sulfhydryl-modified surface with 2,2'-dithiodipyridine or other disulfides.

[0122] MSP (e.g., MSR) surface modifications containing carboxylic acid groups can be prepared, for example, by treating the surface with a carboxylic acid-containing trialkoxysilane compound or by treating the MSP with a trialkoxysilane compound having a functional group that can be chemically converted to a carboxylic acid. For example, MSP can be treated with 3-cyanopropyltriethoxysilane and then hydrolyzed with sulfuric acid.

[0123] Epoxide-containing MSP (eg, MSR) surface modifications have at least one epoxide, which can be prepared, for example, by treating the MSP with an epoxide-containing trialkoxysilane compound, such as glycidoxypropyltriethoxysilane.

[0124] The surface modification having a hydrophobic moiety has at least one moiety intended to reduce solubility in water or increase solubility in organic solvents. Examples of hydrophobic moieties include long chain alkyl groups (e.g., C8 to C 20 alkyl group), fatty acid esters (e.g., C1-C 22 Alkyl acid esters) and C6-C 10 It includes aromatic rings having carbon atoms.

[0125] In some embodiments, the reaction of the MSP (e.g., MSR) with the trialkoxysilyl reagent is carried out at ambient or room temperature. In other embodiments, the reaction is carried out at elevated temperatures. In further embodiments, the temperature of the reaction is about 40°C to about 120°C, about 50°C to about 100°C, about 60°C to about 80°C, about 70°C to about 80°C, or about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C.

[0126] Viral Vectors In some embodiments, the compositions described herein may comprise the mesoporous silica particles and a viral vector described herein.

[0127] The viral vector can be any viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. By way of example, the viral vector can be an adenovirus, a lentivirus, a retrovirus, an adeno-associated virus, or a herpes virus. In some embodiments, the viral vector is a lentivirus vector or an adenovirus vector.

[0128] Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, since they allow long-term stable integration and transmission of transgenes to daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of being less immunogenic. Retroviral vectors can also be, for example, gamma retroviral vectors. Gamma retroviral vectors can include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, such as a gene encoding a CAR. Gamma retroviral vectors can lack viral structural genes, such as gag, pol, and env. Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen focus forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV) and vectors derived therefrom. Other gammaretroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun;3(6):677-713.

[0129] In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the present invention is an adenoviral vector (A5 / 35). In another embodiment, expression of the nucleic acid encoding the CAR can be achieved using transposons such as sleeping beauty, CRISPR, CAS9 and zinc finger nucleases. See June et al. 2009 Nature Reviews Immunology 9.10:704-716, incorporated herein by reference.

[0130] In some embodiments, the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. In some embodiments, the nucleotide sequence expresses a chimeric antigen receptor (CAR), an engineered TCR, a cytokine, a chemokine, an shRNA that blocks an inhibitory molecule, or an mRNA that induces expression of a protein. In some embodiments, the protein is a CAR that includes an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a signaling domain. In some embodiments, the signaling domain is a CD3 zeta signaling domain.

[0131] In some embodiments, the nucleotide sequence in the viral vector expresses a peptide engineered to target a tumor antigen. In some embodiments, the peptide is 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, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, 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, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, 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, mutThe peptide targets a tumor antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and any combination thereof. In some embodiments, the peptide is a chimeric antigen receptor (CAR) or an engineered TCR. Such peptides are described in more detail below in the section entitled "General Description of Chimeric Antigen Receptor Technology."

[0132] Composition of mesoporous silica particles and viral vectors Compositions comprising a first population of mesoporous silica particles and a viral vector are also described herein. In some embodiments, the MSPs (e.g., MSRs) further comprise a surface covering the pores and / or nanochannels or a plurality of functional groups adsorbed or covalently attached to the surface. In some embodiments, the functional groups are -OH (hydroxyl), amines, carboxylic acids, phosphonates, halides, azides, alkynes, epoxides, sulfhydryls, disulfides, polyethyleneimines, hydrophobic moieties, or salts thereof. In some embodiments, the functional groups (i.e. -OH (hydroxyl), amines, carboxylic acids, phosphonates, halides, azides, alkynes, epoxides, sulfhydryls, disulfides, polyethyleneimines, hydrophobic moieties, or salts thereof) can be directly attached to the surface of the MSPs. In some embodiments, the functional groups are C1-C 20 The functional group is covalently attached to the MSP (e.g., MSR) surface via an alkyl linker. In other embodiments, the functional group is covalently attached to the MSP surface via a polyethylene glycol linker. In certain embodiments, the polyethylene glycol linker has the formula (O(CH2-CH2-) 1~25 In certain embodiments, the surface modification has a C1 to C 20 It is alkylperhaloalkyl or C1-C20 alkylperfluoroalkyl.

[0133] In some embodiments, the MSP (e.g., MSR) is surface-modified with a primary, secondary, tertiary, or quaternary amine. In certain embodiments, the mesoporous silica rod is modified with polyethyleneimine. In certain embodiments, the polyethyleneimine is branched or unbranched. In alternative embodiments, the polyethyleneimine group has an average molecular weight in the range of about 1000-20,000 Daltons (Da) as measured by gel permeation chromatography (GPC). In some embodiments, the polyethyleneimine group has an average molecular weight of about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, or about 10,000 Da as measured by gel permeation chromatography (GPC).

[0134] In some embodiments, the viral vector is conjugated to the mesoporous silica particles. As described herein, "conjugated to" means attached or attached by any means described herein. In some embodiments, the viral vector is electrostatically or covalently conjugated to the mesoporous silica particles. In some embodiments, the electrostatic binding between the mesoporous silica particles and the viral vector is due to the opposite surface charge of the viral vector and the mesoporous silica particles. For example, without being bound by theory, mesoporous silica particles surface-modified with positively charged polyethyleneimine or primary, secondary, tertiary or quaternary ammonium groups can be conjugated to a negatively surface-charged viral vector. Thus, in some embodiments, the viral vector is negatively charged and the mesoporous silica particles are positively charged. In some embodiments, the covalent binding between the mesoporous silica particles and the viral vector is achieved by methods known to those skilled in the art, with or without a linker. For example, but not limited to, the linker can be a polyethylene glycol, an alkyl group, a polymer, a polyamide bond, etc.

[0135] In some aspects, what is provided herein includes pharmaceutical compositions comprising mesoporous silica particles as described herein, formulated for use in producing a population of immune effector cells, such as T lymphocyte cells. In some embodiments, the T lymphocyte cells are transduced with CAR. In some embodiments, the MSPs are conjugated to a viral vector as described herein. In some embodiments, the MSPs for use in producing a population of immune effector cells (e.g., T lymphocyte cells) can be surface-modified as described herein.

[0136] In some embodiments, the composition is suitable for use as an injectable composition comprising mesoporous silica particles and a viral vector, where the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. In some embodiments, the viral vector is conjugated to the mesoporous silica particles described herein.

[0137] In the mesoporous silica particle compositions described herein, the MSP (e.g., MSR) may be present in a concentration of 0.01-1000 μg / ml. In alternative embodiments, the concentration of MSP or MSR in the compositions described herein may be 0.1-500 μg / ml, 0.5-100 μg / ml, 1-90 μg / ml, 1-80 μg / ml, 1-70 μg / ml, 1-60 μg / ml, 1-50 μg / ml, or 1-40 μg / ml.

[0138] In certain embodiments, MSP (e.g., MSR) may be present at a concentration of about 1 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 110 μg / ml, 120 μg / ml, 130 μg / ml, 140 μg / ml or 150 μg / ml.

[0139] In general, the compositions described herein will be administered in any of the conventional and accepted manners known in the art, either alone or in combination with one or more therapeutic agents, in therapeutically effective amounts as described above.

[0140] The compositions for injection may be aqueous isotonic suspensions. The compositions may be sterilized and / or contain adjuvants such as preservatives, stabilizing agents, wetting agents or emulsifying agents, dissolution promoters, salts for adjusting the osmotic pressure and / or buffers. In addition, they may also contain other therapeutically effective substances.

[0141] The pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, but appropriate doses may be determined by clinical trials.

[0142] In some embodiments, the pharmaceutical composition is substantially free, e.g., absent at detectable levels, of contaminants selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cells or plasmid components, bacteria, and fungi. In some embodiments, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes Group A.

[0143] The pharmaceutical composition (or formulation) for administration may be packaged in a variety of ways depending on the method used to administer the compositions described herein. Typically, an article for distribution includes a container having the pharmaceutical formulation therein in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-evident assembly to prevent inadvertent access to the contents of the package. Additionally, the container is labeled with a label that describes the contents of the container. The label may also include appropriate warnings.

[0144] In some embodiments, the compositions described herein further comprise a T cell stimulatory compound or a tumor antigen. In some embodiments, the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to a first population of mesoporous silica particles. In additional or alternative embodiments, the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to a second population of mesoporous silica particles. In further embodiments, the T cell stimulatory compound or tumor antigen is IL-2, IL-15, GM-CSF, anti-CD2 mAb, anti-CD3 mAb, anti-CD28 mAb, neo-antigen peptides, peptides from a common antigen such as TRP2, gp100, tumor cell lysate, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or a combination thereof. Adsorption to an MSP (eg, MSR) surface is generally understood as the attachment of a molecule to a surface.

[0145] In embodiments in which the T cell stimulating compound or tumor antigen is conjugated to the second population of mesoporous silica particles, the T cell stimulating compound or tumor antigen can be conjugated to a lipid bilayer on the surface of the second population of mesoporous silica particles. Methods for creating lipid bilayers on mesoporous silica particles are known. See, for example, WO 2018 / 013797. Briefly, liposomes containing a predetermined amount of a label, such as biotin, are used to coat the MSPs. The label can then be used to attach the T cell stimulating compound using a complementary label, such as streptavidin. Lipids used to create liposomes are known to those skilled in the art and include, but are not limited to, vesicle-forming lipids having two hydrocarbon chains, typically acyl chains, and a polar head group. This class includes phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where the length of the two hydrocarbon chains is usually about 14-22 carbon atoms and has different degrees of unsaturation. In some embodiments, the lipid is a relatively unsaturated phospholipid (having one, two, or three double bonds in the hydrocarbon chain). In some embodiments, the lipid is a phosphatidylcholine. Phosphatidylcholine is a phospholipid that incorporates choline as a head group and combines glycerophosphate with two fatty acids. In some embodiments, the phosphatidylcholine is palmitoylphosphatidylcholine, or oleoylphosphatidylcholine, or 1-palmitoyl, 2-oleoylphosphatidylcholine. Multiple types of lipids may be used in preparing liposomal compositions. The selection of lipids and ratios can be varied to achieve a desired degree of fluidity or rigidity and / or to control stability. When using multiple types of lipids in the preparation of a liposomal composition, an appropriate amount of a relatively unsaturated lipid (such as PC) should be used to form stable liposomes. In some embodiments, at least 45-50 mol % of the lipid used in the formulation is PC.Liposomes may also contain lipids derivatized with hydrophilic polymers such as polyethylene glycol (PEG).Suitable hydrophilic polymers include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol, polyaspartamide and hydrophilic peptide sequences.Methods for preparing lipids derivatized with hydrophilic polymers are known (see, for example, U.S. Pat. No. 5,395,619, which is incorporated herein by reference).

[0146] In some embodiments, the first or second population of mesoporous silica particles further comprises a cytokine. The cytokine may be, but is not limited to, IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, or transforming growth factor beta (TGF-β) or an agonist, mimetic, variant, functional fragment, or combination thereof. In certain embodiments, the cytokine is conjugated or adsorbed to the first or second population of mesoporous silica particles. In embodiments where the cytokine is adsorbed to the second population of mesoporous silica particles, the second population of MSPs (e.g., MSRs) may be further coated with a lipid bilayer as described above.

[0147] method In some embodiments, the present invention provides contacting the T lymphocytes with a composition comprising a first population of mesoporous silica particles (e.g., MSR) and a viral vector; wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.

[0148] In some embodiments, the contacting occurs in vitro. In some embodiments, the T lymphocytes are activated before or after contacting with the mesoporous silica particles.

[0149] In some embodiments, the present invention provides a method of genetically transducing T lymphocytes with a recombinant polynucleotide in vivo, comprising: administering a composition comprising a first population of mesoporous silica particles (e.g., MSR) and a viral vector to a subject having one or more T lymphocytes. Including, Viral vectors include expression vectors that contain a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed; When the composition contacts one or more T lymphocytes, the T lymphocytes are genetically transduced with the recombinant polynucleotide.

[0150] In some embodiments, the present invention provides a method of expanding a T lymphocyte population in vitro, comprising: (a) a composition comprising a first population of mesoporous silica particles (e.g., MSR) and a viral vector for providing a transduced T lymphocyte population; and (b) contacting the transduced T lymphocyte population with a T cell stimulatory compound or a tumor antigen and, optionally, a cytokine. wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.

[0151] In some embodiments of the presently described methods, the methods result in an increase in the percentage of T lymphocytes in the population.

[0152] In some embodiments, the invention relates to a method of expanding a chimeric antigen receptor (CAR) T cell population, comprising contacting the CAR-T cell population with mesoporous silica particles (e.g., MSR) conjugated to a targeting moiety, wherein the targeting moiety is complementary to the CAR.

[0153] In some embodiments, the present invention provides a method for selectively expanding a proportion of T lymphocytes in a culture, comprising: (a) a composition comprising a first population of mesoporous silica particles (e.g., MSR) and a viral vector for providing a transduced T lymphocyte population; and (b) contacting the transduced T lymphocyte population with a T cell stimulatory compound or a tumor antigen and, optionally, a cytokine. wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.

[0154] In some embodiments, the cultures comprise different effector cell types including NK cells, monocytes, B cells. In certain embodiments, the percentage of T lymphocytes is enhanced by about 10%, 20%, 30%, 40%, or 50% compared to the percentage of T lymphocytes prior to contact with the MSP composition. In some embodiments, the population of cells is expanded for a period of 8 days or less.

[0155] In some embodiments, the invention is a method of delivering a viral vector to a desired site of action in a subject, comprising administering to the subject a composition comprising a first population of mesoporous silica particles and a viral vector, wherein the composition of mesoporous silica particles (e.g., MSR) and the viral vector is as described above.

[0156] In some aspects, in the methods recited herein, mesoporous silica particles can be surface modified with multiple functional groups adsorbed or covalently attached to the surface covering the pores and / or nanochannels or to the surface of the MSP (e.g., MSR) as described herein. In some embodiments, the functional groups are -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, disulfide, polyethyleneimine, hydrophobic moiety or salt thereof. In some embodiments, the functional groups (i.e. -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, disulfide, polyethyleneimine, hydrophobic moiety or salt thereof) can be separated from the silica surface by a linker. In some embodiments, the functional groups are C1-C 20 The functional group is covalently attached to the MSP or MSR surface via an alkyl linker. In other embodiments, the functional group is covalently attached to the MSP (e.g., MSR) surface via a polyethylene glycol linker. In certain embodiments, the surface modification is a C1-C 20 Alkyl perhaloalkyl or C1-C 20 It is an alkyl perfluoroalkyl.

[0157] In another aspect, in the methods described herein, the viral vector is as described herein. In the methods of the present invention, the viral vector can be conjugated to mesoporous silica particles (e.g., MSR) as described herein. In some embodiments, the electrostatic bond between the mesoporous silica particles and the viral vector is due to the oppositely charged viral vector and mesoporous silica particles. For example, without being bound by theory, mesoporous silica particles surface-modified with positively charged polyethyleneimine or primary, secondary, tertiary or quaternary ammonium groups can be conjugated to a negatively charged viral vector. Thus, in some embodiments, the viral vector is negatively charged and the surface-modified mesoporous silica particles are positively charged. In some embodiments, the covalent bond between the mesoporous silica particles and the viral vector is achieved by methods known to those skilled in the art, with or without a linker. For example, but not limited to, the linker can be a polyethylene glycol, an alkyl group, a polymer, a polyamide bond, etc.

[0158] In some methods recited herein, the nucleotide sequence expresses a chimeric antigen receptor (CAR), an engineered TCR, a cytokine, a chemokine, an shRNA that blocks an inhibitory molecule, or an mRNA that induces expression of a protein. In certain embodiments, the expressed nucleotide sequence expresses a CAR.

[0159] In some methods described herein, the T lymphocytes can be activated by contacting the T lymphocytes with a T cell stimulating compound or a tumor antigen. Examples of T cell stimulating compounds are provided herein. In some embodiments, the T cell stimulating compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles or both populations of MSPs (e.g., MSRs). In other embodiments, the T cell stimulating compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles. In certain embodiments, the T cell stimulating compound or tumor antigen is directly conjugated to the lipid envelope on the second population of mesoporous silica particles or the surface of the second population of mesoporous silica particles. The preparation of lipid envelopes on the surface of MSPs is known and described herein. See, for example, WO 2018 / 013797.

[0160] The methods described herein may further comprise contacting the T lymphocytes with a cytokine. In some embodiments, the cytokine is in a medium containing the MSP (e.g., MSR) or is conjugated or adsorbed to the first or second population of mesoporous silica particles or both populations. In some embodiments, the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, or transforming growth factor beta (TGF-β), or an agonist, mimetic, variant, functional fragment, or combination thereof.

[0161] In some embodiments, the method further comprises expanding the population of T cells after transduction. The T cells / T lymphocytes can be expanded by the methods described herein. In some embodiments, the population of cells is expanded for a period of 8 days or less.

[0162] In yet another embodiment, a population of cells is expanded in vitro for 5 days and the resulting cells exhibit higher proinflammatory IFN-γ and / or GM-CSF levels compared to the same cells expanded in culture for 9 days under the same culture conditions.

[0163] Without being bound by theory, it is believed that the methods described herein preserve the lentivirus during the CAR-T cell manufacturing process. The stimulatory capabilities of the material system allow for unique capabilities from reagents currently used in CAR T cell manufacturing by allowing for antigen-specific stimulation of CAR T cells, which may enhance CAR T cell function when transferred into the body, or may be used to selectively stimulate and expand CAR T cells compared to non-CAR T cells in culture to increase the purity of the CAR T cell product.

[0164] In some embodiments, the present invention is a method of delivering an active agent to a desired site of action in a subject, comprising administering to the subject a composition comprising mesoporous silica particles conjugated to polyethyleneimine. In certain embodiments, the polyethyleneimine is covalently attached to the mesoporous silica particles. In some embodiments, the polyethyleneimine group has an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, or about 10,000 Da, as measured by gel permeation chromatography (GPC).

