Chimeric antigen receptors, compositions and applications thereof

Abstract

Provided is application of chimeric antigen receptor (CAR)-modified T (CART) cells in preparing drugs for cancer treatment, the CART cells contain an artificially-introduced costimulatory signal transduction domain, and the CART cell does not contain an artificially-introduced first signal transduction domain.

Claims

1. Chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment, wherein the CART cells comprise a first CAR, wherein the first CAR comprises an antigen binding domain, a transmembrane domain and an intracellular domain, wherein the intracellular domain contains artificially introduced costimulatory signal transduction domains, wherein the CART cells does not contain an artificially introduced first signal transduction domain.

2. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 1, wherein the artificially introduced costimulatory signal transduction domain is selected from one or more of the following intracellular signal transduction domains of costimulatory proteins: 4-1BB (CD137), CD28 , CD27, CD30, OX40, GITR, CD40, BAFFR, ICAM-1, LCK, CD278(ICOS), CD150(SLAMF1), CD270(HVEM), LAT, NKD2CSLP76, TRIM, ZAP70, DAP-10, DAP-12, LFA-1, CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3 or a ligand that specifically binds to CD83 and the like.

3. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 2, wherein the artificially introduced costimulatory signal transduction domain is an intracellular signal transduction domain of 4-1BB or OX40.

4. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 1, wherein the antigen binding domain binds to disease-associated cell surface antigens.

5. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 4, wherein the disease-associated cell surface antigen is selected from immune checkpoint proteins or tumor antigens.

6. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 1, wherein the CART cell further expresses a second CAR and/or secretory polypeptides, wherein the second CAR comprises an antigen binding domain and a transmembrane domain, wherein the secretory polypeptides are constitutively or inducibly expressed.

7. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 6, wherein the antigen binding domain of the second CAR binds to disease-associated cell surface antigens, and preferably, the disease-associated cell surface antigen is a tumor antigen.

8. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 7, wherein the secretory polypeptides are selected from but not limited to: one or two of the followings, immune function regulatory factors, antibodies specifically targeting the tumor antigen, or antibodies specifically targeting the immune checkpoint.

9. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to according to claim 5, wherein the tumor antigen is independently selected from: epidermal growth factor receptor family (EGFR, HER2, HER3, HER4), PD-1, PD-L1 , CTLA-4, 4-1BB(CD137), OX40, CD28, CD40, CD47, CD70, CD80, CD122, GTIR, A2AR, B7-H3(CD276), B7-H4, IDO, KIR, Tim-3, NY-ESO-1, GPC3, CLL-1, BCMA, mucin family (MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUCSB, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC20), CD19, CD20, CD22, CD30, CD33, CD52, chemokine receptor family (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCL27, CCL28, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6), PSMA, CEA, HDAC6, EpCAM, Mesothelin, TERT, TLR, TLR9, TLR4, CD33, GITR, Survivin, CD123, TIGIT, TIM-3, CD73, fibroblast growth factor body (FGFR), vascular endothelial growth factor receptor (FLT1, KDR/Flk-1, VEGFR-3), hepatocyte growth factor receptor (HGFR), nerve growth factor receptor (NGFR), insulin-like growth factor receptor (IGFR), platelet-derived growth factor receptor (PDGFR) or hormone receptor (melanocortin 1 receptor (MC1R, MSHR)) and the like.

10. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 9, wherein the tumor antigen is PSMA, GPC3, CD19, MUC16, EGFR, HER2, CD3 or FAP.

11. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 5, wherein the immune checkpoint protein is selected from: PD-1, PD-L1, CTLA-4, LAG-3, OX40, CD28, CD40, CD47, CD70, CD80, CD122, GTIR, A2AR, B7-H3(CD276), B7-H4, IDO, KIR, Tim-3 or 4-1BB (CD137).

12. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 11, wherein the immune checkpoint protein is PD-1 or PD-L1.

13. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 8, wherein the immune function regulatory factor is selected from: IL-2, IL-12, IL-7, IL-15, IL-18, IL-21, IL-24, 4-1BBL, PD-1 extracellular region, PD-L1 extracellular region, PD-1 mutant, CCL19, MIP-1α, GM-CSF, IFN-α, IFN-β, IFN-γ, TNF-α, M-CSF, TGF-β or TRAIL and the like.

14. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 13, wherein the immune function regulatory factor is IL-2, IL-12, IL-15, IL-18, or PD-1 mutant.

15. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 6, wherein the secretory polypeptides are on the same peptide chain as the first or second CAR, and the secretory polypeptides are linked to the CAR by self-cleaving peptides, or the coding sequence of the secretory polypeptides are linked to the coding sequence of the CAR by promoter and signal peptide coding sequences.

16. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 6, wherein the secretory polypeptides are on different peptide chains from the first or second CAR.

17. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 5, wherein the antigen binding domain is an antibody or an antibody fragment.

18. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 6, wherein the secretory polypeptides are proteins, antibodies, or antibody fragments.

19. The chimeric antigen receptor (CAR)-modified T (CART) cells for use in cancer treatment according to claim 1, wherein the T cells is selected from one or more subsets of specific T cells, such as a tumor-infiltrating lymphocyte (TIL), a cytotoxic T lymphocyte (CTL), a natural killer T (NKT) cell, a δ T cell or a regulatory T cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Drawings that constitute the present application are used to provide further understanding of the present disclosure. The exemplary embodiments and descriptions of the present disclosure thereof are used to explain the present disclosure, and do not constitute improper limitation to the present disclosure. In the drawings:

[0055] FIG. 1 shows expression of CAR on the surface of CART cells targeting PSMA.

[0056] FIG. 2A-2J show expression of CAR on the surface of CARTs cell targeting GPC-3.

