BCMA-targeted engineered immune cells and uses thereof

Abstract

To provide an engineered immune cell targeting BCMA, and uses thereof.SOLUTION: The present invention provides a chimeric antigen receptor (CAR) specifically targeting BCMA, the CAR comprising an antigen-binding domain which is an S-derived scFv and has an antibody heavy chain variable region represented by a specific sequence and an antibody light chain variable region represented by another specific sequence. The present invention also provides a CAR-T cell expressing the CAR, a dual CAR-T cell comprising the S-derived scFv, and related uses thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001]

[0001] The present invention relates to the field of immunotherapy, and more specifically to engineered immune cells that target BCMA and uses thereof. [Background technology]

[0002]

[0002] Multiple myeloma (MM) is a malignant plasma cell neoplasm. The tumor cells originate from plasma cells in the bone marrow, which are cells that develop into the final functional stage of B lymphocytes. Multiple myeloma is an essentially incurable disease characterized by high morbidity and mortality. According to 2017 statistics, there were 30,000 newly diagnosed multiple myeloma patients in the United States, of whom 12,000 will face death. Currently, common treatments for multiple myeloma include cytotoxic drug therapy, protease inhibitors (e.g., bortezomib), lenalidomide, monoclonal antibodies, and corticosteroids. However, all current treatments are partially effective, do not provide sustained relief, and have a high risk of relapse. Therefore, improved treatments for multiple myeloma are considered particularly important.

[0003]

[0003] Therefore, there is a pressing need in the art for an effective, low relapse, and safe treatment for multiple myeloma. Summary of the Invention [Problem to be solved by the invention]

[0004]

[0004] An object of the present invention is to provide engineered immune cells that target BCMA and uses thereof.

[0005] Another object of the present invention is to provide engineered immune cells that target both CD19 and BCMA and uses thereof. [Means for solving the problem]

[0005]

[0006] In a first aspect of the present invention, a chimeric antigen receptor (CAR) or TCR is provided, wherein the antigen-binding domain (scFv) of the CAR or TCR comprises the antibody heavy chain variable region shown in SEQ ID NO: 9 and the antibody light chain variable region shown in SEQ ID NO: 10.

[0006]

[0007] In another preferred embodiment, the scFv further comprises a linker peptide positioned between the heavy chain variable region and the light chain variable region.

[0008] In another preferred embodiment, the scFv has the formula A or B: V H -V L , (A); V L -V H , (B) (In the formula, V H is an antibody heavy chain variable region; V L is an antibody light chain variable region; "-" is a linker peptide or peptide bond) As shown in

[0007]

[0009] In another preferred embodiment, V H And, V L The linker peptide between is 1 to 4, preferably 1 to 4, more preferably 3 to 4 consecutive sequences as shown in SEQ ID NO: 7 (GGGGS).

[0008]

[0010] In another preferred embodiment, the CAR has the following formula I: L-scFv-H-TM-C-CD3ζ (I) (In the formula, each "-" is independently a linker peptide or a peptide bond; L is absent or is a signal peptide sequence; H is absent or is the hinge region; TM is the transmembrane domain; C is a costimulatory signal molecule, CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ) It has the structure shown below.

[0009]

[0011] In a second aspect of the invention there is provided a bispecific CAR or TCR, wherein the bispecific CAR or TCR targets BCMA and a first target, the BCMA-targeting antigen-binding domain (scFv) in the bispecific CAR comprises an antibody heavy chain variable region set forth in SEQ ID NO: 9 and an antibody light chain variable region set forth in SEQ ID NO: 10; The first target is CD138, kappa light chain, NKG2D-ligand, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII , ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GH R, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, claudin 18.2, folate receptor α, folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or a combination thereof In another preferred embodiment, the bispecific CAR or TCR comprises an antigen-binding domain that targets CD19.

[0010]

[0012] In another preferred embodiment, the first target is CD19, and the CD19-targeting antigen-binding domain (scFv) in the bispecific CAR comprises the antibody heavy chain variable region set forth in SEQ ID NO:11 and the antibody light chain variable region set forth in SEQ ID NO:12.