[0165] In some embodiments, the present invention provides a method of providing sustained drug delivery to a subject at a desired site of action, comprising administering to the subject a composition comprising mesoporous silica particles conjugated to polyethyleneimine and an active agent absorbed or adsorbed to the mesoporous silica particles.

[0166] In some embodiments, the active agent is an anti-cancer agent.

[0167] General description of chimeric antigen receptor technology In some embodiments, described herein are methods for using mesoporous silica particles to produce, e.g., activate and / or expand, a population of immune effector cells, e.g., T cells or NK cells, that have been engineered to express a CAR molecule, e.g., one described herein, where the cells have enhanced activity (e.g., proliferation, cytokine release and / or tumor targeting efficacy).

[0168] In some embodiments, the recombinant polypeptide construct encodes a chimeric antigen receptor (CAR) that includes an antigen binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain) (e.g., an intracellular signaling domain that includes a costimulatory domain (e.g., a costimulatory domain described herein) and / or a primary signaling domain (e.g., a primary signaling domain described herein). In some embodiments, the tumor-supporting antigen is an antigen present on stromal cells or myeloid-derived suppressor cells (MDSCs). In other aspects, the invention features polypeptides encoded by such nucleic acids and host cells that include such nucleic acids and / or polypeptides.

[0169] In some embodiments, the nucleotide sequence in the vector expresses a protein engineered to target a tumor antigen.

[0170] In some embodiments, the tumor antigen is CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also referred to as CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family members B cell component mature (BCMA); Tn antigen ((TnAg) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific fetal antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP) ;elongation factor 2 mutated (ELF2M);ephrinB2;fibroblast activation protein alpha (FAP);insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);proteasome (prosome, macropain) subunit, beta, 9 (LMP2);glycoprotein 100 (gp100);oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl);tyrosinase;ephrin type A receptor 2 (EphA2);fucosyl-GM1;sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (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 (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X-chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globo-H glycoceramide (Glo boH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoreceptor beta 3 (ADRB3); pannexin 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 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen 1 recognized by T cells (Melan-A or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17);paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or brother of regulator of imprinted sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1);

[0171] The CARs described herein can include an antigen binding domain (e.g., an antibody or antibody fragment, a TCR or a TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen described herein). In some embodiments, the tumor-supporting antigen is an antigen present on stromal cells or myeloid-derived suppressor cells (MDSCs). Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation. Without wishing to be bound by theory, in some embodiments, CAR-expressing cells destroy tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival.

[0172] In one embodiment, the stromal cell antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) and tenascin. In some embodiments, the FAP-specific antibody competes for binding with or has the same CDR as sibrotuzumab. In an embodiment, the MDSC antigen is selected from one or more of CD33, CD11b, C14, CD15 and CD66b. Thus, in some embodiments, the tumor-supporting antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) or tenascin, CD33, CD11b, C14, CD15 and CD66b.

[0173] 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. 17, 105 (1987)).

[0174] In some instances, scFvs can be produced by methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking together VH and VL domains using a flexible polypeptide linker. The scFv molecule includes a linker (e.g., a Ser-Gly linker) with optimal length and / or amino acid composition. The linker length can greatly affect how the variable regions of the scFv fold and interact. Indeed, when short polypeptide linkers are used (e.g., 5-10 amino acids), intrachain folding is prevented. Intrachain folding is also necessary for the two variable regions to come together and form a functional epitope binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publication No. 2005 / 0100543, U.S. Patent Application Publication No. 2005 / 0175606, U.S. Patent Application Publication No. 2007 / 0014794, and WO 2006 / 020258 and WO 2007 / 024715, which are incorporated herein by reference.

[0175] An scFv may comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence 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 a series of glycine and serine repeats, such as (Gly4Ser)n, where n is one or more positive integers (SEQ ID NO:22). In some embodiments, the linker may be (Gly4Ser)4 (SEQ ID NO:29) or (Gly4Ser)3 (SEQ ID NO:30). Variation in linker length may maintain or enhance activity, resulting in superior efficacy in activity testing.

[0176] In another embodiment, the antigen binding domain is a T cell receptor ("TCR") or a fragment thereof, such as a single chain TCR (scTCR). Methods for generating such TCRs are known in the art. See, for example, Willemsen RA et al, Gene Therapy 7:1369-1377 (2000); Zhang T et al, Cancer Gene Ther 11:487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) (references are incorporated herein in their entirety). For example, a scTCR can be engineered that contains Vα and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is highly useful for cancer-associated targets that are themselves intracellular, but fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC.

[0177] In certain embodiments, the encoded antigen binding domain comprises 10 -4 M~10 -8 It has a binding affinity KD of M.

[0178] In some embodiments, the encoded CAR molecule is 10 -4 M~10 -8 M, for example 10 -5 M~10 -7 M, for example 10 -6 M or 10 -7 Binding affinity K of M D In some embodiments, the antigen binding domain has a binding affinity that is at least 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or 1,000-fold less than a reference antibody, e.g., an antibody described herein. In some embodiments, the encoded antigen binding domain has a binding affinity that is at least 5-fold less than a reference antibody (e.g., the antibody from which the antigen binding domain is derived). In some aspects, such antibody fragments are functional in providing a biological response, including, but not limited to, immune response activation, inhibition of signaling initiation from its target antigen, as will be appreciated by one of skill in the art.

[0179] In some embodiments, the antigen binding domain of the CAR is an scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived.

[0180] In some embodiments, the antigen binding domain (e.g., scFv) of the CAR of the present invention is encoded by a nucleic acid molecule whose sequence is codon-optimized for expression in mammalian cells. In some embodiments, the entire CAR construct of the present invention is encoded by a nucleic acid molecule whose entire sequence is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be coded by various nucleotide sequences. Various codon optimization methods are known in the art, including, for example, at least those disclosed in U.S. Pat. No. 5,786,464 and U.S. Pat. No. 6,114,148.

[0181] In embodiments involving immune effector cells engineered to express a CAR molecule, such as those described herein, it is understood that the method of treatment may further include any of the steps, aspects, or features described below in the section regarding chimeric antigen receptors.

[0182] The cells are preferably immune effector cells. In some embodiments, the cells are T cells. In some embodiments, the cells are NK cells. In embodiments, the invention relates to populations of cells of the invention, such as populations of immune effector cells of the invention. In one embodiment, the populations of cells of the invention comprise cells of the indicated type and may comprise other types (e.g., a population of immune effector cells, such as T cells, engineered to express a CAR molecule, such as those described herein, may comprise T cells engineered to express the CAR molecule and T cells (or other cell types) that are not engineered to express the CAR molecule). In an embodiment, the populations of cells used in the methods of the invention consist essentially of cells of the indicated type. In one embodiment, the populations of cells of the invention are substantially free of other cell types. In an embodiment, the populations of cells of the invention consist of the indicated cell types.

[0183] In any of the foregoing aspects and embodiments, the cells and / or population of cells are or comprise immune effector cells, e.g., the population of immune effector cells comprises, e.g., consists of, T cells or NK cells. In embodiments, the cells are T cells, e.g., CD8+ T cells, CD4+ T cells, or combinations thereof. In certain embodiments, the cells are NK cells.

[0184] In one embodiment, the cells are human cells. In an embodiment, the cells are autologous, e.g., to the subject to whom the cells are administered. In an embodiment, the cells are allogeneic, e.g., to the subject to whom the cells are administered.

[0185] Generally, in the methods described herein, the compositions described herein are administered in the above therapeutically effective amounts, alone or in combination with one or more therapeutic agents, by any of the usual and accepted methods known in the art. In certain embodiments, the compositions are administered by injection. In more specific embodiments, for in vivo administration, the compositions are administered subcutaneously to the subject in need thereof. In other embodiments, the compositions can be administered in the form of an implant at the desired site of action. The site of action can be determined by those skilled in the art according to the needs of the subject.

[0186] CAR target Described herein are viral vectors that transduce immune effector cells (e.g., T cells, NK cells) engineered to contain one or more CARs that direct the immune effector cells to unwanted cells (e.g., cancer cells). This is accomplished through an antigen-binding domain on the CAR that is specific for a cancer-associated antigen. There are two classes of cancer-associated antigens (tumor antigens) that can be targeted by the CARs of the present invention: (1) cancer-associated antigens that are expressed on the surface of cancer cells; and (2) cancer-associated antigens that are themselves intracellular, but where a fragment of such an antigen (peptide) is presented on the surface of cancer cells by the MHC (major histocompatibility complex).

[0187] In some embodiments, the tumor antigen is CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also referred to as CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family members B cell component mature (BCMA); Tn antigen ((TnAg) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific fetal antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP) ;elongation factor 2 mutated (ELF2M);ephrinB2;fibroblast activation protein alpha (FAP);insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);proteasome (prosome, macropain) subunit, beta, 9 (LMP2);glycoprotein 100 (gp100);oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl);tyrosinase;ephrin type A receptor 2 (EphA2);fucosyl-GM1;sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (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 (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X-chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globo-H glycoceramide (Glo boH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoreceptor beta 3 (ADRB3); pannexin 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 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen 1 recognized by T cells (Melan-A or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17);paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or brother of regulator of imprinted sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1);

[0188] CD19 A non-limiting exemplary tumor antigen is CD19. CARs that bind to CD19 are known in the art. For example, those disclosed in WO2012 / 079000 and WO2014 / 153270. Any known CD19 CAR in the art, such as the CD19 antigen binding domain of any known CD19 CAR, can be used according to the present disclosure. For example, LG-740; CD19 CAR described in U.S. Patent No. 8,399,645; U.S. Patent No. 7,446,190; 122(17):2965-2973(2013);Brentjens et al.,Blood,118(18):4817-4828(2011);Kochenderfer et al.,Blood 116(20):4099-102(2010);Kochenderfer et al.,Blood 122(25):4129-39(2013); and 16th Annu Meet Am Soc Gen Cell Ther(ASGCT)(May 15-18,Salt Lake City)2013,Abst 10.

[0189] Non-limiting exemplary CD19 CARs include, for example, the CD19 CARs described herein or those described in Xu et al. Blood 123.24(2014):3750-9; Kochenderfer et al. Blood 122.25(2013):4129-39, Cruz et al. Blood 122.17(2013):2965-73, NCT00586391, NCT01087294, NCT02456350, NCT00840853, NCT02659943, NCT02650999, NCT02640209, NCT01747486, N CT02546739, NCT02656147, NCT02772198, NCT00709033, NCT02081937, NCT00924326, NCT02735083, NCT02794246, NCT02746952, NCT01593 696, NCT02134262, NCT01853631, NCT02443831, NCT02277522, NCT02348216, NCT02614066, NCT02030834, NCT02624258, NCT02625480, NC T02030847, NCT02644655, NCT02349698, NCT02813837, NCT02050347, NCT01683279, NCT02529813, NCT02537977, NCT02799550, NCT026725 01, NCT02819583, NCT02028455, NCT01840566, NCT01318317, NCT01864889, NCT02706405, NCT01475058, NCT01430390, NCT02146924, NCT 02051257, NCT02431988, NCT01815749, NCT02153580, NCT01865617, NCT02208362, NCT02685670, NCT02535364, NCT02631044, NCT0272888 2, NCT02735291, NCT01860937, NCT02822326, NCT02737085, NCT02465983, NCT02132624, NCT02782351, NCT01493453, NCT02652910, NCT02247609, NCT01029366, NCT01626495, NCT02721407, NCT01044069, NCT00422383, NCT01680991, NCT02794961, or NCT02456207, each of which is incorporated by reference in its entirety.

[0190] In some embodiments, the CD19 CAR comprises a fusion polypeptide sequence provided as SEQ ID NO: 12 in WO 2012 / 079000, which provides an scFv fragment of murine origin that specifically binds to human CD19.

[0191] In some embodiments, the CD19 CAR comprises the amino acid sequence provided as SEQ ID NO: 12 in WO 2012 / 079000.

[0192] In some embodiments, the CD19 CAR comprises: [ka] or a sequence substantially homologous thereto.

[0193] In some embodiments, the CD19 CAR comprises: [ka] The amino acid sequence of

[0194] In some embodiments, the CD19 CAR has the amino acid sequence: [ka] and a humanized CD19 CAR comprising:

[0195] In some embodiments, the CD19 CAR comprises a sequence, such as a CDR, VH, VL, scFv or full-length CAR sequence disclosed in Table 1 below, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0196] [Table 1]

[0197] [Table 2]

[0198] [Table 3]

[0199] [Table 4]

[0200] BCMA A non-limiting exemplary tumor antigen is BCMA. CARs that bind to BCMA are known in the art, such as those disclosed in WO2016 / 014565 or WO2019 / 241426. Any known BCMA CAR in the art, such as 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, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, B CMA_EBB-C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB- C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2 or C13F12.1.

[0201] In some embodiments, the BCMA CAR is selected from the group consisting of BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB-C2016-014565. 1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2 or C13F12.1, or a sequence substantially identical thereto (e.g., 95 to 99%).

[0202] In some embodiments, the BCMA CAR comprises a sequence, such as a CDR, VH, VL, scFv or full length CAR sequence disclosed in Tables 2-14, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0203] [Table 5]

[0204] [Table 6]

[0205] [Table 7]

[0206]

Table 8

[0207]

Table 9

[0208]

Table 10

[0209]

Table 11

[0210]

Table 12

[0211]

Table 13

[0212]

Table 14

[0213]

Table 15

[0214]

Table 16

[0215]

Table 17

[0216]

Table 18

[0217]

Table 19

[0218]

Table 20

[0219]

Table 21

[0220]

Table 22

[0221]

Table 23

[0222]

Table 24

[0223]

Table 25

[0224]

Table 26

[0225]

Table 27

[0226]

Table 28

[0227]

Table 29

[0228]

Table 30

[0229]

Table 31

[0230]

Table 32

[0231]

Table 33

[0232]

Table 34

[0233]

Table 35

[0234]

Table 36

[0235]

Table 37

[0236]

Table 38

[0237]

Table 39

[0238]

Table 40

[0239]

Table 41

[0240]

Table 42

[0241]

Table 43

[0242]

Table 44

[0243]

Table 45

[0244]

Table 46

[0245]

Table 47

[0246]

Table 48

[0247]

Table 49

[0248] In some embodiments, a BCMA CAR may be generated using the VH and VL sequences from WO 2012 / 0163805, the contents of which are incorporated herein by reference in their entirety. In some embodiments, a BCMA CAR may be generated using the CDR, VH, VL, scFv or full length CAR sequences from WO 2019 / 241426, the contents of which are incorporated herein by reference in their entirety.

[0249] Other Exemplary Targets Further non-limiting exemplary tumor antigens include CD20, CD22, EGFR, CD123, and CLL-1.

[0250] CARs that bind to CD20 are known in the art, for example those disclosed in WO 2018 / 067992 or WO 2016 / 164731, both of which are incorporated herein by reference. 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 CD20 binding sequences or CD20 CAR sequences are disclosed, for example, in Tables 1-5 of WO 2018 / 067992, both of which are incorporated herein by reference. In some embodiments, the CD20 CAR comprises the CDRs, variable regions, scFv or full length sequences of the CD20 CARs disclosed in WO 2018 / 067992 or WO 2016 / 164731, both of which are incorporated herein by reference.

[0251] CARs that bind to CD22 are known in the art, for example, those disclosed in WO 2018 / 067992 or WO 2016 / 164731. Any known CD22 CAR in the art, such as the CD22 antigen-binding domain of any known CD22 CAR, can be used according to the present disclosure.

[0252] Exemplary CD22 binding sequences or CD22 CAR sequences are disclosed, for example, in Tables 6A, 6B, 7A, 7B, 7C, 8A, 8B, 9A, 9B, 10A and 10B of WO2016164731 and Tables 6-10 of WO2018067992. In some embodiments, the CD22 CAR sequence comprises the CDRs, variable regions, scFv or full length sequence of a CD22 CAR disclosed in WO2018067992 or WO2016164731.

[0253] In embodiments, the CAR comprises an antigen binding domain that binds to CD22 (CD22 CAR). In some embodiments, the antigen binding domain targets human CD22. In some embodiments, the antigen binding domain comprises a single chain Fv sequence as described herein.

[0254] The sequence of the human CD22 CAR is provided below: In some embodiments, the human CD22 CAR is CAR22-65. Human CD22 CAR scFv sequence [ka] Human CD22 CAR heavy chain variable region [ka] Human CD22 CAR light chain variable region [ka]

[0255] [Table 50]

[0256] [Table 51]

[0257] CARs that bind to EGFR are known in the art, for example, those disclosed in WO 2014 / 130657, which is incorporated herein by reference. Any known EGFR CAR in the art, for example, the EGFR antigen binding domain of any known EGFR CAR, can be used according to the present disclosure. An exemplary EGFRvIII CAR can include the CDRs, variable regions, scFv or full-length CAR sequences disclosed in WO 2014 / 130657, for example, Table 2 of WO 2014 / 130657, which is incorporated herein by reference.

[0258] CARs that bind to CD123 are known in the art, for example, those disclosed in WO 2014 / 130635 or WO 2016 / 028896. Any known CD123 CAR in the art, such as the CD123 antigen-binding domain of any known CD123 CAR, can be used according to the present disclosure, for example, CAR1 to CAR8 disclosed in WO 2014 / 130635; or CAR123-1 to CAR123-4 and hzCAR123-1 to hzCAR123-32 disclosed in WO 2016 / 028896. The amino acid and nucleotide sequences encoding the CD123 CAR molecule and antigen binding domain (e.g., comprising one, two, three VH CDRs and one, two, three VL CDRs according to Kabat or Chothia) are specified in WO 2014 / 130635 and WO 2016 / 028896.

[0259] CAR that binds to CLL-1 is known in the art.For example, it is disclosed in US Patent Application Publication No. 2016 / 0051651A1, which is incorporated herein by reference.In the art, any known CLL-1 CAR, for example, the CLL-1 antigen binding domain of any known CLL-1 CAR, can be used according to the present disclosure.