[0057] FIG. 3A-3D shows levels of IL-2, IFN-γ, IL-12 and IL-18 secretory by PSMA-targeted CART effector cells after co-incubation with target cells PC3, LnCAP or 22RV1 at a ratio of 1:1. Herein Mock is PBMC cells without genetic modification, 1 is aPSMA BBZ, 2 is aPSMA BBZ+iIL-12, 3 is aPSMA BBZ+iIL-15, 4 is aPSMA BB+iIL-2, 5 is aPSMA BB+iIL-18, and 6 is aPSMA BB+IL-12.

[0058] FIG. 4 shows the level of IL-2 or aPD-1 scfv secretory by GPC-3-targeted CART cells after co-incubation with target cells A431 or HepG2 at a ratio of 1:1. In FIG. 4A, Mock is PBMC cells without genetic modification, 1 is aGPC-3BBZ, 2 is aGPC-3 BB+iaPD-1, 3 is aGPC-3 BB+iaPD-L1, 4 is aGPC-3 BB-PGK+aPD-1, 5 is aGPC-3 BB-P2A+iaPD-1, and 6 is aGPC-3 BB+iIL-2; and in FIG. 4B, Mock is PBMC cells without genetic modification, 1 is aGPC-3BBZ, 2 is aGPC-3 BB+iaPD1, 3 is aGPC-3 BBZ+iaPD-1, 4 is aGPC-3 BB-P2A+aPD-1, 5 is aGPC-3 Z+iaPD-1, 6 is aGPC-3 BB+iIL-2, 7 is aGPC-3 Z+iIL-2, and 8 is positive control nivolumab. 5 and 7 are CD3Z only CAR.

[0059] FIG. 5 shows killing activity of PMSA-targeted CART cells after co-incubation with target cells LNCaP or PC3 at different ratios, herein Mock is PBMC cells without genetic modification.

[0060] FIG. 6 shows killing activity of GPC-3-targeted CART cells after co-incubation with a target cell HepG2 or A431 at different ratios, herein Mock is PBMC cells without genetic modification.

[0061] FIG. 7 is a schematic diagram of CAR.

[0062] FIG. 8 shows the killing activity of CAR-T cells containing only costimulatory signal transduction domain with the first activation signal mediated by tumor-specific BiTE.

[0063] FIG. 9 shows the killing effect of patient derived CART cells expressing aPSMA BBZ towards tumor cells in vitro.

[0064] FIG. 10 shows the activation of NF-κb responsive promoter by TNFα, Phorbol myristate acetate (PMA) and 4-1bbL.

[0065] FIG. 11 shows that CART cells expressing aPSMA BBZ could effectively inhibit the growth of a PSMA-positive solid tumors in mice.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0066] The present disclosure is described in detail herein by reference to the following definitions and embodiments. The contents of all patents and publications mentioned herein, including all sequences disclosed in these patents and publications, are expressly incorporated herein by reference.

[0067] The term “chimeric antigen receptor” or the term “CAR” is an artificially engineered receptor. From extracellular to intracellular, the “CAR” of the present disclosure includes: a) antigen binding domain; b) transmembrane domain; and c) intracellular costimulatory signal transduction domain., “CAR” of the present disclosure includes an artificially introduced costimulatory signal transduction domain, and does not contain an artificially introduced first signal transduction domain. The “chimeric antigen receptor-modified T (CART) cells” or “CAR-modified T cells” of the present disclosure have the following characteristics: the CART cells contain a natural, unmodified first signal transduction domain CD3zeta, and an artificially introduced costimulatory signal transduction domain, wherein the natural, unmodified first signal is provided by the natural TCR-CD3 complex on the surface of the CART cells.

[0068] The term “first signal” refers to T cell activation signal provided by the binding of the TCR-CD3 complex on the surface of the T cells to antigenic peptide-MHC molecules. Different from the first signal of traditional CART cells (the first to the fourth generation) provided by CD3zeta on CAR and TCR on T cells, the “first signal” of the chimeric antigen receptor-modified T cells of the present disclosure is provided by the natural TCR-CD3 complex on the surface of T cells.

[0069] The term “costimulatory signal” refers to the second signal provided by binding of costimulatory molecules (such as CD28) on the surface of T cell to its ligand (such as B7). The “second signal” of the chimeric antigen receptor-modified T cell of the present disclosure is provided by the artificially introduced costimulatory signal transduction domain on CAR.

[0070] The term “signal transduction domain” refers to a functional portion of a protein, and the functional portion exerts the following functions by transmitting information inside the cell: regulating cell activities by generating a second messenger or as an effector in response to such messengers.

[0071] The term “first signal transduction domain” refers to a signal transduction domain that regulates the primary activation of the TCR-CD3 complex in a stimulatory manner or in an inhibitory manner. The primary signal transduction domain that acts in the stimulatory manner may contain a signal transduction motif called immunoreceptor tyrosine-based activation motif (ITAM). Illustrative examples of ITAM containing the first signal transduction domain include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3ε, CD3δ, CD3zeta, CD22, CD79a, CD79b, and CD66d.

[0072] The term “intracellular signal transduction domain of costimulatory protein” may be the intracellular portion of a costimulatory protein, which may contain all the intracellular part or all the natural intracellular signal transduction domain of, or its functional fragment or derivative thereof. “Costimulatory protein” refers to some adhesion molecules on the surface of immune cells (such as T cells). These adhesion molecules bind to their ligands to activate the immune cells (such as T cells), thus enhancing the proliferation and cytokine secretion as well as persistence of the activated immune cells. “costimulatory proteins” include, but are not limited to: MHC class-I molecule, BTLA and Toll ligand receptor, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS(CD278), 4-1BB(CD137), GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1 , ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and ligand that specifically binds to CD83.