[0011]

[0013] In another preferred embodiment, the first target is CD19, and the antigen-binding domain (scFv) targeting CD19 in the bispecific CAR comprises an antibody heavy chain variable region set forth in any one of SEQ ID NOs: 21 to 30 and an antibody light chain variable region set forth in any one of SEQ ID NOs: 31 to 36.

[0012]

[0014] The specific sequences are as follows: CD19 antibody heavy chain variable region (H9) shown in SEQ ID NO: 21 QVQLQESGPGLVKPSETLSLTTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDTSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS; CD19 antibody heavy chain variable region (H1) shown in SEQ ID NO: 22 QVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARHYYYGGSYAMDYWGQGTTVTVSS; CD19 antibody heavy chain variable region (H8) shown in SEQ ID NO: 23 QVKLQESGPGLVKPSETLSLTTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRVTISKDTSKSQVFLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS; CD19 antibody heavy chain variable region (H10) shown in SEQ ID NO: 24 QVKLQESGPGLVKPSETLSLTTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDTSKSQVFLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS; CD19 antibody heavy chain variable region (H2) shown in SEQ ID NO: 25 QVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTTVTVSS; CD19 antibody heavy chain variable region (H3) shown in SEQ ID NO: 26 QVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRLTISKDTSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTTVTVSS; CD19 antibody heavy chain variable region (H4) shown in SEQ ID NO: 27 QVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDTSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTTVTVSS; CD19 antibody heavy chain variable region (H5) shown in SEQ ID NO: 28 QVQLQESGPGLVKPSQTLSVTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDTSKNQVSLKLSSLTAADTAVYYCAKHYYYYGGSYAMDYWGQGTTVTVSS; CD19 antibody heavy chain variable region (H6) shown in SEQ ID NO: 29 QVQLQESGPGLVKPSQTLSVTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNAALKSRLTISKDTSKNQVSLKLSSLTAADTAVYYCAKHYYYGGSYAMDYWGQGTTVTVSS; CD19 antibody heavy chain variable region (H7) shown in SEQ ID NO: 30 QVKLQESGPGLVKPSETLSLTTCTVSGVSLPDYGVSWIRQPPGKGLEWLGVIWGSETTYYNSALKSRLTISKDTSKNQFSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS; CD19 antibody light chain variable region (L5) shown in SEQ ID NO: 31 DIQMTQSPSSLSASVGDRVTISCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPYTFGGGTKLEIK; CD19 antibody light chain variable region (L1) shown in SEQ ID NO: 32 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGGGTKLEIK; CD19 antibody light chain variable region (L6) shown in SEQ ID NO: 33 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGGAVKLLIYHTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPYTFGGGTKLEIK; CD19 antibody light chain variable region (L2) shown in SEQ ID NO: 34 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPYTFGGGTKLEIK; CD19 antibody light chain variable region (L3) shown in SEQ ID NO: 35 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQGNTLPYTFGGGTKLEIK CD19 antibody light chain variable region (L4) shown in SEQ ID NO: 36 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQANTLPYTFGGGTKLEIK

[0015] In another preferred embodiment, the bispecific CAR comprises both an antigen-binding domain that targets a first target and an antigen-binding domain that targets BCMA.

[0013]

[0016] In another preferred embodiment, the bispecific CAR has the following formula II: L-scFv1-I-scFv2-H-TM-C-CD3ζ (II) (In the formula, each "-" is independently a linker peptide or a peptide bond; L is absent or is a signal peptide sequence; I is a flexible linker; H is absent or is the hinge region; TM is the transmembrane domain; C is a costimulatory signal molecule, CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; One of scFv1 and scFv2 is an antigen-binding domain that targets a first target, and the other is an antigen-binding domain that targets BCMA. It has a structure as shown below.

[0014]

[0017] In another preferred embodiment, scFv1 and scFv2 may be in tandem or in a leap structure, independently of each other.

[0018] In another preferred embodiment, scFv1 is an antigen-binding domain that targets a first target and scFv2 is an antigen-binding domain that targets BCMA.