[0260] In some embodiments, the CAR comprises a CLL-1 CAR or antigen binding domain according to Table 2 of WO 2016 / 014535, which is incorporated herein by reference. Amino acid and nucleotide sequences encoding the CLL-1 CAR molecule and antigen binding domain (e.g., comprising one, two, three VH CDRs and one, two, three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 014535.

[0261] CARs that bind to CD33 are known in the art. For example, those disclosed in US Patent Publication No. 2016 / 0096892A1 and WO 2016 / 014576, which are incorporated herein by reference. Any known CD33 CAR in the art, such as the CD33 antigen-binding domain of any known CD33 CAR, can be used according to the present disclosure. For example, CAR33-1 to CAR33-9 disclosed in WO 2016 / 014576.

[0262] In some embodiments, the CAR comprises a CD33 CAR or antigen binding domain according to Tables 2 or 9 of WO 2016 / 014576, which is incorporated herein by reference. Amino acid and nucleotide sequences encoding CD33 CAR molecules and antigen binding domains (e.g., comprising one, two, three VH CDRs and one, two, three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 014576.

[0263] CARs that bind to mesothelin are known in the art, such as those disclosed in WO2015090230 and WO2017112741, which are incorporated herein by reference, that bind to human mesothelin, such as those disclosed in Tables 2, 3, 4, and 5 of WO2017112741. Any mesothelin CAR known in the art, such as the mesothelin antigen-binding domain of any known mesothelin CAR, can be used in accordance with the present disclosure.

[0264] CARs that bind to GFR ALPHA-4 are known in the art, for example, those disclosed in WO 2016 / 025880. Any known GFR ALPHA-4 CAR in the art, for example, the GFR ALPHA-4 antigen binding domain of any known GFR ALPHA-4 CAR, can be used according to the present disclosure. The amino acid and nucleotide sequences encoding the GFR ALPHA-4 CAR molecule and antigen binding domain (e.g., including one, two, three VH CDRs and one, two, three VL CDRs according to Kabat or Chothia) are specified in WO 2016 / 025880.

[0265] Antigen-binding domain structure 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. 17, 105 (1987)).

[0266] In some instances, scFvs can be produced by methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking together VH and VL domains using a flexible polypeptide linker. The scFv molecule includes a linker (e.g., a Ser-Gly linker) with optimal length and / or amino acid composition. The linker length can greatly affect how the variable regions of the scFv fold and interact. Indeed, when short polypeptide linkers are used (e.g., 5-10 amino acids), intrachain folding is prevented. Intrachain folding is also necessary for the two variable regions to come together and form a functional epitope binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publication No. 2005 / 0100543, U.S. Patent Application Publication No. 2005 / 0175606, U.S. Patent Application Publication No. 2007 / 0014794, and WO 2006 / 020258 and WO 2007 / 024715, which are incorporated herein by reference.

[0267] An scFv may comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence 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 a series of glycine and serine repeats, such as (Gly4Ser)n, where n is one or more positive integers (SEQ ID NO:22). In some embodiments, the linker may be (Gly4Ser)4 (SEQ ID NO:29) or (Gly4Ser)3 (SEQ ID NO:30). Variation in linker length may maintain or enhance activity, resulting in superior efficacy in activity testing.

[0268] In another embodiment, the antigen binding domain is a T cell receptor ("TCR") or a fragment thereof, such as a single chain TCR (scTCR). Methods for generating such TCRs are known in the art. See, for example, Willemsen RA et al, Gene Therapy 7:1369-1377 (2000); Zhang T et al, Cancer Gene Ther 11:487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) (references are incorporated herein in their entirety). For example, a scTCR can be engineered that contains Vα and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is highly useful for cancer-associated targets that are themselves intracellular, but fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC.

[0269] In certain embodiments, the encoded antigen binding domain comprises 10 -4 M~10 -8 It has a binding affinity KD of M.

[0270] In some embodiments, the encoded CAR molecule is 10 -4 M~10 -8 M, for example 10 -5 M~10 -7 M, for example 10 -6 M or 10 -7 Binding affinity K of M D In some embodiments, the antigen binding domain has a binding affinity that is at least 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or 1000-fold less than a reference antibody, e.g., an antibody described herein. In some embodiments, the encoded antigen binding domain has a binding affinity that is at least 5-fold less than a reference antibody (e.g., the antibody from which the antigen binding domain is derived). In some aspects, such antibody fragments are functional in providing a biological response, including, but not limited to, immune response activation, inhibition of signaling initiation from its target antigen, as will be appreciated by one of skill in the art.

[0271] In some embodiments, the antigen binding domain of the CAR is an scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived.

[0272] In some embodiments, the antigen binding domain (e.g., scFv) of the CAR described herein is encoded by a nucleic acid molecule whose sequence is codon-optimized for expression in mammalian cells. In some embodiments, the entire CAR construct of the present invention is encoded by a nucleic acid molecule whose entire sequence is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be coded by various nucleotide sequences. Various codon optimization methods are known in the art, including, for example, the methods disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

[0273] Particular 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 "Target" and below.

[0274] CD19 In some embodiments, the antigen binding domain binds to CD19 and has the same or similar binding specificity as the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997). In some embodiments, the antigen binding domain binds to CD19 and comprises an scFv fragment described in Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997).

[0275] In some embodiments, the antigen binding domain (e.g., a humanized antigen binding domain) binds to CD19 and comprises a sequence from Table 3 of WO 2014 / 153270, which is incorporated herein by reference. WO 2014 / 153270 also describes methods for assaying the binding and efficacy of various CAR constructs.

[0276] Humanization of the murine CD19 antibody is desirable for clinical conditions where murine-specific residues can induce a human anti-mouse antigen (HAMA) response in patients undergoing CART19 treatment, i.e., treatment with T cells transduced with a CAR19 construct. The production, characterization, and efficacy of humanized CD19 CAR sequences are described in WO 2014 / 153270, which is incorporated by reference in its entirety, including Examples 1-5 (pages 115-159).

[0277] In some embodiments, the antigen binding domain comprises the parent murine scFv sequence of the CAR19 construct provided in WO 2012 / 079000, which is incorporated herein by reference. In some embodiments, the antigen binding domain comprises an scFv that binds CD19 and is described in WO 2012 / 079000.

[0278] BCMA Exemplary antigen binding domains that bind BCMA are described in WO 2012 / 0163805, WO 2017 / 021450, WO 2017 / 011804, WO 2017 / 025038, WO 2016 / 090327, WO 2016 / 130598, WO 2016 / 210293, WO 2016 / 090320, and the like. FRET, WO 2016 / 014789, WO 2016 / 094304, WO 2016 / 154055, WO 2015 / 166073, WO 2015 / 188119, WO 2015 / 158671, U.S. Pat. No. 9,243,058, U.S. Pat. No. 8,920,776, U.S. Pat. No. 9,273,141, U.S. Pat. No. 7 US Patent Publication No. 2007 / 0049735, US Patent Publication No. 2015 / 0284467, US Patent Publication No. 2015 / 0051266, US Patent Publication No. 2015 / 0344844, US Patent Publication No. 2016 / 0131655, US Patent Publication No. 2016 / 0297884, US Patent Publication No. 2016 / 0297885 Detailed description, U.S. Patent Application Publication No. 2017 / 0051308, U.S. Patent Application Publication No. 2017 / 0051252, U.S. Patent Application Publication No. 2017 / 0051252, International Publication No. 2016 / 020332, International Publication No. 2016 / 087531, International Publication No. 2016 / 079177, International Publication No. 2015 / 172800, International Publication No. 2017 / 008169, U.S. Patent No. 9,340,No. 621, U.S. Patent Application Publication No. 2013 / 0273055, U.S. Patent Application Publication No. 2016 / 0176973, U.S. Patent Application Publication No. 2015 / 0368351, U.S. Patent Application Publication No. 2017 / 0051068, U.S. Patent Application Publication No. 2016 / 0368988, and U.S. Patent Application Publication No. 2015 / 0232557, which are incorporated by reference in their entireties. In some embodiments, the antigen binding domain is one or more of the BCMA antigen binding domains disclosed therein.

[0279] In some embodiments, the antigen binding domain comprises a human antibody or human antibody fragment that binds BCMA. In some embodiments, the antigen binding domain comprises one or more (e.g., all three) of the LC CDR1, LC CDR2 and LC CDR3 of a human anti-BCMA binding domain described herein (e.g., Tables 2-14) and / or one or more (e.g., all three) of the HC CDR1, HC CDR2 and HC CDR3 of a human anti-BCMA binding domain described herein (e.g., Tables 2-14). In some embodiments, the human anti-BCMA binding domain comprises a human VL described herein (e.g., Tables 2, 6 and 10) and / or a human VH described herein (e.g., Tables 2, 6 and 10). In some embodiments, the antigen binding domain is an scFv comprising a VL and a VH of the amino acid sequences 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) to an amino acid sequence provided in Tables 2, 6 and 10, but not more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence with 95-99% identity to an amino acid sequence of Tables 2, 6 and 10; and / or a VH comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions, e.g., conservative substitutions) to an amino acid sequence provided in Tables 2, 6 and 10, but not more than 30, 20 or 10 modifications (e.g., substitutions, e.g., conservative substitutions), or a sequence with 95-99% identity to an amino acid sequence of Tables 2, 6 and 10.

[0280] In certain embodiments, the antigen binding domain described herein comprises: (1) one, two, or three light chain (LC) CDRs selected from: (i) an LC CDR1 of SEQ ID NO: 54, an LC CDR2 of SEQ ID NO: 55, and an LC CDR3 of SEQ ID NO: 56; and / or (2) one, two, or three heavy chain (HC) CDRs from one of the following: (i) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 84; (ii) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 46; (iii) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 68; or (iv) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45, and HC CDR3 of SEQ ID NO: 76 Includes.

[0281] In some embodiments, the antigen binding domain described herein comprises: (1) one, two, or three light chain (LC) CDRs from one of the following: (i) an LC CDR1 of SEQ ID NO: 95, an LC CDR2 of SEQ ID NO: 131, and an LC CDR3 of SEQ ID NO: 132; (ii) an LC CDR1 of SEQ ID NO: 95, an LC CDR2 of SEQ ID NO: 96, and an LC CDR3 of SEQ ID NO: 97; (iii) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 114, and LC CDR3 of SEQ ID NO: 115; or (iv) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 114, and LC CDR3 of SEQ ID NO: 97; and / or (2) one, two, or three heavy chain (HC) CDRs from one of the following: (i) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 130, and HC CDR3 of SEQ ID NO: 88; (ii) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 87, and HC CDR3 of SEQ ID NO: 88; or (iii) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 109, and HC CDR3 of SEQ ID NO: 88 Includes.

[0282] In some embodiments, the antigen binding domain described herein comprises: (1) one, two, or three light chain (LC) CDRs from one of the following: (i) an LC CDR1 of SEQ ID NO: 147, an LC CDR2 of SEQ ID NO: 182, and an LC CDR3 of SEQ ID NO: 183; (ii) an LC CDR1 of SEQ ID NO: 147, an LC CDR2 of SEQ ID NO: 148, and an LC CDR3 of SEQ ID NO: 149; or (iii) LC CDR1 of SEQ ID NO: 147, LC CDR2 of SEQ ID NO: 170, and LC CDR3 of SEQ ID NO: 171; and / or (2) one, two, or three heavy chain (HC) CDRs from one of the following: (i) HC CDR1 of SEQ ID NO: 179, HC CDR2 of SEQ ID NO: 180, and HC CDR3 of SEQ ID NO: 181; (ii) HC CDR1 of SEQ ID NO: 137, HC CDR2 of SEQ ID NO: 138, and HC CDR3 of SEQ ID NO: 139; or (iii) HC CDR1 of SEQ ID NO: 160, HC CDR2 of SEQ ID NO: 161, and HC CDR3 of SEQ ID NO: 162 Includes.

[0283] In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 comprise the amino acid sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 44, 45, 76, 54, 55 and 56, respectively.

[0284] In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 comprise the amino acid sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 47, 48, 76, 57, 58 and 59, respectively.

[0285] In some embodiments, the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 comprise the amino acid sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 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 sequence of SEQ ID NO: 49, 50, 77, 60, 58 and 56, respectively.

[0286] Other Exemplary Targets Exemplary antigen binding domains that bind to CD20 are described in WO 2016 / 164731 and WO 2018 / 067992, which are incorporated herein by reference, and in some embodiments are antigen binding domains of one or more of the CD20 antigen binding domains disclosed therein.

[0287] Exemplary antigen binding domains that bind to CD22 are described in WO 2016 / 164731 and WO 2018 / 067992, which are incorporated by reference herein.

[0288] In some embodiments, the antigen binding domain comprises a HC CDR1, a HC CDR2, and a HC CDR3 of any of the heavy chain binding domain amino acid sequences listed in Table 15. In embodiments, the antigen binding domain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. In embodiments, the antigen binding domain comprises a LC CDR1, a LC CDR2, and a LC CDR3 amino acid sequence listed in Table 16.

[0289] In some embodiments, the antigen binding domain comprises one, two or all of a LC CDR1, a LC CDR2 and a LC CDR3 of any of the light chain binding domain amino acid sequences listed in Table 16 and one, two or all of a HC CDR1, a HC CDR2 and a HC CDR3 of any of the heavy chain binding domain amino acid sequences listed in Table 15.

[0290] Exemplary antigen binding domains that bind to EGFRvIII are described in WO 2014 / 130657.

[0291] Exemplary antigen binding domains that bind CD123 are described in WO 2014 / 130635 and WO 2016 / 028896, which are incorporated by reference herein.

[0292] In some embodiments, the antigen binding domain comprises a sequence from Tables 1-2 of WO 2014 / 130635, which is incorporated herein by reference.

[0293] In some embodiments, the antigen binding domain comprises a sequence from Tables 2, 6, and 9 of WO 2016 / 028896, which is incorporated by reference herein.

[0294] Exemplary antigen binding domains that bind to CLL-1 are disclosed in WO 2016 / 014535, which is incorporated herein by reference.

[0295] In some embodiments, the antigen binding domain comprises one, two, three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, HC CDR3, HC CDR4, HC CDR5, HC CDR6, HC CDR7, HC CDR8, HC CDR9, HC CDR10, HC CDR11, HC CDR12, HC CDR13, HC CDR14, HC CDR15, HC CDR16, HC CDR17, HC CDR18, HC CDR19 ...9, HC CDR16, HC CDR19, HC CDR19, HC CDR15, HC CDR16, HC CDR19, HC CDR18, HC CDR19, HC CDR19, HC CDR19, HC CDR10, HC CDR11, HC CDR12, HC CDR13, HC CDR14, HC CDR15, HC CDR16, HC CDR17, HC CDR18, HC CDR19, HC CDR19, HC CDR19, HC CDR15, HC CDR16, HC CDR19, HC CDR16, HC CDR19, HC CDR16, HC CDR19, HC CDR19, HC CDR16, HC CDR19, HC CDR16, HC CDR17, HC CDR18, HC CDR19, HC CDR19, HC CDR19, HC CDR19, HC CDR19, HC CDR19, and / or one, two, three (e.g., all three) light chain CDRs, LC CDR2 and HC CDR3 from an antibody described herein (e.g., an antibody described in WO 2015 / 142675, U.S. Patent Application Publication No. 2015-0283178-A1, U.S. Patent Application Publication No. 2016-0046724-A1, U.S. Patent Application Publication No. 2014 / 0322212A1, U.S. Patent Application Publication No. 2016 / 0068601A1, U.S. Patent Application Publication No. 2016 / 0051651A1, U.S. Patent Application Publication No. 2016 / 0096892A1, U.S. Patent Application Publication No. 2014 / 0322275A1, or WO 2015 / 090230, which are incorporated by reference herein). LC CDR1, LC CDR2 and LC CDR3. In some embodiments, the antigen binding domain comprises the heavy chain variable region and / or the light chain variable region of an antibody listed above.

[0296] In embodiments, the antigen binding domain is an antigen binding domain described in WO 2015 / 142675, US Patent Publication Nos. 2015-0283178-A1, 2016-0046724-A1, US Patent Publication No. 2014 / 0322212A1, US Patent Publication No. 2016 / 0068601A1, US Patent Publication No. 2016 / 0051651A1, US Patent Publication No. 2016 / 0096892A1, US Patent Publication No. 2014 / 0322275A1 or WO 2015 / 090230, which are incorporated herein by reference.

[0297] Exemplary target antigens that can be targeted using CAR-expressing cells include, but are not limited to, CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4, among others, as described, for example, in 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.

[0298] In some embodiments, the antigen binding domain of any of the CARs described herein (e.g., any of CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4) comprises one, two, three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2, and HC CDR3, from an antibody listed above, and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3, from an antigen binding domain listed above. In some embodiments, the antigen binding domain comprises the heavy chain variable region and / or the light chain variable region of an antibody listed or described above.

[0299] 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 listed above, and / or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2, and LC CDR3, from an antibody listed above. In some embodiments, the antigen binding domain comprises a heavy chain variable region and / or a light chain variable region of an antibody listed or described above.

[0300] Bispecific CAR In certain embodiments, the antigen binding domain is a bi- or multispecific molecule (e.g., a multispecific antibody molecule). In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In some embodiments, the first and second epitopes are the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody that has binding specificity for a first epitope and a half antibody that has binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody or fragment thereof that has binding specificity for a first epitope and a half antibody or fragment thereof that has binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises an scFv or fragment thereof that has binding specificity for a first epitope and an scFv or fragment thereof that has binding specificity for a second epitope.

[0301] In some embodiments, the antibody molecule is a multispecific (e.g., bispecific or trispecific) antibody molecule. Such molecules include, for example, bispecific fusion proteins, such as expression constructs comprising two scFvs with a hydrophilic helical peptide linker between them and a complete constant region, such as those described in US Pat. No. 5,637,481; minibody constructs with linked VL and VH chains further linked by a peptide spacer to the antibody hinge and CH3 regions that can dimerize to form a bispecific / multivalent molecule, such as those described in US Pat. No. 5,837,821; No. 5,864,019; and single-chain binding polypeptides having both VH and VL domains linked by a peptide linker combined into multivalent structures by non-covalent or chemical cross-linking, using both scFV or bispecific antibody type formats to form, for example, homobivalent, heterobivalent, trivalent and tetravalent structures, for example, as described in U.S. Pat. No. 5,869,620. The contents of the above-referenced applications are incorporated herein by reference in their entireties.