[0073] The term “antigen binding domain” may have any structures as long as it binds to targetantigen. The domain may include, for example, variable regions of antibody heavy and light chains (for example, 1 to 6 CDRs); a module about 35 amino acids called domain A, which is contained in Avimer (a cell membrane protein existing in vivo) (WO2004/044011 and WO2005/040229); 10Fn3 domain-containing Adnectin that binds to a protein in glycoprotein fibronectin expressed on the cell membrane (WO2002/032925); Affibody which has an IgG-binding scaffold(a triple-helix of 58 amino acids which constituting the protein A) (WO1995/001937); the designed Ankyrin repeat proteins (DARPins), which are exposed on the molecular surface of an ankyrin repeat (AR), wherein the AR has such a structure with repeat stack of subunits containing aa turn with 33 amino acid residues, two antiparallel helix, and a loop (WO2002/020565); Anticalins and the like, which are tetracyclic regions at one side of a centrally twisted barrel structure of eight antiparallel chains that are highly conserved in supporting molecule neutrophil gelatinase-associated lipocalin (NGAL) (WO2003/029462) and concave regions formed by a parallel sheet structure inside a horseshoe-shaped structure formed by repeated stacking of a leucine-rich repeat (LRR) module of a variable lymphocyte receptor (VLR) (it does not have an immunoglobulin structure and is the system for acquired immunity in a jawless vertebrate such as lampreys and hagfish) (WO2008/016854). As a preferred example of the antigen binding domain of the present disclosure, it may be an antigen binding domain containing variable regions of heavy and light chains of an antibody. As an example of such an antigen binding domain, it may be “scFv (single-chain Fv)”, “single-chain antibody”, “Fv”, “scFv2 (single-chain Fv2)”, “Fab” or “F(ab')2” and the like.

[0074] The term “antibody” is used in the broadest sense as long as it exerts the desired biological activity, wherein it may include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments (including but not limited to Fab, Fab′, F(ab′)2, Fv fragment, scFv fragment, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domain, linear antibodies, single domain antibodies such as sdAb (VL or VH), camelid VHH domains, multispecific antibodiesor isolated CDRs or other epitope binding fragments formed by antibody fragments (for example, a bivalent fragment including two Fab fragments linked by disulfide bonds at the hinge region), multispecific antibodies (multiple specific antibodies) (for example, bispecific antibodies (dual specific antibodies), chimeric antibodies, humanized antibodies and other antibodies.