[0015]

[0019] In another preferred embodiment, scFv1 is an antigen-binding domain that targets BCMA and scFv2 is an antigen-binding domain that targets a first target.

[0020] In another preferred embodiment, the sequence of flexible linker I is 1 to 6, preferably 3 to 5, consecutive sequences such as those shown in SEQ ID NO: 7 (GGGGS).

[0016]

[0021] In another preferred embodiment, flexible linker I has the sequence shown in SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

[0022] In another preferred embodiment, the antigen-binding domain that targets the first target has a structure as shown in Formula C or Formula D below:

[0023] V L1 -V H1 (C); V H1 -V L1 (D)

[0024] (In the formula, V L1 is an antibody light chain variable region against the first target; V H1 is the antibody heavy chain variable region against the first target; "-" indicates a linker peptide or peptide bond. (This is the case.)

[0017]

[0025] In another preferred embodiment, the antigen-binding domain targeting CD19 has a structure as shown in Formula C or Formula D below:

[0026] V L1 -V H1 (C); V H1 -V L1 (D)

[0027] (In the formula, V L1 is the antibody light chain variable region against CD19; V H1 is the antibody heavy chain variable region against CD19; "-" is a linker peptide or peptide bond).

[0018]

[0028] In another preferred embodiment, the antigen-binding domain targeting CD19 comprises the heavy and light chain variable regions of the monoclonal antibody FMC63.

[0029] In another preferred embodiment, the heavy chain variable region of the anti-CD19 antibody has the amino acid sequence set forth in SEQ ID NO:11.

[0019]

[0030] In another preferred embodiment, the light chain variable region of the anti-CD19 antibody has the amino acid sequence set forth in SEQ ID NO:12.

[0031] In another preferred embodiment, the BCMA-targeting antigen-binding domain has a structure as shown in Formula A or Formula B below: V H -V L , (A); V L -V H , (B) (In the formula, V H is an antibody heavy chain variable region; V L is an antibody light chain variable region; "-" is a linker peptide or peptide bond).

[0020]

[0032] In another preferred embodiment, scFv1 comprises the antibody heavy chain variable region set forth in SEQ ID NO: 11 and the antibody light chain variable region set forth in SEQ ID NO: 12, and scFv2 comprises the antibody heavy chain variable region set forth in SEQ ID NO: 9 and the antibody light chain variable region set forth in SEQ ID NO: 10.

[0021]

[0033] In another preferred embodiment, scFv1 comprises the antibody heavy chain variable region set forth in SEQ ID NO: 9 and the antibody light chain variable region set forth in SEQ ID NO: 10, and scFv2 comprises the antibody heavy chain variable region set forth in SEQ ID NO: 11 and the antibody light chain variable region set forth in SEQ ID NO: 12.

[0022]

[0034] In another preferred embodiment, the scFv1 and / or scFv is a murine, human, chimeric human and murine, or fully humanized single chain antibody variable region fragment.

[0035] In another preferred embodiment, the bispecific CAR has the following formula III or III':LV L3 -scFv3-V H3 -H-TM-C-CD3ζ (III) LV H3 -scFv3-V L3 -H1-TM-C-CD3ζ (III') (In the formula, each "-" is independently a linker peptide or a peptide bond; The elements L, H, TM, C, and CDζ are as described above; scFv3 is the antigen-binding domain that targets BCMA, and V H3 is the antibody heavy chain variable region against the first target, and V L3 is an antibody light chain variable region against a first target; or scFv3 is the antigen-binding domain that targets the first target, V H3 is the antibody heavy chain variable region against BCMA, and V L3 is the antibody light chain variable region against BCMA).

[0023]

[0036] In another preferred embodiment, the scFv3 comprises the antibody heavy chain variable region set forth in SEQ ID NO:9 and the antibody light chain variable region set forth in SEQ ID NO:10.

[0037] In another preferred embodiment, V H3 has an antibody heavy chain variable region set forth in SEQ ID NO: 9, and V L3 has the antibody light chain variable region shown in SEQ ID NO:10.