[0302] Within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH1) upstream of its VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall bispecific antibody molecule has the arrangement VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL1) upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the arrangement VL1-VH1-VH2-VL2. Optionally, a linker is placed between two antibodies or antibody fragments (e.g., scFvs), for example between VL1 and VL2 when the construct is arranged as VH1-VL1-VL2-VH2, or between VH1 and VH2 when the construct is arranged as VL1-VH1-VH2-VL2. The linker can be a linker as described herein, for example a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 691). In general, the linker between two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. Optionally, the linker is placed between the VL and VH of the first scFv. Optionally, the linker is placed between the VL and VH of the second scFv. In constructs with multiple linkers, any two or more of the linkers can be the same or different. Thus, in some embodiments, the bispecific CAR comprises a VL, a VH and optionally one or more linkers in an arrangement as described herein.

[0303] 1. Transmembrane domain With regard to the transmembrane domain, in various embodiments, the chimeric molecules (e.g., CARs) described herein can be designed to include a transmembrane domain that is linked to the extracellular domain of the chimeric molecule. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In some embodiments, the transmembrane domain is associated with one of the other domains of the chimeric protein (e.g., CAR), e.g., in some embodiments, the transmembrane domain can be from the same protein from which the signaling domain, the costimulatory domain, or the hinge domain is derived. In another embodiment, the transmembrane domain is not from the same protein from which any other domain of the chimeric protein (e.g., CAR) is derived. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or a different surface membrane protein, e.g., to minimize interactions with other members of the receptor complex. In some embodiments, the transmembrane domain can homodimerize with another CAR on the cell surface of a CAR-expressing cell. In a different embodiment, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interactions with the binding domain of a natural binding partner present on the same CAR-expressing cell.

[0304] The transmembrane domain can be from natural or recombinant sources. When the source is natural, the domain can be from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain can transmit a signal to the intracellular domain whenever the CAR binds to the target. Particularly useful transmembrane domains in the present invention can include at least the transmembrane regions of, for example, the alpha, beta or zeta chains of the T cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain is selected from the group consisting of, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG It may include at least the transmembrane domain of AM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D or NKG2C.

[0305] In some instances, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. For example, in some embodiments, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge or an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge. In some embodiments, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 4. In some aspects, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO: 12.

[0306] In some embodiments, the encoded transmembrane domain comprises the amino acid sequence of a CD8 transmembrane domain having at least one, two or three modifications but not more than 20, 10 or 5 modifications to the amino acid sequence of SEQ ID NO: 12, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the encoded transmembrane domain comprises the sequence of SEQ ID NO: 12.

[0307] In other embodiments, the nucleic acid encoding the CAR molecule comprises a nucleotide sequence of a CD8 transmembrane domain comprising, for example, the sequence of SEQ ID NO: 13, or a sequence having 95-99% identity thereto.

[0308] 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 the sequence of SEQ ID NO: 5 or SEQ ID NO: 7, or a sequence with 95-99% identity to SEQ ID NO: 5 or 7, corresponding to a CD8 hinge or an IgG4 hinge, respectively.

[0309] In some embodiments, the hinge or spacer comprises an IgG4 hinge. For example, in some embodiments, the hinge or spacer comprises the amino acid sequence [ka] In some embodiments, the hinge or spacer comprises: [ka] The hinge is encoded by the nucleotide sequence of

[0310] In some embodiments, the hinge or spacer comprises an IgD hinge. For example, in some embodiments, the hinge or spacer comprises the amino acid sequence [ka] In some embodiments, the hinge or spacer comprises: [ka] The hinge is encoded by the nucleotide sequence of

[0311] In some embodiments, the transmembrane domain may be recombinant and, if so, comprises predominantly hydrophobic residues such as leucine and valine, hi some embodiments, triplets of phenylalanine, tryptophan and valine can be found at each end of the recombinant transmembrane domain.

[0312] Optionally, a short oligo- or polypeptide linker of 2-10 amino acids in length can form the bond between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 10). In some embodiments, the linker is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 11).

[0313] In some embodiments, the hinge or spacer comprises a KIR2DS2 hinge.

[0314] 2. Signaling Domain In embodiments of the invention having an intracellular signaling domain, such domain may comprise, for example, one or more of a primary signaling domain and / or a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a sequence encoding a primary signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain.

[0315] The intracellular signaling sequences within the cytoplasmic portion of the CAR of the present invention may be linked to each other randomly or in a specific order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length, may form the linkage between the intracellular signaling sequences. In some embodiments, a glycine-serine doublet may be used as a suitable linker. In some embodiments, a single amino acid, e.g., alanine, glycine, may be used as a suitable linker.

[0316] In some embodiments, the intracellular signaling domain is designed to include two or more, e.g., two, three, four, five or more, costimulatory signaling domains. In some embodiments, the two or more, e.g., two, three, four, five 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.

[0317] Primary signaling domains The primary signaling domain controls the primary activation of the TCR complex in either a stimulatory or inhibitory direction. The primary intracellular signaling domain that acts in a stimulatory direction may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. In CAR, such domains are used for the same purpose.

[0318] Examples of ITAM-containing primary intracellular signaling domains that are particularly useful in the present invention include those of CD3 zeta, common FcRγ (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In some embodiments, a CAR of the invention comprises an intracellular signaling domain, such as the primary signaling domain of CD3 zeta.

[0319] In some embodiments, the encoded primary signaling domain comprises a functional signaling domain of CD3 zeta. The encoded CD3 zeta primary signaling domain can comprise an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications to SEQ ID NO: 18 or SEQ ID NO: 20, or a sequence with 95-99% identity to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, the encoded primary signaling domain comprises the sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In other embodiments, the nucleic acid sequence encoding the primary signaling domain comprises the sequence of SEQ ID NO: 19 or SEQ ID NO: 21, or a sequence with 95-99% identity thereto.

[0320] Costimulatory Signaling Domains In some embodiments, the encoded intracellular signaling domain comprises a costimulatory signaling domain. For example, the intracellular signaling domain can comprise a primary signaling domain and a costimulatory signaling domain. In some embodiments, the encoded costimulatory signaling domain is selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD22 6), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46 and NKG2D.

[0321] 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 SEQ ID NO: 14 or SEQ ID NO: 16, or a sequence with 95-99% identity to SEQ ID NO: 14 or SEQ ID NO: 16. In some embodiments, the encoded costimulatory signaling domain comprises the sequence of SEQ ID NO: 14 or SEQ ID NO: 16. In other embodiments, the nucleic acid sequence encoding the costimulatory signaling domain comprises the sequence of SEQ ID NO: 15 or SEQ ID NO: 17, or a sequence with 95-99% identity thereto.

[0322] 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.

[0323] In some embodiments, the nucleic acid sequence encoding the intracellular signaling domain comprises the sequence of SEQ ID NO: 15 or SEQ ID NO: 17, or a sequence having 95-99% identity thereto, and the sequence of SEQ ID NO: 19 or SEQ ID NO: 21, or a sequence having 95-99% identity thereto.

[0324] 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.

[0325] In some embodiments, the intracellular signaling domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of CD28. In some embodiments, the intracellular signaling domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of 4-1BB. In some embodiments, the signaling domain of 4-1BB is the signaling domain of SEQ ID NO: 14. In some embodiments, the signaling domain of CD3 zeta is the signaling domain of SEQ ID NO: 18.

[0326] In some embodiments, the intracellular signaling domain is designed to comprise a signaling domain of CD3 zeta and a signaling domain of CD27. In some embodiments, the signaling domain of CD27 comprises the amino acid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 16). In some embodiments, the signaling domain of CD27 comprises the amino acid sequence of [ka] It is encoded by the nucleic acid sequence of

[0327] Inhibitory domain In some embodiments, the vector comprises a nucleic acid sequence encoding a CAR, e.g., a CAR described herein, and a nucleic acid sequence encoding an inhibitory molecule comprising an inhKIR cytoplasmic domain; a transmembrane domain, e.g., a KIR transmembrane domain; and an inhibitor cytoplasmic domain, e.g., an ITIM domain, e.g., an inhKIR ITIM domain. In some embodiments, the inhibitory molecule is a naturally occurring inhKIR or a sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology with a naturally occurring inhKIR, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues.

[0328] In some embodiments, the nucleic acid sequence encoding the inhibitory molecule comprises a SLAM family cytoplasmic domain; a transmembrane domain, e.g., a SLAM family transmembrane domain; and an inhibitor cytoplasmic domain, e.g., a SLAM family domain, e.g., a SLAM family ITIM domain. In some embodiments, the inhibitory molecule is a naturally occurring SLAM family member or is a sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology with a naturally occurring SLAM family member, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues.

[0329] 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 of the vector further comprises a poly(A) tail, e.g., a polyA tail. In some embodiments, the nucleic acid sequence of the vector further comprises a 3'UTR, e.g., a 3'UTR comprising at least one repeat of a 3'UTR derived from human β-globulin, e.g., as described herein. In some embodiments, the nucleic acid sequence of the vector further comprises a promoter, e.g., a T2A promoter.

[0330] promoter In some embodiments, the vector further comprises a promoter. In some embodiments, the promoter is selected from an EF-1 promoter, a CMV IE gene promoter, an EF-1 alpha promoter, a ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter. In some embodiments, the promoter is an EF-1 promoter. In some embodiments, the EF-1 promoter comprises the sequence of SEQ ID NO:1.

[0331] In some embodiments of the invention, immune effector cells, such as T cells, can be obtained from a unit of blood drawn from a subject using any technique known to one of skill in the art, such as Ficoll™ separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically includes T cells, monocytes, granulocytes, B cells, lymphocytes, including other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction, and optionally the cells can be suspended in a buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, can lack magnesium, or can lack many, if not all, divalent cations.

[0332] [Table 52]

[0333] [Table 53]

[0334] [Table 54]

[0335] [Table 55]

[0336] [Table 56]

[0337] In vitro CAR-T production While the methods contemplated herein involve in vivo transduction of cells, challenges with in vitro production are also recognized.

[0338] In some embodiments, cells transduced with a viral vector as described herein are expanded, for example, by the methods described herein. In some embodiments, the cells are expanded in culture for a period of several hours (e.g., about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 18 hours, 21 hours) to about 14 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days). In some embodiments, the cells are expanded for a period of 4 to 9 days. In some embodiments, the cells are expanded for a period of 8 days or less, for example, 7 days, 6 days, or 5 days. In some embodiments, the cells are expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. Potency can be defined, for example, by various T cell functions, such as proliferation, target cell killing, cytokine production, activation, migration, or a combination thereof. In some embodiments, the cells are grown for 5 days and exhibit at least a 1-fold, 2-fold, 3-fold, or 4-fold increase in cell doublings upon antigen stimulation compared to the same cells grown in culture for 9 days under the same culture conditions. In some embodiments, the cells are grown in culture for 5 days and the resulting cells exhibit high proinflammatory cytokine production, e.g., IFN-γ and / or GM-CSF levels, compared to the same cells grown in culture for 9 days under the same culture conditions. In some embodiments, the cells grown for 5 days exhibit at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more increase in proinflammatory cytokine production, e.g., IFN-γ and / or GM-CSF levels in pg / ml, compared to the same cells grown in culture for 9 days under the same culture conditions.

[0339] The initial activation process in the absence of calcium may lead to enhanced activation. As will be readily appreciated by those skilled in the art, the washing steps may be accomplished by semi-automated "flow-through" centrifugation (e.g., Cobe 2991 cell processor, Baxter CytoMate or Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, saline solutions with or without PlasmaLyte A or other buffering agents. Alternatively, undesirable elements of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0340] It is recognized that the in vitro methods of the present application can utilize culture media conditions comprising 5% or less, e.g., 2%, human AB serum, and can use known culture media conditions and compositions, such as those described in Smith et al., “Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement” Clinical & Translational Immunology (2015) 4, e31; doi:10.1038 / cti.2014.31.

[0341] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation. The isolated T cells may be further used in the methods described herein.

[0342] The methods described herein can include the selection of a specific subpopulation of immune effector cells, e.g., T cells, e.g., a T regulatory cell depleted population, CD25+ depleted cells, e.g., using, e.g., negative selection techniques described herein. Preferably, the T regulatory depleted cell population comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

[0343] In some embodiments, T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody or fragment thereof, or a CD25-binding ligand, IL-2. In some embodiments, the anti-CD25 antibody or fragment thereof, or CD25-binding ligand is conjugated to or otherwise coated on a substrate, e.g., a bead. In some embodiments, the anti-CD25 antibody or fragment thereof is conjugated to a substrate as described herein.

[0344] In some embodiments, T regulatory cells, e.g., CD25+ T cells, are removed from the population using a CD25 depletion agent from Miltenyi™. In some embodiments, the ratio of cells to CD25 depletion agent is 1×10 7 Cells per 20 μL, or 1 x 10 7 Cells per 15 μL, or 1 x 10 7 Cells per 10 μL, or 1 x 10 7 5 μL of cells or 1 x 10 7 2.5 μL per cell, or 1 x 10 7 In some embodiments, for example for T regulatory cell, e.g., CD25+ depletion, 500 million cells / ml are used. In further embodiments, cell concentrations of 600, 700, 800, or 900 million cells / ml are used.

[0345] In some embodiments, the immune effector cell population to be depleted comprises about 6×10 9 In other embodiments, the immune effector cell population to be depleted comprises about 1×10 9 ~1×10 10 (and any integer value therebetween) CD25+ T cells. In some embodiments, the resulting T regulatory depleted cell population comprises 2×10 9 T regulatory cells, e.g., CD25+ cells or less (e.g., 1×10 9 , 5×10 8 , 1×10 8 , 5×10 7 , 1×10 7 or less than CD25+ cells).

[0346] In some embodiments, T regulatory cells, e.g., CD25+ cells, are removed from the population using the CliniMAC system with a depletion tubing set, e.g., tubing 162-01. In some embodiments, the CliniMAC system is run in a depletion setting, e.g., DEPLETION2.1.

[0347] Without wishing to be bound by a particular theory, it is contemplated that a reduction in the levels of negative regulators of immune cells (e.g., unwanted immune cells, such as T cells) in a subject prior to apheresis or during the manufacture of a CAR-expressing cell product may be achieved by reducing the levels of negative regulators of immune cells (e.g., unwanted immune cells, such as T cells) in the subject prior to apheresis or during the manufacture of a CAR-expressing cell product. REG A reduction in the number of T cells may reduce the subject's risk of recurrence. REG Methods for depleting cells are known in the art. REG Methods for reducing cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25 depletion, and combinations thereof.

[0348] In some embodiments, the method of production includes the step of preparing a CAR-expressing cell prior to production of the T REG For example, the manufacturing method can include contacting a sample, e.g., an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or fragment thereof or a CD25-binding ligand) to reduce (e.g., deplete) the number of cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product. REG This involves depleting the cells.

[0349] In some embodiments, the subject is treated with T REG In some embodiments, the subject is pretreated with one or more therapies that reduce the T REGMethods for reducing cells include, but are not limited to, administering to the subject one or more of cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof may occur before, during, or after infusion of the CAR-expressing cell product.

[0350] In some embodiments, the subject is pretreated with cyclophosphamide prior to harvesting of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of the subject relapsing to CAR-expressing cell treatment. In some embodiments, the subject is pretreated with an anti-GITR antibody prior to harvesting of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of the subject relapsing to CAR-expressing cell treatment.

[0351] In some embodiments, the cell population to be removed is not regulatory T cells or tumor cells, but cells that otherwise negatively affect the proliferation and / or function of CART cells, such as cells expressing CD14, CD11b, CD33, CD15, or other markers expressed by potentially immunosuppressive cells. In some embodiments, such cells are intended to be removed simultaneously with the regulatory T cells and / or tumor cells, or after said depletion, or in a different order.

[0352] The methods described herein may include two or more selection steps, e.g., two or more depletion steps. Enrichment of a T cell population by negative selection can be achieved, for example, by a combination of antibodies directed to surface markers unique to the cells to be negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on the cells to be negatively selected. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail may include antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.

[0353] The methods described herein further include depletion from the population of cells expressing tumor antigens, e.g., tumor antigens that do not include CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, thereby providing a T regulatory depleted, e.g., CD25+ depleted and tumor antigen depleted cell population suitable for expression of a CAR, e.g., a CAR as described herein. In some embodiments, tumor antigen expressing cells are depleted simultaneously with T regulatory, e.g., CD25+ cells. For example, anti-CD25 antibodies or fragments thereof and anti-tumor antigen antibodies or fragments thereof can be attached to the same substrate, e.g., beads, which can be used to remove the cells, or anti-CD25 antibodies or fragments thereof and anti-tumor antigen antibodies or fragments thereof can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, depletion of T regulatory cells, e.g., CD25+ cells, and depletion of tumor antigen expressing cells are sequential, e.g., can occur in either order.

[0354] Also provided are methods that include removing from a population one or more of cells expressing a checkpoint inhibitor, e.g., a checkpoint inhibitor as described herein, e.g., PD1+ cells, LAG3+ cells, and TIM3+ cells, thereby providing a T regulatory depleted, e.g., CD25+ depleted, and checkpoint inhibitor depleted, e.g., PD1+, LAG3+, and / or TIM3+ depleted cell population. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA, and LAIR1. In some embodiments, checkpoint inhibitor expressing cells are removed simultaneously with T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-check point inhibitor antibody or fragment thereof can be attached to the same bead, which can be used to remove the cells, or an anti-CD25 antibody or fragment thereof and an anti-check point inhibitor antibody or fragment thereof can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of check point inhibitor-expressing cells are sequential, e.g., can occur in either order.

[0355] The methods described herein may include a positive selection step. For example, T cells may be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient for positive selection of the desired T cells. In some embodiments, the time is about 30 minutes. In further embodiments, the time ranges from 30 minutes to 36 hours or longer and any integer value therebetween. In further embodiments, the time is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another embodiment, the time is 10 to 24 hours, such as 24 hours. Longer incubation times may be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, the use of longer incubation times may increase the efficiency of capture of CD8+ T cells. Thus, simply by binding the T cells to the CD3 / CD28 beads for a shorter or longer time and / or by increasing or decreasing the bead to T cell ratio (as further described herein), a subpopulation of T cells is preferentially selected at the beginning of the culture or at other times during the process. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, a subpopulation of T cells is preferentially selected at the beginning of the culture or at other desired times during the process. In some embodiments, a T cell population can be selected that expresses one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B and perforin or other suitable molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, for example, by methods described in WO 2013 / 126712.