[0075] “Disease-associated cell surface antigen” of the present disclosure refers to cell surface antigens that are associated with disease treatment and diagnosis, expressed on the cell surface, and may be targeted by CAR. The “disease-associated cell surface antigen” of the present disclosure includes CD antigens, immune checkpoint antigen, tumor-associated antigens, tumor-specific antigens, virus-induced tumor antigens, and may be selected from but not limited to the following antigens: CD19, PD-1, PD-L1, HER2, STAT3, CTLA-4, IDO, NY-ESO-1, CD40, CSF1R, BCMA, MUC1, ADORA2A, CD20, GD2, TLR7, VVT1, IFNAR1, CD47, Neoantigen, EGFR, LAG- 3. OX40, PSMA, Mesothelin, TERT, TLR, TLR9, 4-1BB, IL2R, TLR4, CD33, GITR, HPV E6, Survivin, CD123, TIGITTIM-3, CD73, HPV E7, TLR3, CD38, EBV, STING, CD22, GPC3, HDAC1, CXCR4, GMCSFR, CD30, CEACAM5, HDAC6, HPV, CD3, MAGE-A3, TNF, PSA, CD25, CEA, EPCAM, CMV, IL12, PRAME, IL12R, 5T4, Beta catenin, CCR2, PMEL, CXCL12, IGF1, CD46, CXCR1, GMCSF, IL15R, ROR1, TGFBR2, CCR4, FLT-3, FOLR1, GCSFR, ICOS, JAK2, KRAS, VISTA, CD133, CD27, CD39, CEACAM6, NKG2D, STATS, TGFB1, TLR2, USP7, ANG1, ANG2, B7-H3, CLEC12A, IL13RA2, RIG-I, TRP2, VEGF, AFP, Alpha-Gal, COX-2, EPHA2, gp96, MUC16, p53, TGF-β, CD138, CDw136, CS1, CXCR2, EGFRvIII, Gelactin-3, Globo H, GR, IFNAR2, IFNGR1, IL6, JAK1, MLANA, RAS, SLAMF7, TDO, TGFB2, TLR8, ALK, Arginase, CCR1, CD56, CD70, FAP, GD3, IDH1, IL6R, IRAK4, MAGE-A4, MERTK, MIF, PSCA, PTGER4, SIRPA, TGFB, TGFBR1, ACPP, ADORA2B, AR, Brachyury, CA19-9, CD32, CEACAM1, Gastrin, HDAC, HPV L2, IFNAR, IFNGR, IGF1R, IGF2, IL15, IL17R, IL1B, IL7R, JAK, MAGE-A, MAGE-A1, MAGE-A6, P38, RORC, TLR5, VEGFR2, ADORA3, ATRT, B7-H4, c-KIT, CCR7, CD11b, CD135, CD171, CD174, CDH3, CX3CR1, Gelactin-1, GM3, HLA-A2, HSP70, IL10, IL17, IL2RB, JAK3, MDA5, NKG2A, PBF, PVRIG, SPAM1, URLC10, VEGFR1, ABCB5, ADABP, ADAM17, ADP, AEG1, Alpha-lactalbumin, AMHR2, ASPH, AXL, BCL2, BTE6-LX-8b, BTE6-X-15-7, Carbohydrate antigens, CCL20, CCL3, CCNB1, CD147, CD155, CD16, CD162, CD16a, CD200, CD21, CD28 , CD44, CD52, CD54, CD7, CD80, CD88, Claudin 18, cMET, COX2, CSF1, CTCFL, CXCR5, CXCR7, E1A, EIF2AK3, ERG, FGF2, FN1, GC, GM2, gpA33, HBV, Hemagglutinin, HER3, HILPDA, HLA-DR, HMW-MAA, HP59, HPV 16, HPV E617, HPV L1, HSP105, HSP65, HVEM, Hyaluronan, IL13RA1, IL2, IL21R, IL8, KIF20A, KIR2DL1, KIR2DL3, LXR, MAGE-A10, MAGE-C2, Mammaglobin A, MAPK, MICA, MiHA, MMP-11, MVP, Myeloblastin, N-Myc, NKp46, NLRP3, NR2F6, Oncofetal Antigen, P2RX7, RhoC, SIM-2, SSTR2, SSX2, STAT1, STn, TAG72, TAMA, TFDP3, TGFBR, TSA, TYK2, Tyrosinase, VEGFA, 5′Nucleotidase (Ecto 5′Nucleotidase or CD73 or NT5E or EC 3.1.3.5), ADAM9, AIM2, B7-H6, BAFF-R, BAI1, BARD1, BOB-1, CA9, Cancer testis antigen, CB2, CBLB, CC R9, CD13, CD130, CD150, CD160, CD200R1, CD267, CD29, CD3E, CD4, CD51, CD8, CGEN-XXXX, Claudin 6, CLEC2D, COX, COX-1, CPEB4, CPEG4, CRBN, CRLF2, CSPG4, CTA, CXCL1, CXCR3, Cytosine deaminase, DCK, DKK1, DLL3, DR3, DR5, EBNA3C, EGF, EGFR5, ELVAL4, EPHA3, EPS8, EVI1, FAIM-3, FasR, FCU1, FLT3, FOLR, FOXM1, FSHR, Galectin-3, GaINAc, GARP, Gelactin-9, Gelatcin-1/3/9, GLD18, GNRHR, GP160, GP73, H3.3K27M, HAGE, HDAC2, HDAC8, HPV16E6, HPV16E7, HSP, Hypoxia, ICAM, ICAM7, IDO1, IFNG, IFNGR2, IGF2R, IGFBP2, IGK, IL10RA, IL12RB1, IL13, IL13R, IL13Ralpha2, IL15RA, IL17A, IL17B, IL1A, IL1R1, IL1R3, IL21, IL22R, IL27R, IL2RA, IL35, IL9R, Integrin beta-7, IRAK1, ITGB5, Kappa myeloma antigen, KIR2DL2, Kynurenine, L1CAM, Lambda Myeloma Antigen, LAMP, LLO, LXRA, LXRB, Mas receptor, MG7, MHCI, MHCII, MIC, MOSPD2, MRP-3, MRP1, MRP3765, muGNTP01, MYB, MYBL2, NFAT, NGcGM3, Nrf2, p38MAP Kinase (EC 2.7.11.24), P55, PAM4, PAP, PASD1, PCDH18, PD-L2, PI3K-delta, POTE, PPT, Protein tolemerase, PTGER2, RANKL, RBL001, RNF43, ROR2, S100A9, SEREX, SLAMF1, STAT, TACSTD2, TASTD2, TDO2, TEM, thymidine kinase, Thymidylate synthase, TIE2, TIMP3, TM4SF5, TOP1, TRBC1, TRBC2, TRIF, Tryptophan, TSHR, TWEAK, UTA2-1, VDBP, VRP, VSIG-4, XAGE1, XAGE1A, ZP1, ZP3.

[0076] “Immune function regulatory/modulatory factor” refers to any cytokine or molecule that can regulate the process or outcome of an immune response. The “immune function regulatory factor” includes immune function stimulating factors and immune function inhibiting factors. The “immune function regulatory factor” includes but is not limited to: IL-2, IL-12, IL-7, IL-15, IL-18, IL-21, IL-24, 4-1BBL, PD-1 extracellular region, PD-L1 extracellular region, CCL19, MIP-1a, GM-CSF, IFN-α, IFN-β, IFN-γ, TNF-α, M-CSF, TGF-β and TRAIL and the like.

[0077] “Expression vector” of the present disclosure refers to a vector containing a recombinant polynucleotide which contains an expression control sequence operably linked to a coding nucleotide sequence. The expression vector includes sufficient cis-acting element for expression, and other expression elements may be provided by host cells or in an in vitro expression system. The expression vector includes all those known in the art, including cosmids, plasmids (for example, naked or contained in a liposome) and viruses (for example, lentivirus, retrovirus, adenovirus and adeno-associated virus) that are incorporated with the recombinant polynucleotide.

[0078] The term “lentiviral vector” refers to a vector derived from at least a part of the lentiviral genome, and specifically includes a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of the lentiviral vector that may be used in clinical practice include, but are not limited to Lentivector® gene delivery technology from Oxford BioMedica, and LLENTIMAX vector system from Lentigen. Non-clinical types of lentiviral vectors may also available and may be known to those skilled in the art.

[0079] “Constitutive” expression includes the state in which a gene product is produced in a living cell under most or all physiological conditions of the cell.

EMBODIMENT

[0080] It should be noted that, in the case without conflicting, the embodiments in the present application are merely illustrative, and are not intended to limit the present disclosure in any manner.