[0024]

[0038] In another preferred embodiment, the scFv3 comprises the antibody heavy chain variable region set forth in SEQ ID NO: 11 and the antibody light chain variable region set forth in SEQ ID NO: 12, and H3 has an antibody heavy chain variable region set forth in SEQ ID NO: 9, and V L3 has the antibody light chain variable region shown in SEQ ID NO:10.

[0025]

[0039] In another preferred embodiment, the scFv3 comprises the antibody heavy chain variable region set forth in SEQ ID NO: 9 and the antibody light chain variable region set forth in SEQ ID NO: 10, and H3 has an antibody heavy chain variable region set forth in SEQ ID NO: 11, and V L3 has the antibody light chain variable region shown in SEQ ID NO:12.

[0026]

[0040] In another preferred embodiment, the CAR has the structure shown in FIG.

[0041] In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0027]

[0042] In another preferred embodiment, L is a signal peptide derived from CD8.

[0043] In another preferred embodiment, L has the amino acid sequence shown in SEQ ID NO:16 or SEQ ID NO:1.

[0028]

[0044] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof. In another preferred embodiment, H is each independently a hinge region from CD8.

[0029]

[0045] In another preferred embodiment, H has the amino acid sequence set forth in SEQ ID NO:8.

[0046] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof. In another preferred embodiment, the TM is each independently a transmembrane region derived from CD8 or CD28. In another preferred embodiment, the transmembrane region derived from CD8 has the amino acid sequence set forth in SEQ ID NO:7.

[0030]

[0047] In another preferred embodiment, the transmembrane region from CD28 has the amino acid sequence shown in SEQ ID NO:6.

[0048] In another preferred embodiment, C is a costimulatory signal molecule of a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof. In another preferred embodiment, C is a costimulatory signal molecule derived from CD28 and / or 4-1BB.

[0031]

[0049] In another preferred embodiment, the 4-1BB-derived costimulatory signal molecule has the amino acid sequence shown in SEQ ID NO:5.

[0050] In another preferred embodiment, the CD28-derived costimulatory signal molecule has the amino acid sequence shown in SEQ ID NO:4.

[0032]

[0051] In another preferred embodiment, CD3ζ has the amino acid sequence shown in SEQ ID NO:3.

[0052] In another preferred embodiment, the CAR (preferably at the C-terminus or N-terminus) is It also contains a suicide element.

[0033]

[0053] In another preferred embodiment, the cell suicide element is linked to L or CD3ζ of the CAR or bispecific CAR via T2A.

[0054] In a third aspect of the invention, there is provided a nucleic acid molecule, which encodes a CAR or TCR according to the first aspect of the invention or a bispecific CAR or TCR according to the second aspect of the invention.

[0034]

[0055] In a fourth aspect of the invention, there is provided a vector, the vector comprising a nucleic acid molecule according to the third aspect of the invention.

[0056] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, a transposon, or a combination thereof.

[0035]

[0057] In another preferred embodiment, the vector is a lentiviral vector.

[0058] In a fifth aspect of the invention, there is provided a host cell, which comprises a vector according to the fourth aspect of the invention exogenously integrated into a chromosome, or a nucleic acid molecule according to the third aspect of the invention, or which expresses a CAR or TCR according to the first aspect of the invention or a bispecific CAR or TCR according to the second aspect of the invention.

[0036]

[0059] In a sixth aspect of the invention, there is provided an engineered immune cell, which comprises a vector according to the fourth aspect of the invention exogenously integrated into a chromosome, or a nucleic acid molecule according to the third aspect of the invention, or which expresses a CAR or TCR according to the first aspect of the invention or a bispecific CAR or TCR according to the second aspect of the invention.