[0356] To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) to ensure maximum contact between the cells and the beads. For example, in some embodiments, a concentration of 10 billion cells / ml, 9 billion cells / ml, 8 billion cells / ml, 7 billion cells / ml, 6 billion cells / ml, or 5 billion cells / ml is used. In some embodiments, a concentration of 1 billion cells / ml is used. In some further embodiments, a concentration of 75, 80, 85, 90, 95, or 100 million cells / ml of cells is used. In further embodiments, a concentration of 125 or 150 million cells / ml can be used.

[0357] High concentrations can be used to increase cell yield, cell activation and cell proliferation. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells, or from samples where many tumor cells are present (e.g., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are desirable to obtain. For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which normally have weak CD28 expression.

[0358] In some embodiments, it may be desirable to use a low concentration of cells. By significantly diluting the mixture of T cells and a surface (e.g., a particle such as a bead), the interaction between the particles and the cells is minimized. This selects for cells that express high amounts of the desired antigen that binds to the particles. For example, CD4+ T cells express high levels of CD28 and are captured more efficiently than CD8+ T cells at dilute concentrations. In some embodiments, the concentration of cells used is 5×10 6 In other embodiments, the concentration used is about 1×10 5 / ml~1×10 6 / ml and any integer value therebetween.

[0359] In other embodiments, the cells can be incubated on a rotator at various speeds for various lengths of time at 2-10° C. or at room temperature.

[0360] T cells for stimulation may also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogenous product by removing granulocytes and to some extent monocytes from the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this regard, one method uses PBS containing 20% ​​DMSO and 8% human serum albumin or culture medium containing 10% Dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, and then the cells are frozen at a rate of 1° / min to −80° C. and stored in a liquid nitrogen storage tank in the vapor phase. Other methods of controlled freezing as well as uncontrolled freezing directly to −20° C. or liquid nitrogen can also be used.

[0361] In some embodiments, cryopreserved cells are thawed as described herein, washed, and allowed to rest at room temperature for 1 hour prior to activation using the methods of the invention.

[0362] In the context of the present invention, collection of blood samples or apheresis products from subjects at a time period prior to the time when the expanded cells described herein may be required is also contemplated. Thus, a source of expanded cells can be collected at any time required, and desired cells, such as T cells, can be isolated and frozen for subsequent use in immune effector cell therapy for any number of diseases and conditions that would benefit from immune effector cell therapy, such as those described herein. In some embodiments, blood samples or apheresis are generally collected from healthy subjects. In some embodiments, blood samples or apheresis are generally collected from healthy subjects who are at risk of developing a disease, but have not yet developed the disease, and the cells of interest are isolated and frozen for subsequent use. In some embodiments, T cells can be expanded, frozen, and used at a later time. In some embodiments, samples are collected from patients immediately after diagnosis of a particular disease described herein, but prior to any treatment. In further embodiments, cells are isolated from a blood sample or apheresis from the subject prior to any number of relevant treatment modalities, including, but not limited to, treatment with antibodies or other immunoablative agents such as natalizumab, efalizumab, antivirals, chemotherapeutics, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and irradiation.

[0363] In further embodiments of the present invention, T cells are obtained directly from a patient after a treatment that leaves the subject with functional T cells. It has been observed that after certain cancer treatments, particularly with drugs that damage the immune system, the quality of T cells obtained may be optimal or improved in terms of their ability to expand ex vivo immediately after the treatment, while the patient usually recovers from the treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for engraftment and enhanced in vivo expansion. Therefore, in the context of the present invention, it is contemplated to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period. Furthermore, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in the subject that favor the repopulation, recirculation, regeneration, and / or proliferation of certain cell types, particularly during a defined time frame after treatment. Examples of cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0364] In some embodiments, the T cell population is diacylglycerol kinase (DGK) deficient. DGK deficient cells are cells that do not express DGK RNA or protein, or that have reduced or inhibited DGK activity. DGK deficient cells can be generated by genetic methods to reduce or block DGK expression, such as administering RNA interference agents, such as siRNA, shRNA, miRNA. Alternatively, DGK deficient cells can be generated by treatment with DGK inhibitors as described herein.

[0365] In some embodiments, the T cell population is Ikaros-deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein or have reduced or inhibited Ikaros activity, and Ikaros-deficient cells can be generated by genetic methods to reduce or block Ikaros expression, such as administration of RNA interference agents, such as siRNA, shRNA, miRNA. Alternatively, Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, such as lenalidomide.

[0366] In embodiments, the T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK- and Ikaros-deficient cells can be produced by any of the methods described herein.

[0367] In some embodiments, the NK cells are obtained from a subject. In another embodiment, the NK cells are an NK cell line, such as the NK-92 cell line (Conkwest).

[0368] In certain exemplary embodiments, the subject is subjected to leukapheresis, in which leukocytes are harvested and enriched or depleted ex vivo to select and / or isolate cells of interest, such as T cells. These T cell isolates can be expanded by the methods described herein. The subject in need of treatment can then be subjected to standard treatment of high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after or at the same time as transplantation, the subject receives an infusion of expanded CAR T cells prepared by the methods of the present invention. In further embodiments, the expanded cells are administered before or after surgery.

[0369] Further manifestations of drugs It is understood that in the embodiments contemplated herein, additional agents may be encoded in the vectors described herein above. Accordingly, these agents are described below in relation to the CAR-expressing cells.

[0370] In another embodiment, the CAR-expressing immune effector cells described herein can further express another agent, e.g., an agent that enhances the activity of the CAR-expressing cell. For example, in some embodiments, the agent can be an agent that inhibits an inhibitory molecule. Examples of inhibitory molecules include PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta, e.g., those described herein. In some embodiments, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, bound to a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In some embodiments, the agent comprises a first polypeptide of an inhibitory molecule such as, for example, PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, or a fragment of any of these, and a second polypeptide that is an intracellular signaling domain described herein (e.g., a second polypeptide that is a costimulatory domain (e.g., 41BB, CD27 or CD28 as described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain as described herein). In some embodiments, the agent comprises a first polypeptide of PD-1 or a fragment thereof, and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28, CD27, OX40 or 4-IBB signaling domain as described herein and / or a CD3 zeta signaling domain as described herein).

[0371] In some embodiments, the CAR-expressing immune effector cells described herein may further comprise a second CAR, e.g., a different antigen binding domain of the second CAR, e.g., directed to the same target (e.g., a target as described above) or a different target. In some embodiments, the second CAR comprises an antigen binding domain directed to a target expressed in the same cancer cell type as the target of the first CAR. In some embodiments, the CAR-expressing immune effector cells comprise a first CAR that targets a first antigen and comprises an intracellular signaling domain having a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different antigen and comprises an intracellular signaling domain having a primary signaling domain but not a costimulatory signaling domain. Without wishing to be bound by theory, placement of a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27, or OX-40, on the first CAR and a primary signaling domain, e.g., CD3 zeta, on the second CAR restricts CAR activity to cells in which both targets are expressed. In some embodiments, the CAR-expressing immune effector cells comprise a first CAR comprising, for example, an antigen binding domain, a transmembrane domain, and a costimulatory domain that targets a target described above, and a second CAR comprising an antigen binding domain, a transmembrane domain, and a primary signaling domain that targets an antigen other than the antigen targeted by the first CAR (e.g., an antigen expressed on the same cancer cell type as the first target). In another embodiment, the CAR-expressing immune effector cells comprise, for example, a first CAR comprising, for example, an antigen binding domain, a transmembrane domain, and a primary signaling domain that targets a target described above, and a second CAR comprising an antigen binding domain, a transmembrane domain, and a primary signaling domain that targets an antigen other than the antigen targeted by the first CAR (e.g., an antigen expressed on the same cancer cell type as the first target), and a secondary CAR comprising an antigen binding domain, a transmembrane domain, and a costimulatory signaling domain for an antigen.

[0372] In some embodiments, the CAR-expressing immune effector cells include CARs described herein, such as CARs against the targets described above, and inhibitory CARs. In some embodiments, the inhibitory CARs include an antigen-binding domain that binds to an antigen found on normal cells, such as normal cells that also express the target, but not on cancer cells. In some embodiments, the inhibitory CARs include an antigen-binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of the inhibitory CAR can be the intracellular domain of PD1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR beta.

[0373] In some embodiments, the immune effector cell (e.g., T cell, NK cell) comprises a first CAR comprising an antigen binding domain that binds to a tumor antigen described herein and a second CAR comprising the PD1 extracellular domain or a fragment thereof.

[0374] In some embodiments, the cells further comprise an inhibitory molecule as described above.

[0375] In some embodiments, the second CAR in the cell is an inhibitory CAR, which comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. The inhibitory molecule may be selected from one or more of PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, and CEACAM-5. In some embodiments, the second CAR molecule comprises the extracellular domain of PD1 or a fragment thereof.

[0376] In one embodiment, the second CAR molecule in the cell further comprises an intracellular signaling domain comprising a primary signaling domain and / or an intracellular signaling domain.

[0377] In other embodiments, the intracellular signaling domain in the cell comprises a primary signaling domain comprising a functional domain of CD3 zeta and a costimulatory signaling domain comprising a functional domain of 4-1BB.

[0378] In some embodiments, the antigen binding domain of the first CAR molecule comprises an scFv and the antigen binding domain of the second CAR molecule does not comprise an scFv, for example, the antigen binding domain of the first CAR molecule comprises an scFv and the antigen binding domain of the second CAR molecule comprises a camelid VHH domain.

[0379] CAR structure In the embodiments contemplated herein, it is understood that one or more CAR conformations can be regulated by the vectors described herein above. Accordingly, these conformations are described below in relation to CAR-expressing cells.

[0380] Split CAR In some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in more detail in WO2014 / 055442 and WO2014 / 055657. Briefly, the split CAR system comprises a cell expressing a first CAR with a first antigen-binding domain and a costimulatory domain (e.g., 41BB), and the cell also expresses a second CAR with a second antigen-binding domain and an intracellular signaling domain (e.g., CD3 zeta). When the cell encounters the first antigen, the costimulatory domain is activated and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell killing activity is initiated. Thus, the CAR-expressing cell is only fully activated in the presence of both antigens.

[0381] Multiple CARs In some embodiments, the CAR-expressing cells described herein may further comprise a second CAR, e.g., a different antigen binding domain of the second CAR, e.g., to the same target or a different target (e.g., a target other than a cancer associated antigen described herein or a different cancer associated antigen described herein). In some embodiments, the second CAR comprises an antigen binding domain to a target expressing the same cancer cell type as the cancer associated antigen. In some embodiments, the CAR-expressing cells comprise a first CAR that targets a first antigen and comprises an intracellular signaling domain having a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different antigen and comprises an intracellular signaling domain having a primary signaling domain but not a costimulatory signaling domain. Without wishing to be bound by theory, placement of a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27, or OX-40, on the first CAR and a primary signaling domain, e.g., CD3 zeta, on the second CAR restricts CAR activity to cells in which both targets are expressed. In some embodiments, a CAR expressing cell comprises a first cancer associated antigen CAR comprising an antigen binding domain that binds to a target antigen described herein, a transmembrane domain, and a costimulatory domain, and a second CAR that targets a different target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and comprises an antigen binding domain, a transmembrane domain, and a primary signaling domain. In another embodiment, a CAR expressing cell comprises a first CAR comprising an antigen binding domain that binds to a target antigen described herein, a transmembrane domain, and a primary signaling domain, and a second CAR that targets an antigen other than the first target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and comprises an antigen binding domain to an antigen, a transmembrane domain, and a costimulatory signaling domain.

[0382] In some embodiments, the invention comprises a first and a second CAR, wherein the antigen-binding domain of one of the first and second CARs does not comprise a variable light chain domain and a variable heavy chain domain. In some embodiments, the antigen-binding domain of one of the first and second CARs is an scFv and the other is not an scFv. In some embodiments, the antigen-binding domain of one of the first and second CARs comprises a single VH domain, such as a camelid, shark or lamprey single VH domain or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of the first and second CARs comprises a nanobody. In some embodiments, the antigen-binding domain of one of the first and second CARs comprises a camelid VHH domain.

[0383] Once the methods described herein have been performed, various assays can be used to evaluate activity, for example, the ability to expand T cells after antigen stimulation in appropriate in vitro and animal models, sustained T cell proliferation in the absence of restimulation, and anti-cancer activity. Assays for evaluating the efficacy of the CAR of the present invention are known to those skilled in the art and are generally described below.

[0384] Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Very briefly, T cells expressing CAR (CD4 + and CD8 + A 1:1 mixture of T cells is expanded in vitro for over 10 days, followed by lysis and SDS-PAGE under reducing conditions. CARs containing the full-length TCR-zeta cytoplasmic domain and the endogenous TCR-zeta chain are detected by Western blotting using an antibody against the TCR-zeta chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to allow assessment of covalent dimer formation.

[0385] CAR after antigen stimulation + In vitro proliferation of T cells can be measured by flow cytometry.

[0386] Sustained CAR in the absence of restimulation + T cell proliferation can also be measured. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, mean T cell volume (fl) is measured using a Coulter Multisizer III particle counter, Nexcelom Cellometer Vision or Millipore Scepter on day 8 of culture after stimulation with αCD3 / αCD28 coated magnetic beads on day 0 and transduction with the indicated CAR on day 1.

[0387] Animal models can also be used to measure CART activity. For example, the use of CARs specific for human cancer-associated antigens described herein to treat primary human pre-B ALL in immunodeficient mice. + Xenograft models using T cells can be used, see, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009).

[0388] Dose-dependent CAR treatment response can be assessed. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after leukemia is established in mice treated with CAR T cells, an equal number of mock-transduced T cells, or no T cell treatment on day 21. Mice from each group are randomly bled for determination of peripheral blood cancer-associated antigen ALL blast counts as described herein and sacrificed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.

[0389] Assessment of cell proliferation and cytokine production has been previously described, for example, in Milone et al., Molecular Therapy 17(8):1453-1464 (2009).

[0390] Cytotoxicity can be assessed by standard 51Cr release assays. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009).

[0391] Imaging techniques can be used to assess the specific trafficking and proliferation of CARs in tumor-bearing animal models. Such assays are described, for example, in Barrett et al., Human Gene Therapy 22:1575-1586 (2011).

[0392] Other assays, including those described in the Examples section herein as well as those known in the art, can also be used to evaluate CARs according to the present invention.

[0393] Treatment In some embodiments, the invention is a method of treating a subject having a disease, injury, or condition associated with elevated expression of a tumor antigen, the method comprising: administering to a subject a composition comprising a first population of mesoporous silica particles and a viral vector. wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence expressing a chimeric antigen receptor (CAR) engineered to target a tumor antigen.

[0394] In yet another aspect, the invention features a method of treating a subject having a disease associated with expression of a tumor antigen (e.g., an antigen described herein), the method including administering to the subject an effective amount of a composition including a mesoporous silica particle described herein and a viral vector, where the viral vector includes an expression vector including a recombinant polynucleotide including an expression control sequence operably linked to a nucleotide sequence expressing a chimeric antigen receptor (CAR) engineered to target the tumor antigen.

[0395] In some embodiments, the MSP is rod-shaped (MSR). In some embodiments, the MSP (e.g., MSR) further comprises a plurality of functional groups adsorbed or covalently attached to the surface covering the pores and / or nanochannels or to the surface of the MSP or MSR. As used herein, a "functional group" defines a chemical moiety that is linked to the surface of the MSR (e.g., MSP) directly or via a linker. In some embodiments, the functional group is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, disulfide, polyethyleneimine, hydrophobic moiety, or salt thereof. In some embodiments, the functional group (i.e., -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, disulfide, polyethyleneimine, hydrophobic moiety, or salt thereof) can be separated from the silica surface by a linker. In some embodiments, the functional group is a C1-C 20 In other embodiments, the functional group is covalently attached to the MSP surface via a polyethylene glycol linker. In certain embodiments, the polyethylene glycol linker has the formula (O(CH2-CH2-) 1~25 In certain embodiments, the surface modification has a C1 to C 20 Alkyl perhaloalkyl or C1-C 20 It is an alkyl perfluoroalkyl.

[0396] In some embodiments of the described method, the electrostatic bond between the mesoporous silica particles and the viral vector is due to the oppositely charged viral vector and mesoporous silica particles. For example, without being bound by theory, mesoporous silica particles (e.g., MSR) surface-modified with positively charged polyethyleneimine or primary, secondary, tertiary or quaternary ammonium groups can be conjugated or attached to a negatively charged viral vector. Thus, in some embodiments, the viral vector is negatively charged and the mesoporous silica particles (e.g., MSR) are positively charged. In some embodiments, the covalent bond between the mesoporous silica particles (e.g., MSR) and the viral vector is achieved by methods known to those skilled in the art, with or without a linker. For example, but not limited to, the linker can be a polyethylene glycol, an alkyl group, a polymer, a polyamide bond, etc.

[0397] In some embodiments of the method, the composition further comprises a T cell stimulatory compound or a tumor antigen conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles or both populations of MSPs (e.g., MSR). Alternatively, the method comprises administering the second population of mesoporous silica particles, e.g., in combination with, simultaneously with, or immediately after, administration of the first population of MSPs (e.g., MSR). Alternatively, the second population of MSPs (e.g., MSR) can be administered an extended period of time after administration of the first population of MSPs.

[0398] In some embodiments, the methods include administering a cytokine, where the cytokine is conjugated or adsorbed to the first or second population of mesoporous silica particles.

[0399] In some embodiments, the second population of MSPs (e.g., MSRs) is administered to the subject simultaneously (e.g., administered on the same day) or shortly after (e.g., 1, 2, 3, 4, 5, 6, or 7 days after administration) of the first population of MSPs. In other embodiments, the cytokine is administered to the subject extended period of time (e.g., at least 2, 3, 4, 6, 8, 10 or more weeks) after administration of the first population of MSPs or after evaluation of the subject's response to the cells.