Embodiment

Embodiment 1: Construction of Recombinant Lentiviral Vector of Chimeric Antigen Receptor

[0081] Recombinant vectors containing the following sequences are constructed (expression framework from 5-end to 3-end):

[0082] 1) Vector Targeting PSMA:

[0083] EF1a promoter-CD8α signal peptide-aPSMA scfv-CD8 hinge-CD8 transmembrane region-4-1BB (aPSMA BB)

[0084] EF1a promoter-CD8α signal peptide-aPSMA scfv-CD8 hinge-CD8 transmembrane region-4-1BB-CD3Z (aPSMA BBZ)

[0085] 2) Vector Targeting PSMA and Inducibly Secreting IL-12:

[0086] EF1a promoter-CD8α signal peptide-aPSMA scfv-CD8 hinge-CD8 transmembrane region-4-1BB-6XNFAT promoter-IgG kappa signal peptide-IL-12 (P40-35) (aPSMA BB+iIL-12)

[0087] EF1a promoter-CD8α signal peptide-aPSMA scfv-CD8 hinge-CD8 transmembrane region-4-1BB-CD3Z-6XNFAT promoter-IgG kappa signal peptide-IL-12(P40-35) (aPSMA BBZ+iIL-12)

[0088] 3) Vector Targeting PSMA and Inducibly Secreting IL-12:

[0089] EF1a promoter-CD8α signal peptide-aPSMA scfv-CD8 hinge-CD8 transmembrane region-4-1BB-6XNFAT promoter-IgG kappa signal peptide-IL-15 (aPSMA BB+iIL-15)

[0090] EF1a promoter-CD8α signal peptide-aPSMA scfv-CD8 hinge-CD8 transmembrane region-4-1BB-CD3Z-6XNFAT promoter-IgG kappa signal peptide-IL-15 (aPSMA BBZ+iIL-15)

[0091] 4) Vector Targeting PSMA and Inducibly Secreting IL-18:

[0092] EF1a promoter-CD8α signal peptide-aPSMA BB-CD8 hinge-CD8 transmembrane region-4-1BB-6XNFAT promoter-IgG kappa signal peptide-IL-18 (aPSMA BB+iIL-18)

[0093] 5) Vector Targeting PSMA and Inducibly Secreting IL-2:

[0094] EF1a promoter-CD8α signal peptide-aPSMA scfv-CD8 hinge-CD8 transmembrane region-4-1BB-6XNFAT promoter-IgG kappa signal peptide-IL-2 (aPSMA BB+iIL-2)

[0095] 6) Vector Targeting PSMA and Constitutively Secreting IL-12:

[0096] EF1a promoter-CD8a signal peptide-aPSMA scfv-CD8 hinge-CD8 transmembrane region-4-1BB-PGK promoter-IgG kappa signal peptide-IL-12 (P40-35) (aPSMA BB-PGK+IL-12)

[0097] 7) Vector Targeting GPC3:

[0098] EF1a promoter-CD8α signal peptide-aGPC3 scfv-CD8 hinge-CD8 transmembrane region-4-1BB (aGPC-3BB)

[0099] EF1a promoter-CD8α signal peptide-aGPC3 scfv-CD8 hinge-CD8 transmembrane region-4-1BB-CD3Z (aGPC-3BBZ)

[0100] 8) Vector Targeting GPC3 and Inducibly Secreting aPD-1:

[0101] EF1 a promoter-CD8α signal peptide-aGPC3scfv-CD8 hinge-CD8 transmembrane region-4-1BB-6XNFAT promoter-IgG kappa signal peptide-aPD-1 scfv (aGPC-3BB+iaPD-1)

[0102] EF1a promoter-CD8α signal peptide-aGPC3 scfv-CD8 hinge-CD8 transmembrane region-4-1BB-CD3Z-6XNFAT promoter-IgG kappa signal peptide-aPD-1 scfv (aGPC-3BBZ+iaPD-1)

[0103] 9) Vector Targeting GPC3 and Inducibly Secreting aPD-L1:

[0104] EF1a promoter-CD8α signal peptide-aGPC3scfv-CD8 hinge-CD8 transmembrane region-4-1BB-6XNFAT promoter-IgG kappa signal peptide-aPD-L1 scfv (aGPC-3BB+iaPD-L1)

[0105] EF1a promoter-CD8α signal peptide-aGPC3scfv-CD8 hinge-CD8 transmembrane region-4-1BB-CD3Z-6XNFAT promoter-IgG kappa signal peptide-aPD-L1 scfv (aGPC-3BBZ+iaPD-L1)

[0106] 10) Vector Targeting GPC3 and Constitutively Secreting aPD-1:EF1a Promoter-CD8α Signal Peptide-aGPC3scfv-CD8hinge-CD8 Transmembrane Region-4-1BB-PGK Promoter-IgG Kappa Signal Peptide-aPD-1 scfv (aGPC-3BB-PGK+aPD-1)

[0107] 11) Vector Targeting GPC3 and Constitutively Secreting aPD-1:

[0108] EF1a promoter-CD8α signal peptide-aGPC3scfv-CD8 hinge-CD8 transmembrane region-4-1BB-P2A-IgG kappa signal peptide-aPD-1 scfv (aGPC-3BB-P2A+aPD-1)

[0109] 12) Vector Targeting GPC3 and Inducibly Secreting IL-2: EF1a Promoter-CD8α Signal Peptide-aGPC3scfv-CD8 Hinge-CD8 Transmembrane Region-4-1BB-6XNFAT Promoter-IgG Kappa Signal Peptide-iIL-2 (aGPC-3BB+iIL-2)

[0110] 13) Vector Targeting GPC3 and Constitutively Secreting IL-12 (p40-35), and aPD-1:

[0111] EF1a promoter-CD8α signal peptide-aGPC3scfv-CD8 hinge-CD8 transmembrane region-4-1BB-PGK promoter-IgG kappa signal peptide-IL-12(p40-35)-P2A-IgG kappa signal peptide-aPD-1 scfv (aGPC-3BB-PGK+IL-12-P2A+aPD-1)

[0112] 14) Vector Targeting GPC3 and Inducibly Secreting IL-12 (p40-35) and Constitutively Secreting aPD-1:

[0113] EF1 a promoter-CD8α signal peptide-aGPC3scfv-CD8 hinge-CD8 transmembrane region-4-1BB-6XNFAT promoter-IgG kappa signal peptide-IL-12 (p40-35)-P2A-IgG kappa signal peptide-aPD-1 scfv (aGPC-3BB+iIL-12-P2A+aPD-1)

[0114] The above sequences are synthesized and cloned into the lentiviral expression plasmid pLenti, and verified by sequencing. The nucleic acid and amino acid sequences corresponding to each part are shown in the table below.

TABLE-US-00001 Nucleic acid Aminoa cid sequence sequence SEQ ID NO: SEQ ID NO: EF1apromoter   1 / PGK promoter   2 / 6XNFAT promoter   3 / IL-2 promoter   4 / IgG kappasignal peptide   5   6 SP-Kappa 128477   7   8 CD8 hinge region and transmembrane region   9  10 CD3Z signal domain  11  12 4-1BB signal domain  13  14 IL-2  15  16 IL-12(P40-35)  17  18 IL-15  19  20 IL-18  21  22 aGPC3scfv  23  24 aPD-1 scfv  25  26 aPD-L1 scfv  27  28 P2A  29  30 aPSMA scfv  31 / aPSMA BB  32 / aPSMA BBZ  33 / aPSMA BB + ilL-12  34 / aPSMABBZ + ilL-12  35 / aPSMA BB + ilL-15  36 / aPSMABBZ + ilL-15  37 / aPSMA BB + ilL-18  38 / aPSMA BB-ilL-2  39 / aPSMABB-IL-12  40 / aGPC-3BB  41 / aGPC-3BBZ  42 / aGPC-3BB + iaPD-1  43 / aGPC-3BBZ + iaPD-1  44 / aGPC-3 BB + iaPD-L1  45 / aGPC-3BBZ + iaPD-L1  46 / aGPC-3BB-PGK + aPD-1  47 / aGPC-3BB-P2A + aPD-1  48 / aGPC-3BB + ilL-2  49 / GPC-3BB-PGK + IL-12-P2A + aPD-1  50 / aGPC-3BB + ilL-12-P2A + aPD-1  51 / aCD19 scfv  52  53 aCD19BBZ  54  55 aMUC16 scfv  56  57 aFAP scfv  58  59 OX40  60  61 aPD-1 HCDR1 /  62 aPD-1 HCDR2 /  63 aPD-1 HCDR3 /  64 aPD-1 LCDR1 /  65 aPD-1 LCDR2 /  66 aPD-1 LCDR3 /  67 aPD-L1 HCDR1 /  68 aPD-L1 HCDR2 /  69 aPD-L1 HCDR3 /  70 aPD-L1 LCDR1 /  71 aPD-L1 LCDR2 /  72 aPD-L1 LCDR3 /  73 aPSMA HCDR1 /  74 aPSMA HCDR2 /  75 aPSMA HCDR3 /  76 aPSMA LCDR1 /  77 aPSMA LCDR2 /  78 aPSMA LCDR3 /  79 aGPC3 HCDR1 /  80 aGPC3 HCDR2 /  81 aGPC3 HCDR3 /  82 aGPC3 LCDR1 /  83 aGPC3 LCDR2 /  84 aGPC3 LCDR3 /  85 aCD19 HCDR1 /  86 aCD19HCDR2 /  87 aCD19HCDR3 /  88 aCD19HCDR4 /  89 aCD19 LCDR1 /  90 aCD19LCDR2 /  91 aCD19LCDR3 /  92 aMUC16HCDR1 /  93 aMUC16HCDR2 /  94 aMUC16HCDR3 /  95 aMUC16LCDR1 /  96 aMUC16LCDR2 /  97 aMUC16LCDR3 /  98 aFAP HCDR1 /  99 aFAP HCDR2 / 100 aFAP HCDR3 / 101 aFAP LCDR1 / 102 aFAP LCDR2 / 103 aFAP LCDR3 / 104 aGPC3 VH / 105 aGPC3 VL / 106 aPSMA VH / 107 aPSMA VL / 108 aPD-1 VH / 109 aPD-1 VL / 110 aPD-L1 VH / 111 aPD-L1 VL / 112 aCD19 VH / 113 aCD19 VL / 114 aMUC16 VH / 115 aMUC16 VL / 116 aFAP VH / 117 aFAP VL / 118 aEGFR VH / 119 aEGFR VL / 120 aHER2 VH / 121 aHER2 VL / 122 aCD3 VH / 123 aCD3 VL / 124 aFAP scfv-aCD3 / 125 aHER2-aCD3 / 126 aCD3 HCDR1 / 127 aCD3 HCDR2 / 128 aCD3 HCDR3 / 129 aCD3 LCDR1 / 130 aCD3 LCDR2 / 131 aCD3 LCDR3 / 132 aEGFR HCDR1 / 133 aEGFR HCDR2 / 134 aEGFR HCDR3 / 135 aEGFR LCDR1 / 136 aEGFR LCDR2 / 137 aEGFR LCDR3 / 138 aHER2 HCDR1 / 139 aHER2 HCDR2 / 140 aHER2 HCDR3 / 141 aHER2 LCDR1 / 142 aHER2 LCDR2 / 143 aHER2 LCDR3 / 144 CD28 intracellular signaltransduction 145 146 domain LAG3 intracellular signaltransduction 147 148 domain NF-.sub.Kbresponse element.1 149 / NF-.sub.Kbresponse element.2 150 / NF-.sub.Kbresponse element.3 151 / NF-.sub.Kbresponse element.4 152 / NF-.sub.Kbbasal promoter.1 153 / NF-.sub.Kbbasal promoter.2 154 / NF-.sub.Kbbasal promoter.3 155 / NF-.sub.Kb response promoter 156 /