[0037]

[0060] In another preferred embodiment, the immune cells are (a) PD-1 gene expression in immune cells is silenced; (b) the immune cells are T cells, and TCR gene expression in the T cells is silenced; (c) immune cells express exogenous cell suicide elements; (d) the immune cells express or secrete a PD-1 antibody, a PD-L1 antibody, a CD47 antibody, a Tim3 antibody, a Lag3 antibody, a Tigit antibody, an OX40 antibody, an ICOS antibody, IL7, CXCL19, IL21, IL15, IL2, IL18, or a combination thereof; and (e) a cytokine-related signaling pathway in an immune cell is enhanced, and the cytokine is selected from the group consisting of IL7, CXCL19, IL21, IL15, IL2, IL18, or a combination thereof. The present invention has one or more characteristics selected from the group consisting of:

[0038]

[0061] In another preferred embodiment, the engineered immune cells are (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) Chimeric antigen receptor NK cells (CAR-NK cells) is selected from the group consisting of:

[0039]

[0062] In another preferred embodiment, the immune cells express an exogenous cell suicide element.

[0063] In another preferred embodiment, the CAR and the cell suicide element are co-expressed in the immune cell.

[0040]

[0064] In another preferred embodiment, the CAR and the cell suicide element are linked by a self-cleaving element.

[0065] In another preferred embodiment, the cell suicide element is located at the N-terminus or C-terminus of the CAR.

[0041]

[0066] In another preferred embodiment, the self-cleaving element comprises a 2A sequence or an IRES sequence, preferably P2A and T2A.

[0067] In another preferred embodiment, the cell suicide element is selected from the group consisting of HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or a combination thereof.

[0042]

[0068] In another preferred embodiment, the cell suicide element has the following formula IV: L2-DF (IV) (In the formula, each "-" is independently a linker peptide or a peptide bond; L2 is an optional signal peptide sequence; D is the suicide switch element; F is a transmembrane element) It has a structure as shown below.

[0043]

[0069] In another preferred embodiment, the signal peptide is a signal peptide derived from GM-CSFR.

[0070] In another preferred embodiment, the cell suicide element is selected from the group consisting of a truncated epidermal growth factor receptor (EGFRt), a truncated CD19 (CD19t) gene, an inducible caspase 9 gene (iCasp9), HSV-TK, ΔCD20, mTMPK, or a combination thereof.

[0044]

[0071] In another preferred embodiment, the cell suicide element is EGFRt.

[0072] In another preferred embodiment, the engineered immune cells are used in autoimmune therapy and / or allogeneic immunotherapy.

[0045]

[0073] In another preferred embodiment, the engineered immune cells are capable of killing tumor cells capable of clonal expansion.

[0074] In another preferred embodiment, immune cells expressing a bispecific CAR according to the second aspect of the invention survive longer in vivo compared to immune cells expressing a CAR according to the first aspect of the invention.

[0046]

[0075] In another preferred embodiment, in vivo includes autologous in vivo or allogeneic in vivo.

[0076] In a seventh aspect of the present invention there is provided an engineered immune cell comprising: the immune cells comprise a first expression cassette and a second expression cassette that are exogenous, wherein the first expression cassette is used to express a first CAR or a first exogenous TCR that targets a first target, and the second expression cassette is used to express a second CAR or a second exogenous TCR that targets BCMA; or the immune cell expresses a first CAR or a first exogenous TCR that targets a first target and a second CAR or a second exogenous TCR that targets BCMA; the BCMA-targeting antigen-binding domain (scFv) of the second CAR or second exogenous TCR comprises the antibody heavy chain variable region set forth in SEQ ID NO: 9 and the antibody light chain variable region set forth in SEQ ID NO: 10; The first target is CD138, kappa light chain, NKG2D-ligand, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, C D28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD 81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMS A, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GI TR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, claudin 18.2, folate receptor α, folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or a combination thereof is selected from the group consisting of:

[0047]

[0077] In another preferred embodiment, the first target is CD19, and the antigen-binding domain (scFv) targeting CD19 in the first CAR comprises the antibody heavy chain variable region set forth in SEQ ID NO: 11 and the antibody light chain variable region set forth in SEQ ID NO: 12.

[0048]

[0078] In another preferred embodiment, the second CAR is a CAR according to the first aspect of the invention.

[0079] In another preferred embodiment, the first CAR and the second CAR are located on the cell membrane of an immune cell.