[0400] In some aspects, in the methods recited herein, mesoporous silica particles can be surface modified as described herein. In some embodiments, the MSP (e.g., MSR) further comprises a plurality of functional groups adsorbed or covalently attached to the surface covering the pores and / or nanochannels or to the surface of the MSP or MSR. As used herein, "functional group" defines a chemical moiety that is linked to the surface of the MSR or MSP, either directly or via a linker. In some embodiments, the functional group is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, disulfide, polyethyleneimine, hydrophobic moiety, or salt thereof. In some embodiments, the functional group (i.e. -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, disulfide, polyethyleneimine, hydrophobic moiety, or salt thereof) can be separated from the silica surface by a linker. In some embodiments, the functional group is a C1-C 20 The functional group is covalently attached to the MSP (e.g., MSR) surface via an alkyl linker. In other embodiments, the functional group is covalently attached to the MSP (e.g., MSR) surface via a polyethylene glycol linker. In certain embodiments, the polyethylene glycol linker has the formula (O(CH2-CH2-) 1~25 In certain embodiments, the surface modification has a C1 to C 20 Alkyl perhaloalkyl or C1-C 20 It is an alkyl perfluoroalkyl.

[0401] In another aspect, in the methods described herein, the viral vector can be conjugated to mesoporous silica particles (e.g., MSR) as described herein. In some embodiments, the electrostatic bond between the mesoporous silica particles and the viral vector is due to the oppositely charged viral vector and mesoporous silica particles. For example, without being bound by theory, mesoporous silica particles (e.g., MSR) surface-modified with positively charged polyethyleneimine or primary, secondary, tertiary or quaternary ammonium groups can be conjugated or attached to a negatively charged viral vector. Thus, in some embodiments, the viral vector is negatively charged and the mesoporous silica particles are positively charged. In some embodiments, the covalent bond between the mesoporous silica particles and the viral vector is achieved by methods known to those skilled in the art, with or without a linker. For example, but not limited to, the linker can be a polyethylene glycol, an alkyl group, a polymer, a polyamide bond, etc.

[0402] In certain embodiments, the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence expressing a chimeric antigen receptor (CAR) engineered to target a tumor antigen. Exemplary CARS are described herein.

[0403] In some embodiments of any of the methods or uses, the disease associated with a tumor antigen, e.g., a tumor antigen described herein, is selected from a proliferative disease, such as a cancer, or a malignancy, or a precancerous condition, such as myelodysplasia, myelodysplastic syndrome, or preleukemia, or is a non-cancer related indication associated with expression of a tumor antigen described herein. In some embodiments, the disease is a cancer described herein, e.g., a cancer described herein as being associated with a target described herein. In some embodiments, the disease is a hematological cancer. In some embodiments, the hematological cancer is leukemia. In some embodiments, the cancer is one or more acute leukemias, including but not limited to B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL); one or more chronic leukemias, including but not limited to B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma. The disease associated with expression of the tumor antigens described herein includes, but is not limited to, atypical and / or atypical cancers, malignancies, precancerous conditions or proliferative disorders expressing the tumor antigens described herein, selected from the group consisting of: marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and additional hematological cancers or conditions including "preleukemia," a diverse collection of hematological conditions combining ineffective production (or dysplasia) of blood cells in the bone marrow. In another embodiment, the disease associated with the tumor antigens described herein is a solid tumor.

[0404] In embodiments, the cancer is colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, The cancer is selected from the group consisting of adrenal gland carcinoma, soft tissue sarcoma, urethral carcinoma, penile carcinoma, childhood solid tumors, bladder carcinoma, kidney or ureter carcinoma, renal pelvis carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

[0405] In some embodiments, the cancer that can be treated with the CAR-expressing cells of the present invention is multiple myeloma.Generally, myeloma cells are considered to be negative for the expression of cancer-associated antigens described herein by flow cytometry.Thus, in some embodiments, for example, the CD19 CAR described herein can be used to target myeloma cells.In some embodiments, the CAR therapy of the present invention can be used in combination with one or more additional therapies, such as lenalidomide treatment.

[0406] In various embodiments, the immune effector cells (e.g., T cells, NK cells) or progeny thereof generated by the methods described herein and administered to a patient persist in the patient for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, or 5 years following administration of the T cells or NK cells to the patient.

[0407] The present invention also includes a type of cellular therapy in which immune effector cells (e.g., T cells, NK cells) are modified to transiently express a chimeric antigen receptor (CAR), e.g., by in vitro or in vivo transcribed RNA. The resulting cells can kill tumor cells in a subject or patient. Thus, in various embodiments, the immune effector cells (e.g., T cells, NK cells) are present for less than one month, e.g., three weeks, two weeks, one week, after administration of the compositions described herein.

[0408] Without being bound to any particular theory, the anti-tumor immune response elicited by CAR-modified immune effector cells (e.g., T cells, NK cells) can be an active or passive immune response, or alternatively, a direct versus indirect immune response. In some embodiments, CAR-transduced immune effector cells (e.g., T cells, NK cells) exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the cancer-associated antigens described herein, resist inhibition of soluble cancer-associated antigens described herein, mediate bystander cell death, and mediate regression of established human tumors. For example, antigen-low tumor cells in a heterogeneous field of tumors expressing the cancer-associated antigens described herein can be subject to indirect destruction by immune effector cells (e.g., T cells, NK cells) redirected to the cancer-associated antigens described herein that have previously responded to nearby antigen-positive cancer cells.

[0409] In some embodiments, the fully human CAR-modified immune effector cells (e.g., T cells or NK cells) of the present invention are a type of vaccine for ex vivo immunization and / or in vivo therapy in a mammal. In some embodiments, the mammal is a human.

[0410] In some embodiments, the CAR-expressing cells of the present invention may be used to treat proliferative disorders such as cancer or malignant tumors or precancerous conditions such as myelodysplasia, myelodysplastic syndromes, or preleukemia. Additionally, disorders associated with the cancer-associated antigens described herein include, but are not limited to, atypical and / or atypical cancers, malignant tumors, precancerous conditions, or proliferative disorders that express, for example, the cancer-associated antigens described herein. Non-cancer-related indications associated with expression of the cancer-associated antigens described herein include, but are not limited to, for example, autoimmune disorders (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation.

[0411] The CAR-modified immune effector cells of the present invention, (e.g., T cells, NK cells), may be administered as a pharmaceutical composition, alone or in combination with other components, such as diluents and / or IL-2 or other cytokines or cell populations.

[0412] blood cancer Hematological cancer conditions are types of cancer such as leukemia, lymphoma and malignant lymphoproliferative conditions that affect the blood, bone marrow and lymphatic system.

[0413] Leukemia can be classified as acute and chronic leukemia. Acute leukemia can be further classified as acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Chronic leukemia includes chronic myeloid leukemia (CML) and chronic lymphoid leukemia (CLL). Another related condition is myelodysplastic syndrome (MDS, formerly known as "preleukemic conditions"), a diverse collection of hematological conditions united by the defective production (or dysplasia) of myeloid blood cells, with the risk of progressing to AML.

[0414] Lymphomas are a group of blood cell tumors that arise from lymphocytes. Representative lymphomas include non-Hodgkin's lymphoma and Hodgkin's lymphoma.

[0415] The invention also provides a method for inhibiting or reducing the proliferation of a cancer associated antigen as described herein, the method comprising contacting a population of cells comprising a cancer associated antigen as described herein with a composition comprising mesoporous silica particles and a viral vector. In certain embodiments, the MSPs are surface modified as described herein. In other embodiments, the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Exemplary nucleotide sequences express a chimeric antigen receptor (CAR), an engineered TCR, a cytokine, a chemokine, an shRNA that blocks an inhibitory molecule, or an mRNA that induces expression of a protein. In some embodiments, the CAR-expressing T cells or NK cells of the invention reduce the content, number, amount or percentage of cells and / or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95% or at least 99% in a subject or animal model having myeloid leukemia or another cancer associated with cells expressing a cancer associated antigen as described herein, compared to a negative control. In some embodiments, the subject is a human.

[0416] Combination Therapy As used herein, administering "in combination" means delivering two (or more) different treatments to a subject during the course of the subject's illness, e.g., two or more treatments are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eradicated or before the treatments are discontinued for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second treatment begins, such that there is an overlap in the administration period. This may be referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., an equal effect is seen with less of the second treatment, or the second treatment alleviates symptoms to a greater extent than is seen when the second treatment is administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In some embodiments, the reduction in symptoms or other parameters associated with the delivery disorder is greater than that observed with delivery of one treatment in the absence of the other treatment. The effect of the two treatments can be partially additive, fully additive, or greater than additive. Delivery can be such that the effect of the first treatment delivered is still detectable upon delivery of the second treatment.

[0417] In some embodiments, the method or use is practiced in combination with an agent that enhances the effectiveness of immune effector cells, such as an agent described herein.

[0418] In some embodiments of the methods or uses described herein, the mesoporous silica rod composition is administered with an agent that enhances the effectiveness of immune effector cells, such as one or more of a protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an inhibitor of immune inhibitory molecules; or a T REG It is administered in combination with an agent that reduces the level or activity of the cells.

[0419] In some embodiments of the methods or uses described herein, the protein phosphatase inhibitor is an SHP-1 inhibitor and / or an SHP-2 inhibitor.

[0420] In other embodiments of the methods or uses described herein, the kinase inhibitor is selected from one or more of a CDK4 inhibitor, a CDK4 / 6 inhibitor (e.g., palbociclib), a BTK inhibitor (e.g., ibrutinib or RN-486), an mTOR inhibitor (e.g., rapamycin or everolimus (RAD001)), an MNK inhibitor, or a dual P13K / mTOR inhibitor. In some embodiments, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK).

[0421] In other embodiments of the methods or uses described herein, the agent that inhibits immune inhibitory molecules comprises an antibody or antibody fragment that inhibits expression of an inhibitory molecule, an inhibitory nucleic acid, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN) or a zinc finger endonuclease (ZFN).

[0422] In another embodiment of the methods or uses described herein, the agent that reduces the level or activity of TREG cells is selected from cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof.

[0423] In some embodiments of the methods or uses described herein, the immune inhibitory molecule is selected from the group consisting of PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3 and CEACAM-5.

[0424] In other embodiments, the agent that inhibits an inhibitory molecule comprises a first polypeptide comprising the inhibitory molecule or a fragment thereof and a second polypeptide that provides a positive signal to a cell, wherein the first and second polypeptides are expressed on a CAR-containing immune cell, and wherein (i) the first polypeptide comprises PD1, PD-L1, CTLA-4, TIM-3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, and CEACAM-5, or fragments thereof; and / or (ii) the second polypeptide comprises an intracellular signaling domain comprising a primary signaling domain and / or a costimulatory signaling domain. In some embodiments, the primary signaling domain comprises a functional domain of CD3 zeta; and / or the costimulatory signaling domain comprises a functional domain of a protein selected from 41BB, CD27, and CD28.

[0425] In other embodiments, the cytokine is selected from IL-7, IL-15, or IL-21, or a combination thereof.

[0426] In another embodiment, the immune effector cells comprising a CAR molecule and a second, e.g., any of the combination therapies disclosed herein (e.g., an agent that increases the efficacy of the immune effector cells), are administered substantially simultaneously or sequentially.

[0427] In other embodiments, immune cells containing a CAR molecule are administered in combination with a molecule that targets GITR and / or modulates GITR function. In some embodiments, the molecule that targets GITR and / or modulates GITR function is administered prior to the CAR-expressing cell or population of cells or prior to apheresis.

[0428] In some embodiments, lymphocyte infusion, e.g., allogeneic lymphocyte infusion, is used to treat cancer, wherein the lymphocyte infusion comprises at least one CAR-expressing cell of the present invention. In some embodiments, autologous lymphocyte infusion is used to treat cancer, wherein the autologous lymphocyte infusion comprises at least one CAR-expressing cell as described herein.

[0429] In some embodiments, the cell is a T cell, and the T cell is diacylglycerol kinase (DGK) deficient. In some embodiments, the cell is a T cell, and the T cell is Ikaros deficient. In some embodiments, the cell is a T cell, and the T cell is both DGK and Ikaros deficient.

[0430] In an embodiment of any of the above methods or uses, there may be further administration of an agent that treats a disease associated with expression of a tumor antigen, such as any of the second or third line therapies disclosed herein. Further exemplary combinations include one or more of the following:

[0431] In another embodiment, there may be further administration of another agent, such as a kinase inhibitor and / or a checkpoint inhibitor as described herein. For example, there may be further administration of an agent that enhances the activity of the CAR-expressing cell.

[0432] For example, in some embodiments, an agent that enhances the activity of a CAR-expressing cell can be an agent that inhibits an inhibitory molecule (e.g., an immune inhibitor molecule). Examples of inhibitory molecules include PD1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta.

[0433] In some embodiments, the agent that inhibits the inhibitory molecule is an inhibitory nucleic acid, which is a dsRNA, siRNA or shRNA. In embodiments, the inhibitory nucleic acid is linked to a nucleic acid that encodes a component of the CAR molecule. For example, the inhibitory molecule can be expressed on a CAR-expressing cell.

[0434] In another embodiment, the agent that inhibits an inhibitory molecule is, e.g., a molecule described herein, e.g., an agent that comprises a first polypeptide, e.g., an inhibitory molecule, linked to a second polypeptide that provides a positive signal to a cell, e.g., an intracellular signaling domain described herein. In some embodiments, the agent comprises a first polypeptide of an inhibitory molecule such as, for example, PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide that is an intracellular signaling domain described herein (e.g., a second polypeptide that is a costimulatory domain (e.g., 41BB, CD27 or CD28 as described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain as described herein). In some embodiments, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain as described herein and / or a CD3 zeta signaling domain as described herein).

[0435] In some embodiments, the CAR-expressing immune effector cells of the invention, e.g., T cells or NK cells, are administered to a subject who has undergone a prior stem cell transplant, e.g., an autologous stem cell transplant.

[0436] In some embodiments, the CAR-expressing immune effector cells of the invention, such as T cells or NK cells, are administered to a subject who has previously received melphalan.

[0437] In some embodiments, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent that increases the efficacy of the cells expressing the CAR molecule, e.g., an agent described herein.

[0438] In some embodiments, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent that mitigates one or more side effects associated with administration of cells expressing a CAR molecule, e.g., an agent described herein.

[0439] In some embodiments, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent that treats a disease associated with a cancer associated antigen described herein, e.g., an agent described herein.

[0440] In some embodiments, for example, cells expressing two or more CAR molecules described herein are administered to a subject in need thereof to treat cancer. In some embodiments, for example, a population of cells comprising a CAR-expressing cell described herein is administered to a subject in need thereof to treat cancer.

[0441] In some embodiments of the methods or uses described herein, the CAR molecule is administered in combination with another agent. In some embodiments, the agent can be a kinase inhibitor, such as a CDK4 / 6 inhibitor, a BTK inhibitor, an mTOR inhibitor, an MNK inhibitor, or a dual PI3K / mTOR inhibitor, and combinations thereof. In some embodiments, the kinase inhibitor is a CD4 inhibitor, such as a CDK4 inhibitor described herein, such as 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one, hydrochloride (also called palbociclib or PD0332991), such as a CDK4 / 6 inhibitor. In some embodiments, the kinase inhibitor is a BTK inhibitor, such as ibrutinib, such as a BTK inhibitor described herein. In some embodiments, the kinase inhibitor is an mTOR inhibitor, such as rapamycin, a rapamycin analog, OSI-027, such as an mTOR inhibitor described herein. The mTOR inhibitor can be, for example, an mTORC1 inhibitor and / or an mTORC2 inhibitor, for example, an mTORC1 inhibitor and / or an mTORC2 inhibitor described herein. In some embodiments, the kinase inhibitor is an MNK inhibitor, for example, an MNK inhibitor described herein, such as 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine. The MNK inhibitor can be, for example, an MNK1a, MNK1b, MNK2a, and / or MNK2b inhibitor. The dual PI3K / mTOR inhibitor can be, for example, PF-04695102.

[0442] In some embodiments of the methods or uses described herein, the kinase inhibitor is aloisine A; flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066; 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one, hydrochloride (P2 76-00;1-Methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265);Indisulam (E7070);Roscovitine (CYC202);Palbociclib (PD0332991);Dinaciclib (SCH727965);N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide (BMS 387032;4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (MLN8054);5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322);4-( 2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519); 4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438); and XL281 (BMS908662).

[0443] In some embodiments of the methods or uses described herein, the kinase inhibitor is a CDK4 inhibitor, e.g., palbociclib (PD0332991), and palbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg, or 125 mg) daily for a period of time, e.g., daily for 14 to 21 days of a 28 day cycle, or daily for 7 to 12 days of a 21 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib are administered.

[0444] In some embodiments of the methods or uses described herein, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In some embodiments, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK) and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.

[0445] In some embodiments of the methods or uses described herein, the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765), and ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg, or 560 mg) per day for a period of time, e.g., daily for a 21 day cycle or daily for a 28 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib are administered.

[0446] In some embodiments of the methods or uses described herein, the kinase inhibitor is a BTK inhibitor that does not inhibit the kinase activity of ITK, e.g., RN-486, and RN-486 is administered at a dose of about 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg (e.g., 150 mg, 200 mg, or 250 mg) per day for a period of time, e.g., daily for a 28 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7 or more cycles of RN-486 are administered.

[0447] In some embodiments of the methods or uses described herein, the kinase inhibitor is temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 4,9]Hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); semapimod; (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502); and N 2 -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartyl-L-serine- (SEQ ID NO: 692), inner salt (SF1126); and XL765.

[0448] In some embodiments of the methods or uses described herein, the kinase inhibitor is an mTOR inhibitor, such as rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a period of time, such as daily for a 21 day cycle or daily for a 28 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are administered. In some embodiments, the kinase inhibitor is an mTOR inhibitor, such as everolimus, and the everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for a period of time, such as daily for a 28 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus are administered.

[0449] In some embodiments of the methods or uses described herein, the kinase inhibitor is an MNK inhibitor selected from CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo[3,4-d]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

[0450] In some embodiments of the methods or uses described herein, the kinase inhibitor is 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF-04691502); N-[4-[[4-(dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-morpholinyl-1,3 ,5-triazin-2-yl)phenyl]urea (PF-05212384, PKI-587); 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinol linyl]-3-pyridinyl}benzenesulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one maleic acid (NVP-BGT226); 3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol (PI-103 ); 5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765).