Embodiment 2: Lentiviral Package and Preparation of CAR-T Cells

[0115] The Lentiviral packaging cell line 293T cells were seeded in a 10 cm cell culture dish pre-coated with poly-L-lysine, and transfected with the confluence rate of 70%-80% at 37° C., 5% CO.sub.2. The lentiviral expression plasmid prepared in Embodiment 1 and helper plasmids psPax2 and pMD2.G were co-transfected into 293T cells with transfection reagent PEI in a mass ratio of 4:3:1, and further cultured at 37° C., 5% CO.sub.2. After 8-12 hours, supernatant was removed and fresh medium added. Supernatant was collected after further 48 hours' culture and centrifuged at 4° C., 2000 rpm for 10 minutes to remove cell debris, the supernatant was further filtered with a 0.45 μm sterile filter and placed in a low-temperature refrigerator at −80° C. for storage or directly used for T cell infection.

[0116] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors with lymphocyte separation solution, transferred to a cell culture flask and cultured at 37° C., 5% CO.sub.2 for 1 hour to remove adherent cells. Suspended PBMCs were adjusted to 2×10.sup.6/mL and activated for 2 days with anti-CD3 antibody OKT-3 and anti-CD28 antibody 15E8. After activation, an appropriate number of cells were mixed with fresh or thawed virus solution as mention above as well as with protamine at a final concentration of 8 μ/mL. The mixed cells were subjected to centrifuge at 32° C., 1000×G for 1 hour in a horizontal rotor centrifuge(the centrifuge was set with increase speed of 9 and a decrease speed of 0). After discarding the supernatant, cells were further cultured for 8 hours and transferred to fresh medium containing 300 IU/mL IL-2. 72 hours later, CAR expression on cells was detected by flow cytometry. Results were shown in FIG. 1 and FIG. 2A-2J.

Embodiment 3: In Vitro Killing Activity of PSMA-Targeting CART Cells With Secretory Pro-Inflammatory Factor

3.1 Secretion of IL-2, IFN-γ, IL-12 and IL-18 By PSMA-Targeting CAR-T Cells

[0117] Effector cells (such as PSMA-targeted CART with secretory functions in the embodiment) and target cells (such as PC3, LnCAP or 22RV1 cells) are mixed in a 12-well plate at a ratio of 1:1 (both the cell number of the effector cells and target cells in each well was 1*10.sup.6. After incubating at 37° C., 5% CO.sub.2 for 24 hours, the culture supernatant was collected, and the levels of IL-2, IL-12, IFN-γ and IL-18 in the supernatant were detected with Human IL-2 ELISA MAX™ Standard (BioLegend), Human IL-12 ELISA MAX™ Standard (BioLegend), HUMAN IFN GAMMA ELISA RSG (eBioscience) and human interleukin 18 (IL-18) ELISA kits, respectively.

[0118] Results were shown in FIG. 3A-3D. aPSMA-expressing CART cells could effectively secret IL-2 and IFN-γ when incubated with PSMA-positive target cells LnCAP or 22RV1,while no IL-2 or IFN-γ secretion was detected when incubated with PSMA-negative target cells PC3. The cytokines secretion of the CART cells (cells expressing aPSMA BB+iIL-2, aPSMA BB+iIL-18 or aPSMA BB+IL-12) lacking an artificial first signal CD3Z were similar to that of CART cells (cells expressing aPSMA BBZ, aPSMA BBZ+iIL-12, aPSMA BBZ+iIL-15) containing the artificial first signal CD3Z (FIGS. 3A-3D).

3.2 In Vitro Killing Activity of PSMA-Targeting CAR-T Cells

[0119] The effector cells (such as PSMA-targeting CART cells with secretory cytokines in the embodiment) and target cells (such as PC3 or LnCAP cells) were incubated at 37° C., 5% CO.sub.2 at different ratios (20:1, 10:1, 5:1, 2:1, 1:1, and 0.5:1). After 24 hours, cell supernatant was collected, and the levels of LDH was detected by Cyto Tox 96 Non-Radioactive Cytotoxicity Assay (Promega).

[0120] Results were shown in FIG. 5, CART cells expressing aPSMA BBZ or aPSMA BBZ+iIL-12 could effectively exert killing activity on PSMA-positive LnCAP cells while not on PMSA-negative PC3 cells.

Embodiment 4: In Vitro Killing Activity of GPC3-Targeted CART Cells With Secretory Immune-Checkpoint Inhibitors

4.1 Detection of IL-2 and aPD-1 scfv Secretory By CAR-T Cells

[0121] GPC3-targeting effector cells (such as CART cells) and target cells (such as A431 or HepG2 cells) were incubated at a ratio of 1:1 at 37° C., 5% CO.sub.2 After 24 hours, cell supernatant was collected, and the level of IL-2 in the supernatant was detected with Human IL-2 ELISA MAX™ Standard (BioLegend).

[0122] The aPD-1 scfv expression was detected by sandwich ELISA: hPD-1 was coated on a 96-well plate, and incubated at 4° C. overnight. After blocking with PBST (0.5% Tween-20 in phosphate buffered saline (PBS)) containing 2% fetal bovine serum (FBS) at room temperature for 1 hour, the cell supernatant above was added, and then incubated at room temperature for 2 h. After being washed for 4-5 times with PBST containing 2% FBS, goat anti-Human IgG(Fab′)2(HRP) secondary antibody was added and incubated at room temperature for 1 h, followed by washing with PBST containing 2% nonfat dry milk for 4-5 times. Reading at OD450 nm were recorded after TMB reagent (BioLegend) was added for color development.