[0049]

[0080] In another preferred embodiment, a first CAR that targets CD19 and a second CAR that targets BCMA are expressed on the cell membrane of an immune cell.

[0081] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same vector or on different vectors.

[0050]

[0082] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same vector.

[0083] In another preferred embodiment, the first CAR has the following formula V: L-scFv1'-H-TM-C-CD3ζ (V) (In the formula, each "-" is independently a linker peptide or a peptide bond; The elements L, H, TM, C, and CDζ are as described above; scFv1' is the antigen-binding domain that targets CD19) It has the structure shown below.

[0051]

[0084] In another preferred embodiment, the first CAR and the second CAR are linked via a 2A peptide.

[0085] In another preferred embodiment, the 2A peptide has the sequence shown in SEQ ID NO:2.

[0052]

[0086] In another preferred embodiment, the immune cells further comprise a cell suicide element.

[0087] In another preferred embodiment, the cell suicide element and the bispecific CAR are linked (or present in tandem) via T2A.

[0053]

[0088] In another preferred embodiment, the cell suicide element is attached to the first CAR and / or the second CAR via T2A.

[0089] In another preferred embodiment, PD1 gene expression in immune cells is silenced.

[0054]

[0090] In another preferred embodiment, "PD-1 gene expression is silenced" means that the PD-1 gene is not expressed or is under-expressed.

[0055]

[0091] In another preferred embodiment, "low expression" refers to a ratio of the expression level G1 of the PD-1 gene in immune cells to the expression level G0 of the PD-1 gene in normal immune cells (i.e., G1 / G0) of 0.5 or less, preferably a G1 / G0 ratio of 0.3 or less, more preferably 0.2 or less, more preferably 0.1 or less, and most preferably 0.

[0056]

[0092] In another preferred embodiment, "low expression" refers to a ratio of the expression level G1 of the PD-1 gene in CAR-T cells to the expression level G0 of the PD-1 gene in normal T cells (i.e., G1 / G0) of 0.5 or less, preferably a G1 / G0 of 0.3 or less, more preferably 0.2, more preferably 0.1, and most preferably 0.

[0057]

[0093] In an eighth aspect of the invention there is provided a formulation comprising a CAR or TCR according to the first or second aspect of the invention, or an engineered immune cell according to the sixth or seventh aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0058]

[0094] In another preferred embodiment, the formulation is a liquid formulation.

[0095] In another preferred embodiment, the dosage form of the formulation is an injection.

[0096] In another preferred embodiment, the engineered immune cells in the formulation comprise 1 x 10 3 pieces~1×10 8 cells / ml, preferably 1 x 10 4 pieces~1×10 7 The concentration is 100 cells / ml.

[0059]

[0097] In another preferred embodiment, the CAR comprises a bispecific CAR.

[0098] In a ninth aspect of the invention there is provided the use of a CAR or TCR according to the first or second aspect of the invention, or an engineered immune cell according to the sixth or seventh aspect of the invention, in the preparation of a medicament or formulation for preventing and / or treating cancer or tumours.

[0060]

[0099] In another preferred embodiment, the tumor is a hematological tumor.

[0100] In another preferred embodiment, the hematological malignancy is acute myeloid leukemia (AML), multiple myeloid leukemia (MMLL), or multiple myeloid leukemia (MMLL). The cancer is selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0061]

[0101] In another preferred embodiment, the cancer or tumor is multiple myeloma.

[0102] In another preferred embodiment, the cancer or tumor is a lymphoma.

[0103] In another preferred embodiment, the lymphoma is Hodgkin's lymphoma (HL), diffuse large intestinal lymphoma (LV) or diffuse large intestinal lymphoma (LV). The cancer is selected from the group consisting of: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Burkitt's lymphoma (BL), and combined B-cell non-Hodgkin's lymphoma.

[0062]

[0104] In another preferred embodiment, the cancer or tumor is a recurrent cancer or tumor.