[0451] In some embodiments of the methods or uses described herein, there may be further administration of a protein tyrosine phosphatase inhibitor, such as a protein tyrosine phosphatase inhibitor described herein. In some embodiments, the protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, such as sodium stibogluconate, such as an SHP-1 inhibitor described herein. In some embodiments, the protein tyrosine phosphatase inhibitor is an SHP-2 inhibitor.

[0452] In some embodiments of the methods or uses described herein, there may be further administration of another agent, which is a cytokine. The cytokine may be, for example, IL-7, IL-15, IL-21, or a combination thereof. In another embodiment, the CAR molecule is administered in combination with a checkpoint inhibitor, such as a checkpoint inhibitor described herein. For example, in some embodiments, the checkpoint inhibitor inhibits an inhibitory molecule selected from PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta.

[0453] In other embodiments of the methods or uses described herein, there may be further administration of an agent that ameliorates one or more side effects associated with the cells expressing the CAR molecule. The side effects associated with the CAR expressing cells may be selected from cytokine release syndrome (CRS) or hemophagocytic lymphohistiocytosis (HLH).

[0454] The present invention also provides a method of preventing, treating and / or managing a disease associated with cells expressing a cancer associated antigen as described herein (e.g., a hematological or atypical cancer expressing a cancer associated antigen as described herein), the method comprising administering to a subject a composition comprising a first population of mesoporous silica particles and a viral vector, wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence expressing a chimeric antigen receptor (CAR) engineered to target a tumor antigen. In some embodiments, the subject is a human. Non-limiting examples of disorders associated with cells expressing a cancer associated antigen as described herein include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma), and cancers (such as a hematological or atypical cancer expressing a cancer associated antigen as described herein).

[0455] The present invention also provides a method of preventing, treating and / or managing a disease associated with cells expressing a cancer associated antigen as described herein, the method comprising administering to a subject a composition comprising a first population of mesoporous silica particles and a viral vector, wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence expressing a chimeric antigen receptor (CAR) engineered to target a tumor antigen. In some embodiments, the subject is a human.

[0456] The invention provides a method for preventing recurrence of a cancer associated with cells expressing a cancer associated antigen as described herein, the method comprising administering to a subject a composition comprising a first population of mesoporous silica particles and a viral vector, wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence expressing a chimeric antigen receptor (CAR) engineered to target a tumor antigen.

[0457] When an "immunologically effective amount," "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is referred to, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject).

[0458] In some embodiments, it may be desirable to administer activated immune effector cells (e.g., T cells, NK cells) to a subject, then draw blood (or perform apheresis), activate the immune effector cells (e.g., T cells, NK cells) in accordance with the present invention, and reinfuse these activated, expanded, and expanded immune effector cells (e.g., T cells, NK cells) back into the patient. This process can be performed multiple times every few weeks. In some embodiments, immune effector cells (e.g., T cells, NK cells) can be activated from 10cc to 400cc of drawn blood. In some embodiments, immune effector cells (e.g., T cells, NK cells) are activated from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc of drawn blood.

[0459] Administration of the subject compositions may be by any convenient method, including aerosol inhalation, injection, ingestion, infusion, placement, or implantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In some embodiments, the MSP (e.g., MSR) compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the T cell compositions of the invention are administered parenterally. The term "parenteral" administration of a T cell composition includes, for example, intrathecal, epidural, intracranial, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques. In certain embodiments, the T cell compositions are administered intravenously. In some embodiments, the MSP (e.g., MSR) and viral vector compositions may be injected directly into a tumor, lymph node, or site of infection. EXAMPLES

[0460] Example A. Synthesis and post-functionalization of mesoporous silica particles Unless otherwise noted, all reagents were obtained from commercial sources and used as received.

[0461] 1. Exemplary synthesis of mesoporous silica particles Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) average Mn ~5,800 (Pluronic P-123, 80.0 g, 487 mmol; Sigma) surfactant was dissolved in 3 L of 1.6 M HCl at room temperature and heated to 40 °C in a 5 L jacketed flask and mechanically stirred via an overhead stirrer at a speed of 0-600 rpm (but most commonly 300 rpm). Tetraethyl orthosilicate (TEOS, 184 mL, 826 mmol; Sigma) was added in one portion in less than 5 minutes and heated at 40 °C with continued stirring for at least 2 hours, but most commonly 20 hours. The resulting slurry was heated to 80-130 °C (most commonly 100 °C) for 6-72 hours (but most commonly 24 hours) for hydrothermal treatment before cooling to room temperature. The slurry was filtered through a Buchner funnel, washed with deionized water followed by ethanol, and air-dried at room temperature. The resulting silica material was calcined in a furnace with a slow temperature ramp from room temperature to 550°C over 8 hours, then maintained at 550°C for another 8 hours, and then cooled to room temperature to obtain 47 g of mesoporous silica particles.

[0462] The aspect ratio of the microparticles can be changed by changing the stirring speed. To control the pore size of mesoporous materials, it is common to change the conditions of hot water temperature and duration. For details, please refer to J.Chem.Educ.2017,94,91-94 and references therein.

[0463] The final mesoporous materials were characterized by optical microscopy, Malvern Morphologi G3, scanning electron microscopy (SEM) and thermogravimetric analysis (TGA).

[0464] 2. Post-modification of silica particles Example 2(a): Diethyl Ethylphosphonate Functionalized Microparticles Diethyl ethylphosphonate functionalized silica microparticles were prepared by the modified method reported in New J.Chem., 2014, 38, 3853, with partial modifications. Diethylphosphatoethyltriethoxysilane (4.15 mL, 13.03 mmol) was added to a slurry of 2.0 g of mesoporous silica microparticles suspended in 300 mL of toluene. The slurry was stirred and refluxed at 110 degrees Celsius for 14 hours, then cooled to room temperature and filtered. The particles were washed with deionized water, followed by ethanol, and then dried in an oven at 100 degrees Celsius for 20 hours to obtain diethyl ethylphosphonate functionalized particles.

[0465] Example 2(b): Ethylphosphonic Acid Functionalized Microparticles Ethylphosphonic acid functionalized microparticles were prepared by a modified method of the procedure reported in New J. Chem., 2014, 38, 3853. Trimethylsilylchlorosilane (1.388 mL, 10.86 mmol) was added to a slurry of 2.0 g of diethyl ethylphosphonate functionalized microparticles suspended in 150 mL of toluene and heated at 110° C. for 24 hours. The slurry was cooled to room temperature, filtered, washed with deionized water and ethanol, and then dried in an oven at 100° C. for 24 hours. The mesoporous silica particles were then suspended in 100 mL of 12 M HCl and heated at 100° C. for 18 hours. The slurry was cooled to room temperature, filtered, washed with deionized water and ethanol, and then dried in an oven at 100° C. for 24 hours to obtain ethylphosphonic acid functionalized microparticles.

[0466] Example 2(c): Propylamine-functionalized microparticles Propylamine-functionalized microparticles were prepared by a modified method of the procedure reported in Langmuir 2015,31,6457-6462. (3-aminopropyl)trimethoxysilane (3.05 ml, 19.54 mmol; APTMS, Sigma) was added to a slurry of 3.0 grams of mesoporous silica microparticles in 150 mL of reagent grade ethanol. The slurry was refluxed at 75 degrees Celsius for 7 hours. After cooling to room temperature, the slurry was filtered and the particles were washed with deionized water followed by ethanol, then dried in an oven at 100 degrees Celsius for 24 hours.

[0467] Example 2(d): Biotin-functionalized microparticles (+)-Biotin N-succinimidyl ester (246 mg, 0.720 mmol) was added to a slurry of 1.0 g of propylamine-functionalized microparticles in 10.0 mL of pH 7.4 adjusted PBS buffer and stirred at room temperature for 18 h. The slurry was filtered, washed with deionized water and ethanol, and then dried in an oven at 100° C. for 24 h to obtain biotin-functionalized microparticles.

[0468] Example 2(e): Biotin-PEG4 functionalized microparticles PEG4-biotin N-hydroxysuccinimide (106 mg, 0.180 mmol; ThermoFischer EZ-Link NHS-PEG4-biotin) was added to a slurry of 0.25 g of propylamine-functionalized microparticles in 2.5 mL of pH 7.4 adjusted PBS buffer and stirred at room temperature for 18 h. The slurry was filtered, washed with deionized water and ethanol, and then dried in an oven at 100 °C for 24 h to obtain biotin-PEG4-functionalized microparticles.

[0469] Example 2(f): 3(2-pyridyldithio)propionamido)hexanoate functionalized microparticles Succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate (112 mg, 0.360 mmol; LC-SPDP ThermoFischer) was added to a slurry of 0.50 g of propylamine-functionalized microparticles in 2.5 mL of pH 7.4 adjusted PBS buffer and stirred at room temperature for 18 hours. The slurry was filtered, washed with deionized water and ethanol, and then dried in an oven at 100 degrees Celsius for 24 hours to obtain 3(2-pyridyldithio)propionamido)hexanoate-functionalized microparticles.

[0470] Example 2(g): 4-Oxo-4-(propylamino)butanoic acid functionalized microparticles Succinic anhydride (4 g, 40.0 mmol) was added to a slurry of 1.0 g of propylamine-functionalized microparticles in succinic anhydride and stirred at room temperature for 24 hours. The slurry was filtered, washed with deionized water and ethanol, and then dried in an oven at 100° C. for 24 hours to obtain 4-oxo-4-(propylamino)butanoic acid-functionalized microparticles.

[0471] Example 2(h): Propyldiethylenetriamine-functionalized microparticles Trimethoxysilylpropyldiethylenetriamine (1.678 mL, 6.51 mmol) was added to 1.0 g of mesoporous silica microparticles and suspended in 150 mL of reagent grade ethanol. The slurry was refluxed at 75 degrees Celsius for 7 hours. After cooling to room temperature, the slurry was filtered and the particles were washed with deionized water followed by ethanol, then dried in an oven at 100 degrees Celsius for 20 hours to obtain propyldiethylenetriamine functionalized microparticles.

[0472] Example 2(i): 3-propyldihydrofuran-2,5-dione functionalized microparticles (succinic anhydride) 3-(3-(triethoxysilyl)propyl)dihydrofuran-2,5-dione (4.94 mL, 17.37 mmol) was added to a slurry of 3.0 g of mesoporous silica microparticles in 300 mL of toluene. The slurry was heated to 110° C. for 20 hours, then cooled to room temperature, filtered, and washed with deionized water and ethanol. The functionalized microparticles were dried in an oven at 100° C. for 24 hours.

[0473] Example 2(j): Branched Polyethylenimine-Functionalized Microparticles Polyethylenimine (25.1 g, 47.0 mmol; branched, average Mw ~25,000, Sigma) was dissolved in 600 mL of anhydrous DMF and 6.0 g of 3-propyldihydrofuran-2,5-dione functionalized microparticles was added and stirred at room temperature for 20 h. The slurry was filtered and the particles were washed with deionized water followed by ethanol, then dried in an oven at 100 degrees Celsius for 20 h to obtain branched polyethylenimine functionalized microparticles.

[0474] Example 2(k): N,N,N-trimethylolpropane-1-ammonium functionalized microparticles Trimethoxysilylpropyltrimethylammonium chloride (3.61 mL, 6.51 mmol; 50% solution in methanol) was added to a slurry of 1.0 g mesoporous silica microparticles in 150 mL reagent ethanol and heated to 75° C. for 7 hours. After cooling to room temperature, the slurry was filtered and the particles were washed with deionized water followed by ethanol, then dried in an oven at 100° C. for 20 hours to yield N,N,N-trimethylpropane-1-ammonium functionalized microparticles.

[0475] The above procedure was repeated with varying ratios of trimethoxysilylpropyltrimethylammonium chloride to silica microparticles (0.25 mmol of trimethoxysilyltrimethylammonium chloride per gram of microparticles) to affect a change in the ratio of functional densities.

[0476] Example 2(l): Octyl-functionalized microparticles Triethyloxy(octyl)silane (2.05 mL, 6.51 mmol) was added to a slurry of 1.0 g mesoporous silica microparticles in 150 mL reagent ethanol and heated to 75° C. for 7 hours. After cooling to room temperature, the slurry was filtered and the particles were washed with deionized water followed by ethanol, then dried in an oven at 100° C. for 20 hours to obtain octyl-functionalized microparticles.

[0477] Example 2(m): Hexadecyl-functionalized microparticles Hexadecyltrimethoxysilane (2.54 mL, 6.51 mmol) was added to a slurry of 1.0 g mesoporous silica microparticles in 150 mL reagent ethanol and heated to 75° C. for 7 hours. After cooling to room temperature, the slurry was filtered and the particles were washed with deionized water followed by ethanol, then dried in an oven at 100° C. for 20 hours to obtain hexadecyl-functionalized microparticles.

[0478] Example 2(n): 11-azidoundecyl functionalized microparticles (11-azidoundecyl)trimethoxysilane (1.0 g, 3.15 mmol) was added to a slurry of 1.0 g mesoporous silica microparticles in 150 mL reagent ethanol and heated to 75° C. for 7 h. After cooling to room temperature, the slurry was filtered and the particles were washed with deionized water followed by ethanol, then dried in an oven at 100° C. for 20 h to obtain 11-azidoundecyl functionalized microparticles.

[0479] Example 2(o): 3-Azidopropyl-functionalized microparticles (3-azidopropyl)trimethoxysilane (1.0 g, 4.87 mmol) was added to a slurry of 1.0 g mesoporous silica microparticles in 150 mL reagent ethanol and heated to 75° C. for 7 hours. After cooling to room temperature, the slurry was filtered and the particles were washed with deionized water followed by ethanol, then dried in an oven at 100° C. for 20 hours to obtain 3-azidopropyl-functionalized microparticles.

[0480] Example 2(p): 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl-functionalized microparticles Triethoxy(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane (2.499 mL, 6.51 mmol) was added to a slurry of 1.0 g mesoporous silica microparticles in 150 mL reagent ethanol and heated to 75° C. for 7 hours. After cooling to room temperature, the slurry was filtered and the particles were washed with deionized water followed by ethanol, then dried in an oven at 100° C. for 20 hours to yield 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl functionalized microparticles.

[0481] Example B. Validation of MSR surface modifications for virus binding To verify the binding of lentivirus to MSPs, various MSPs were prepared with varying surface chemistry (Figure 1). Dried MSR batches were resuspended at 10 mg / ml in ice-cold Tris-NaCl-EDTA buffer pH 7.5 (NTE buffer). A stock solution of lentivirus (FCT067, Kerafast) expressing green fluorescent protein (GFP) was diluted to 3 × 10 in ice-cold NTE buffer. 6 The MSR suspension and diluted virus were mixed in a 1:1 vol / vol ratio and incubated on ice for 30 min. Control particles were incubated 1:1 vol / vol with virus-free NTE buffer. After incubation, samples were washed once with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at 4°C and then with PBS at 4°C. Samples were then fixed with 4.2% paraformaldehyde in PBS. Samples were stained with an antibody against the viral envelope (anti-VSV-G, 8G5F11 from Kerafast; 1:50 dilution) followed by anti-mouse IgG labeled with Dylight-488 (Invitrogen). Samples were washed twice with PBS and imaged using an Evos fluorescent microscope equipped with a GFP LED light cube (Figure 2). Images showed no detectable binding of the staining reagent to the virus-free MSR. Virus-conjugated rods show various levels of quantitative binding, with trimethylammonium and amine functionalities showing the greatest binding.

[0482] Example C. In Vitro Assay for T Cell Transduction with GFP Lentivirus Using MSR A schematic of the use of MSR for viral transduction of T cells is shown in Figure 3. Naïve human T cells were stimulated with Dynabease T cell activation beads at a bead:cell ratio of 3:1 for 2 days. The beads were removed using a magnet and the cells were transferred to fresh medium. Virus-conjugated MSR was prepared as described above and resuspended at 80 μg / ml in cell culture medium. Serial dilutions of this were performed as shown in Figure 3. This suspension was then transferred to 5 × 10 T cells. 5 The MSR-conjugated virus was mixed 1:1 with 1000 μg / ml and incubated for 4 days. GFP expression was assessed in live singlet cells in culture to evaluate transduction efficiency. The results (Figure 4) show that transduction with MSR-conjugated virus occurred at a higher level than with virus given in culture medium alone. Trimethylammonium-functionalized MSR provided the highest level of transduction.

[0483] Example D. Interaction of T cells with MSRs presenting CD3 / CD28 agonist antibodies, EGFRvIII peptides or BCMA proteins MSRs with surface-immobilized ligands were prepared as described in Cheung, A.S., et al., a scaffold mimicking antigen-presenting cells to enable ex vivo expansion of primary T cells. Nature Biotechnology, 36(2), 160-169. A schematic of this process is shown in Figure 5.

[0484] Briefly, liposomes composed mainly of POPC with 1 mol% PE-biotin were formed using thin film rehydration method and extrusion through a 100 nm polycarbonate membrane. Hydroxyl-functionalized MSR was incubated with the liposomes to allow the formation of a supported lipid bilayer on the MSR surface (Figure 6). To functionalize the MSR with CD3 and CD28 agonist antibodies, the MSR was washed several times with PBS and incubated with streptavidin, followed by binding with biotinylated CD3 and CD28 antibodies. For MSR immobilization of EGFRvIII CAR binding peptide, biotinylated EGFRvIII CAR binding peptide was used (Figure 7). For BCMA CART stimulation, recombinant BCMAFc protein was biotinylated using biotin-NHS and similarly bound to the MSR surface.

[0485] After incubation with the desired ligands, MSRs were washed several times with PBS, resuspended in medium at various concentrations, and incubated with T cells. T cell proliferation was read out using CFSE labeling of T cells and evaluating dye dilution by flow cytometry. Cytokine production was assessed using a multiplex cytokine assay (Mesoscale Delivery V-Plex).

[0486] EGFRvIII CART produced interferon gamma and IL-2 in response to EGFRvIII CAR-binding peptide bound to the surface of MSR, whereas free EGFRvIII CAR-binding peptide in solution, non-stimulatory peptide (OVA) presented on MSR, or unmodified MSR did not elicit a response from the CART (Figure 8). In a separate experiment, the proliferation of EGFRvIII CART was monitored in response to various stimuli using cell counting (Figure 9).