[0123] Results were shown in FIG. 4. The level of IL-2 secretion by CART cells lacking an artificial first signal CD3Z (aGPC-3BB+iaPD1, aGPC-3BB+iaPD-L1, aGPC-3BB-PGK+aPD-1, aGPC-3BB-P2A+iaPD-1, and aGPC-3BB+iIL-2) was similar to that of IL-2 secretory by CART cells containing the artificial first signal CD3Z when incubating with GPC-3 positive target cells HepG2. CART cells lacking the artificial first signal CD3Z while constitutively expressing aPD-1 scfv showed significantly higher level of aPD-1 scfv expression (aGPC-3BB-PGK+aPD-1), compared to CART constructs with inducible a PD-1 scfv secretion (aGPC-3BB+iaPD1, aGPC-3BBZ+iaPD-1, and aGPC-3Z+iaPD-1).

4.2 In Vitro Killing Activity of GPC-3-Targeting CAR-T Cells

[0124] Effector cells (such as GPC-3-targeting CART cells in the embodiment) and target cells (such as HepG2 or A431 cells) were co-incubated at 37° C., 5% CO.sub.2at different ratios (5:1, 3:1, 1:1, 1:3, and 1:5). After 24 hours, cell supernatant was collected, and the LDH level was detected with CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega).

[0125] Results were shown in FIG. 6, CART cells expressing aGPC-3 BBZ or aGPC-3 BBZ+iaPD-1 could effectively exert killing activity on GPC-3 positive HepG2 cells while not on GPC-3 negative A431 cells.

Embodiment 5: In Vitro Killing Activity of PSMA-Targeting CART Cells

[0126] aPSMA BBZ CAR-T cells were incubated with two target cells (LNCaP and PC3) at a ratio of 1:1 (10.sup.4 cells each per well) in a 96-well plate(flat bottom), respectively. Bispecific antibody aPSMA-aCD3 BiTE pre-diluted in a two-fold gradient and PBS (as solvent control)were added, and incubated in a cell incubator (37° C., 5% CO.sub.2) for 16 hours. CytoTox 96° Non-Radioactive Cytotoxicity Assay (Promega™) kit was used to detect the killing activity of CAR-T cells on target cells. As shown in FIG. 8, PSMA-specific CAR-T cells containing only costimulatory signal transduction domain showed specific killing activity on PSMA-positive tumor target cells LnCaP while not on PSMA-negative tumor target cells PC3 in the presence of first activation signal mediated by aPSMA-aCD3 BiTE molecules.

[0127] CAR-T effector cells derived from patient's PBMCs and target cells (such as tumor cells, PC3 cells or LnCAP cells) were mixed at different ratios (1:10, 1:2, 1:1), herein the number of the target cells was 10.sup.4 cells/well. After incubation at 37° C., 5% CO.sub.2 for 24 hours, cell culture supernatant was collected, and LDH level was detected by CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) kit. Results were shown in FIG. 9, patient-derived CART cells expressing aPSMA BBZ could specifically and effectively exert killing activity on PSMA-positive tumor cells in vitro. In contrast, activated T cells without CAR transfection exhibited no killing activity.

Embodiment 6: NF-κb Responsive Promoter Induced By Costimulatory Signal Activation

[0128] Firefly luciferase reporter gene elements driven by NF-κb responsive promoter (from N-terminal to C-terminal: NF-κb responsive promoter-firefly luciferase reporter gene-PGK promoter-green fluorescent protein) were introduced into PBMC cells by lentiviral transduction. The prepared PBMC cells were plated in a 96-well plate at a density of 4×10.sup.4 cells/well, and stimulants (TNFα, Phorbol myristate acetate, 4-1bbL) pre-diluted in a two-fold gradient and PBS (as solvent control) were further added. After incubation (37° C., 5% CO.sub.2) for 6 hours, fluorescence in well was detected by Firefly Luc One-Step Glow Assay Kit (Pierce™), and the induction of luciferase expression were calculated with the solvent control as the base line.

[0129] As shown in FIG. 10, TNFα, Phorbol myristate acetate (PMA) and 4-1bbL could all activate the NF-κb responsive promoter in PBMC cells and drive the expression of downstream genes.

Embodiment 7: In Vivo Study With Mouse Model

[0130] Efficacy studies of PSMA-targeted CAR were performed in 6-week-old male NCG mice. 2×10.sup.6LnCap cells were subcutaneously(SC) injected into the right flank of mice, and the size of tumor was measured with calipers three times a week. When the volume of the tumor reached 100-200 mm.sup.3, CART cells were intravenously injected into the tail (Day 0) (The experiments were divided into two groups: activated T cell injection group, and 5×10.sup.6 CART cell injection group). Tumor size and mouse body weight were measured weekly. The tumor volume was calculated by the following formula: tumor volume=width length*height/2. Five weeks after CART injection, the mice are sacrificed, serum or plasma was collected for cytokine or immune factor analysis and mouse tissues were collected. CART cell population in TILs and PBMCs were analyzed by flow cytometry and immunohistochemistry. For CARTs with secretory function, the levels of the secretory proteins in tumor and blood were further analyzed.

[0131] As were shown in FIG. 11, CART cells expressing aPSMA BBZ exhibited efficient killing activity to PSMA-positive solid tumors in mice, and the activated T cells without CAR transfection at a dose up to 5×10.sup.7showed no killing activity.