[0487] To further analyze the phenotypic proliferation of different T cell subsets, the proliferation of EGFRvIII CARTs was assessed using flow cytometry. CARTs were stained with CFSE and monitored for dye dilution to indicate proliferation by flow cytometry (Figure 10). A similar experiment was performed using functionalized MSRs with BCMAFc protein antigens present on the MSR surface (Figure 11).

[0488] The experimental design shown in FIG. 12 was used to test the co-stimulation and transduction of T cells by viruses using two types of MSRs (MSRs with stimulatory cues and MSRs mixed with lentivirus). One population of MSRs was coated with lipid bilayers and grafted with anti-CD3 / CD28 antibodies as described above. The second population of MSRs was incubated with lentivirus. The results shown in FIG. 13 showed superior transduction levels when T cells were stimulated with anti-CD3 / CD28 agonist antibodies and exposed to virus incubated with PEI-MSR compared to free virus in solution.

[0489] To verify the co-stimulation and transduction of T cells by both cues in the same MSR population, T cells were exposed to either (1) medium containing anti-CD3 / CD28 agonist antibody-loaded lipid-coated stimulatory MSR and virus, (2) PEI-MSR preincubated with anti-CD3 / CD28 agonist antibody-loaded lipid-coated stimulatory MSR and virus, or (3) PEI MSRS adsorbed with anti-CD3 / CD28 agonist antibody and then incubated with virus. After 3 days of culture, the transduction efficiency of T cells was evaluated. Figure 14 shows the effect of stimulatory MSR concentration on MSR under the above conditions (1) and (2) at various amounts of virus. As shown in Figure 14, the overall transduction is enhanced under condition (2) where PEI-MSR is incubated with virus.

[0490] FIG. 15 compares all three conditions, where conditions (1) and (2) are at the highest concentration of stimulatory MSR. As shown in FIG. 15, condition (3), where the stimulatory cue is bound to the PEI-MSR, results in the highest relative transduction efficiency. The same formulation was used to study MSR-mediated transduction of human peripheral blood mononuclear cells (PBMCs). As shown in FIG. 16, transduction in various cell populations as a function of virus concentration is shown at the highest level of stimulation for conditions (1) and (2). FIG. 17 shows the total GFP+ transduced cell fraction and the percentage of each cell population present in the total cell population collected at the highest level of stimulation for conditions (1) and (2).

[0491] Example E. In vivo studies of MSR-induced T cell transduction. A composition of mesoporous silica particles conjugated to a viral vector is injected subcutaneously into mice. Approximately 5-7 days later, MSR adsorbed to a virus encoding an anti-mouse CD19 CAR is injected into this site. Depletion of CD19+ B cells in the blood of the mice will be monitored as an indication that anti-CD19 CARTs have been generated. The presence of these CARTs is confirmed in blood and bone marrow. A detailed histological evaluation of the injection site and drainage of lymph nodes, spleen and liver using in situ hybridization for the CAR transgene is performed to assess leakage of the virus to undesired sites.

[0492] Example F. Drug loading into mesoporous silica microparticles A variety of drugs can be loaded onto the mesoporous silica microparticles. [ka]

[0493] 1. Example 1: Loading of TLR7 agonist onto mesoporous silica microparticles The solution of imiquimod in chloroform is added to a slurry of 100 mg of silica microparticles in 2.0 mL of ...

Claims

1. A composition comprising a first population of mesoporous silica particles and a viral vector.

2. The composition of claim 1 , wherein the viral vector is conjugated to the first population of mesoporous silica particles.

3. The composition of claim 2 , wherein the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles.

4. The composition of claim 2 , wherein the first population of mesoporous silica particles are surface modified.

5. The surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, and optionally C １ ~C ２０ Alkyl or (-O(CH2-CH ２ -) １～２５ The composition of claim 4 , which uses a linker.

6. The composition of claim 5 , wherein the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary, or quaternary amine.

7. 6. The composition of claim 5, wherein the surface modification of the first population of mesoporous silica particles is polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, or about 10,000 Da as measured by gel permeation chromatography (GPC).

8. The composition according to any one of claims 1 to 7, wherein the viral vector is a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus or a lentivirus.

9. The composition of any one of claims 1 to 8, wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.

10. 10. The composition of claim 9, wherein the nucleotide sequence encodes a chimeric antigen receptor (CAR), an engineered TCR, one or more cytokines, one or more chemokines, an shRNA for blocking an inhibitory molecule, or the nucleotide sequence comprises an mRNA for inducing expression of a protein.

11. The composition of claim 10 , wherein the nucleotide sequence encodes a polypeptide engineered to target a tumor antigen.

12. The polypeptides include TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, 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, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, 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, mut12. The composition of claim 11, which targets a tumor antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and any combination thereof.

13. The composition according to any one of claims 10 to 12, wherein the protein is a CAR comprising an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a signaling domain.

14. The composition of claim 13, wherein the signaling domain is a CD3 zeta signaling domain.

15. The composition of any one of claims 1 to 14, further comprising a T cell stimulatory compound or a tumor antigen.

16. 16. The composition of claim 15, wherein the T cell stimulatory compound or the tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles, and the T cell stimulatory compound is IL-2, IL-15, anti-CD2 mAb, anti-CD3 mAb, anti-CD28 mAb, neo-antigen peptides, peptides from a common antigen such as TRP2, gp100, tumor cell lysate, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or combinations thereof.

17. The composition of claim 16, wherein the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles.

18. 17. The composition of claim 16, comprising a second population of mesoporous silica particles, wherein the T cell stimulatory compound or tumor antigen is conjugated to the second population of mesoporous silica particles or to a lipid envelope on the surface of the second population of mesoporous silica particles.

19. The composition of any one of claims 15 to 18, further comprising a cytokine.

20. 20. The composition of claim 19, wherein the cytokine is conjugated or adsorbed to the first or second population of mesoporous silica particles.

21. 21. The composition of claim 19 or 20, wherein the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21 or transforming growth factor beta (TGF-β) or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof or a combination thereof.

22. The composition according to any one of claims 1 to 21, wherein the mesoporous silica particles comprise pores with a diameter of from 2 to 50 nm.

23. The mesoporous silica particles have a diameter of at least about 100 mm. ２ The composition of any one of claims 1 to 22, having a surface area of ​​1 / g.

24. A composition according to any one of claims 1 to 23, which is suitable for injection.

25. contacting the T lymphocytes with a composition comprising a first population of mesoporous silica particles and a viral vector; wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.

26. 26. The method of claim 25, wherein said contacting occurs in vitro.

27. 26. The method of claim 25, wherein the T lymphocytes are activated before or after contact with the first population of mesoporous silica particles.

28. The method of any one of claims 25 to 27, wherein the viral vector is conjugated to the first population of mesoporous silica particles.

29. 29. The method of claim 28, wherein the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles.

30. The method of any one of claims 25 to 29, wherein the first population of mesoporous silica particles is surface modified.

31. The surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, and optionally C １ ~C ２０ Alkyl or (-O(CH ２ -CH ２ -) １～２５ The method of claim 30 , wherein a linker is used.

32. 32. The method of claim 31 , wherein the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary, or quaternary amine.

33. 32. The method of claim 31 , wherein the first population of mesoporous silica particles are surface-modified with polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, or about 10,000 Da, as measured by gel permeation chromatography (GPC).

34. The method according to any one of claims 25 to 33, wherein the viral vector is a lentivirus, a retrovirus or an adenovirus.

35. The method of any one of claims 25 to 34, wherein the nucleotide sequence encodes a chimeric antigen receptor (CAR).

36. 36. The method of claim 35, wherein the CAR is engineered to target a tumor antigen.

37. The method of any one of claims 25 to 36, wherein the T lymphocytes are activated by contacting the T lymphocytes with a T cell stimulatory compound or a tumor antigen.

38. 38. The method of claim 37, wherein the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles.

39. 39. The method of claim 38, wherein the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles.

40. 40. The method of claim 39, wherein the T cell stimulatory compound or tumor antigen is directly conjugated to the second population of mesoporous silica particles or to a lipid envelope on the surface of the second population of mesoporous silica particles.

41. 41. The method of any one of claims 25 to 40, further comprising contacting the T lymphocytes with a cytokine.

42. 42. The method of claim 41, wherein the cytokine is in the culture medium or conjugated or adsorbed to the first or second population of mesoporous silica particles.

43. 43. The method of any one of claims 40 to 42, wherein the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21 or transforming growth factor beta (TGF-β) or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof or a combination thereof.

44. 1. A method for genetically transducing T lymphocytes with a recombinant polynucleotide in vivo, comprising: administering a composition comprising a first population of mesoporous silica particles and a viral vector to a subject having one or more T lymphocytes. Including, The viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed; When said composition contacts one or more T lymphocytes, said T lymphocytes are genetically transduced with said recombinant polynucleotide.

45. 45. The method of claim 44, wherein the viral vector is conjugated to the first population of mesoporous silica particles.

46. 46. ​​The method of claim 45, wherein the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles.

47. The method of any one of claims 44 to 46, wherein the first population of mesoporous silica particles is surface modified.

48. The surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, and optionally C １ ~C ２０ Alkyl or (-O(CH ２ -CH ２ -) １～２５ 48. The method of claim 47, wherein a linker is used.

49. 49. The method of claim 48, wherein the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary, or quaternary amine.

50. 49. The method of any one of claims 44 to 48, wherein the first population of mesoporous silica particles are surface-modified with polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, or about 10,000 Da, as measured by gel permeation chromatography (GPC).

51. The method of any one of claims 44 to 50, wherein the viral vector is a lentivirus, a retrovirus or an adenovirus.

52. 52. The method of any one of claims 44 to 51, wherein the nucleotide sequence encodes a chimeric antigen receptor (CAR).

53. 52. The method of claim 51 , wherein the CAR is engineered to target a tumor antigen.

54. 54. The method of any one of claims 44 to 53, wherein the composition further comprises a T cell stimulatory compound or a tumor antigen conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles.

55. 55. The method of claim 54, wherein the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles.

56. 55. The method of claim 54, wherein the composition comprises a second population of mesoporous silica particles, and the T cell stimulatory compound or tumor antigen is conjugated directly to the second population of mesoporous silica particles or to a lipid envelope on the surface of the second population of mesoporous silica particles.

57. 57. The method of any one of claims 44 to 56, wherein the first or second population of mesoporous silica particles further comprises a cytokine conjugated or adsorbed to the first or second population of mesoporous silica particles.

58. 58. The method of claim 57, wherein the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, or transforming growth factor beta (TGF-β), or an agonist, mimetic, variant, functional fragment, or combination thereof.

59. The method of any one of claims 44 to 58, wherein the subject's T lymphocytes are expanded in vivo.

60. 1. A method for expanding a population of T lymphocytes in vitro, comprising: (a) contacting said population of T lymphocytes with a composition comprising a first population of mesoporous silica particles and a viral vector to provide a transduced population of T lymphocytes; and (b) contacting said transduced T lymphocyte population with a T cell stimulatory compound or a tumor antigen and optionally a cytokine. wherein the viral vector comprises an expression vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.

61. 61. The method of claim 60, wherein the viral vector is conjugated to the first population of mesoporous silica particles.

62. 62. The method of claim 61 , wherein the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles.

63. 63. The method of any one of claims 60 to 62, wherein the first population of mesoporous silica particles is surface modified.

64. The surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, and optionally C １ ~C ２０ Alkyl or (-O(CH ２ -CH ２ -) １～２５ The method of claim 63, wherein a linker is used.

65. 65. The method of claim 64, wherein the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary, or quaternary amine.

66. 66. The method of any one of claims 60 to 65, wherein the first population of mesoporous silica particles are surface-modified with polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, or about 10,000 Da, as measured by gel permeation chromatography (GPC).

67. 67. The method of any one of claims 60 to 66, wherein the viral vector is a lentivirus, a retrovirus or an adenovirus.

68. 68. The method of any one of claims 60 to 67, wherein the nucleotide sequence encodes a chimeric antigen receptor (CAR).

69. 69. The method of claim 68, wherein the CAR is engineered to target a tumor antigen.

70. 70. The method of any one of claims 60 to 69, wherein the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles, and the T cell stimulatory compound or tumor antigen is IL-2, IL-15, anti-CD2 mAb, anti-CD3 mAb, anti-CD28 mAb, neo-antigen peptide, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR or a combination thereof.

71. 71. The method of claim 70, wherein the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles.

72. 71. The method of claim 70, comprising a second population of mesoporous silica particles, wherein the T cell stimulatory compound or tumor antigen is conjugated to the second population of mesoporous silica particles or to a lipid envelope on the surface of the second population of mesoporous silica particles.

73. (c) contacting the T lymphocytes with a cytokine.

73. The method of any one of claims 60 to 72, further comprising: wherein the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21 or transforming growth factor beta (TGF-β) or an agonist, mimetic, variant, functional fragment or combination thereof.

74. 1. A method of treating a subject having a disease, injury, or condition associated with elevated expression of a tumor antigen, comprising: administering to the subject a composition comprising a first population of mesoporous silica particles and a viral vector, wherein the viral vector comprises a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR) that has been engineered to target the tumor antigen. thereby treating said subject.

75. 75. The method of claim 74, wherein the viral vector is conjugated to the first population of mesoporous silica particles.

76. 76. The method of claim 75, wherein the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles.

77. 77. The method of any one of claims 74 to 76, wherein the first population of mesoporous silica particles is surface modified.

78. The surface modification of the first population of mesoporous silica particles is -OH (hydroxyl), amine, carboxylic acid, phosphonate, halide, azide, alkyne, epoxide, sulfhydryl, polyethyleneimine, hydrophobic moiety or salt thereof, and optionally C １ ~C ２０ Alkyl or (-O(CH ２ -CH ２ -) １～２５ The method of claim 77, wherein a linker is used.

79. 80. The method of claim 78, wherein the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary, or quaternary amine.

80. 79. The method of any one of claims 74 to 78, wherein the first population of mesoporous silica particles are surface-modified with polyethyleneimine having an average molecular weight of about 1000-20,000 Da, about 1,200-15,000 Da, about 1,500-12,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 7,000 Da, about 8,000 Da, about 9,000 Da, or about 10,000 Da, as measured by gel permeation chromatography (GPC).

81. The method of any one of claims 74 to 80, wherein the viral vector is a lentivirus, a retrovirus or an adenovirus.

82. 82. The method of any one of claims 74 to 81, wherein the composition further comprises a T cell stimulatory compound or a tumor antigen conjugated or adsorbed to the first population of mesoporous silica particles or the second population of mesoporous silica particles.

83. 83. The method of claim 82, wherein the T cell stimulatory compound or tumor antigen is conjugated or adsorbed to the first population of mesoporous silica particles.

84. 84. The method of claim 83, wherein the composition comprises a second population of mesoporous silica particles, and the T cell stimulatory compound or tumor antigen is conjugated directly to the second population of mesoporous silica particles or to a lipid envelope on the surface of the second population of mesoporous silica particles, and the T cell stimulatory compound or tumor antigen is IL-2, IL-15, anti-CD2 mAb, anti-CD3 mAb, anti-CD28 mAb, neo-antigen peptide, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or a combination thereof.

85. 85. The method of any one of claims 74 to 84, wherein the first or second population of mesoporous silica particles further comprises a cytokine conjugated or adsorbed to the first or second population of mesoporous silica particles.

86. 86. The method of claim 85, wherein the cytokine is IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, or transforming growth factor beta (TGF-β), or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof.

87. A method for delivering a viral vector to a desired site of action in a subject, the method comprising administering to the subject a composition comprising a first population of mesoporous silica particles and the viral vector.

88. 88. The method of claim 87, wherein the viral vector is conjugated to the first population of mesoporous silica particles.

89. 89. The method of claim 88, wherein the viral vector is electrostatically or covalently conjugated to the first population of mesoporous silica particles.

90. 90. The method of any one of claims 87 to 89, wherein the first population of mesoporous silica particles is surface modified.

91. The surface modification of the first population of mesoporous silica particles comprises １～２０ Alkylamines, C １～２０ Carboxylic acid, C １～２０ Azide and substituted or unsubstituted C １～２０ 91. The method of claim 90, wherein said alkyl is alkyl.

92. 92. The method of claim 91, wherein the surface modification of the first population of mesoporous silica particles is a primary, secondary, tertiary, or quaternary amine.

93. 93. The method of any one of claims 87 to 92, wherein the viral vector is a retrovirus, adenovirus, adeno-associated virus, herpes virus or lentivirus.

94. A method according to any one of claims 87 to 93, wherein the first population of mesoporous silica particles comprises pores with a diameter of from 2 to 50 nm.

95. The first population of mesoporous silica particles has a diameter of at least about 100 mm. ２ The method of any one of claims 87 to 94, wherein the surface area is 0.01 to 0.1 g.

96. 1. A method of expanding a chimeric antigen receptor (CAR) T (CAR-T) cell population, comprising contacting the CAR-T cell population with mesoporous silica particles conjugated to a targeting moiety, wherein the targeting moiety is complementary to the CAR.

97. 97. The method of claim 96, wherein the CAR is a protein engineered to target a tumor antigen.

98. The tumor antigens are TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, 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, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 98. The method of claim 96 or 97, wherein the target gene is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-65, IL-65, IL-76, IL-82, IL-83, IL-84, IL-85, IL-85, IL-85, IL-86, IL-87, IL-88, IL-89, IL-89, IL-89, IL-89, IL-89, IL-89, IL-89,

99. A composition according to any one of claims 1 to 24 or a method according to any one of claims 25 to 98, wherein the mesoporous silica particles are in the form of mesoporous silica rods.

100. A composition comprising mesoporous silica particles conjugated to polyethyleneimine.

101. 101. The composition of claim 100, wherein the mesoporous silica particles are in the form of mesoporous silica rods.

102. 102. The composition of claim 100 or 101, further comprising an active agent.

103. The composition of claim 102, wherein the active agent is conjugated or adsorbed to the mesoporous silica particles.

104. 104. A method of delivering an active agent to a desired site of action in a subject, comprising administering to the subject a composition according to claim 102 or 103.

105. 105. The method of claim 104, wherein the composition provides sustained delivery of the active agent to the subject.

106. 104. A method of treating a subject having a disease, injury or condition, comprising administering to the subject a composition according to claim 102 or 103.

107. The method of claim 106, wherein the disease, disorder or condition is associated with a tumor antigen.

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