[0105] In another preferred embodiment, the drug or pharmaceutical agent inhibits tumor cells capable of clonal proliferation. The cancer or tumor is treated by killing the

[0063]

[0106] In another preferred embodiment, the tumor cells capable of clonal proliferation are clone-forming cells, tumor cell precursor cells, and tumor progenitor cells.

[0107] In a tenth aspect of the present invention, a method for producing a nucleic acid molecule according to the third aspect of the present invention comprising the steps of: There is provided a method for preparing an engineered immune cell, comprising the step of transducing an immune cell with a vector according to the fourth aspect of the invention, thereby obtaining an engineered immune cell, wherein the engineered immune cell expresses a CAR or TCR according to the first or second aspect of the invention.

[0064]

[0108] In another preferred embodiment, the immune cell is a T cell or an NK cell.

[0109] In an eleventh aspect of the present invention, a method for producing a medicament for the preparation of ... (1) providing immune cells to be engineered; (2) introducing into the immune cells a first expression cassette for expressing a first CAR that targets a first target; (3) introducing into the immune cells a second expression cassette for expressing a second CAR that targets BCMA, thereby obtaining engineered immune cells. and (c) preparing an engineered immune cell comprising: wherein the BCMA-targeting antigen-binding domain (scFv) of the second CAR comprises an antibody heavy chain variable region set forth in SEQ ID NO: 9 and an antibody light chain variable region set forth in SEQ ID NO: 10; The first target may be CD138, kappa light chain, NKG2D-ligand, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRH R, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, claudin 18.2, folate receptor α, folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or a combination thereof is selected from the group consisting of:

[0065]

[0110] In another preferred embodiment, step (2) is performed before step (3) or after step (3). , may be carried out simultaneously with step (3) or alternately with step (3).

[0111] In another preferred embodiment, the immune cells engineered in step (1) are If R or a second CAR is expressed, step (2) or step (3) may be omitted.

[0066]

[0112] In a twelfth aspect of the present invention, a kit is provided, the kit comprising the sixth or sixth aspect of the present invention. is used to prepare an engineered immune cell according to the seventh aspect, the kit comprising a container and a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention located in the container.

[0067]

[0113] In a thirteenth aspect of the present invention, a kit is provided, the kit comprising the sixth or sixth aspect of the present invention. is used to prepare engineered immune cells according to the seventh aspect, the kit comprising a container and, located in the container: (1) a first nucleic acid sequence, the first nucleic acid sequence comprising a first expression cassette, the first expression cassette being used to express a first CAR that targets a first target; (2) a second nucleic acid sequence, the second nucleic acid sequence comprising a second expression cassette, the second expression cassette being used to express a second CAR that targets BCMA; and wherein the BCMA-targeting antigen-binding domain (scFv) of the second CAR comprises an antibody heavy chain variable region set forth in SEQ ID NO: 9 and an antibody light chain variable region set forth in SEQ ID NO: 10; The first target may be CD138, kappa light chain, NKG2D-ligand, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRH R, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, claudin 18.2, folate receptor α, folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or a combination thereof is selected from the group consisting of:

[0068]

[0114] In another preferred embodiment, the first nucleic acid sequence and the second nucleic acid sequence are contained in the same container. or located in different containers.

[0115] In another preferred embodiment, the first nucleic acid sequence and the second nucleic acid sequence are expressed in the same manner. Located in the vector.

[0069]

[0116] In a fourteenth aspect of the present invention, there is provided a method for the prevention and / or treatment of cancer or tumors, comprising administering to said patient a therapeutically effective amount of the present invention. There is provided a use of the engineered immune cells according to the sixth or seventh aspect of the invention.

[0117] In another preferred embodiment, the cancer or tumor is multiple myeloma.

[0070]

[0118] In a fifteenth aspect of the present invention, there is provided a method of treating a disease, the method comprising administering to a patient a suitable The method comprises administering to a subject in need of treatment an amount of cells according to the sixth or seventh aspect of the invention, or a formulation according to the fifth aspect of the invention.

[0071]

[0119] In another preferred embodiment, the disease is cancer or a tumor.

[0120] In a sixteenth aspect of the present invention, there is provided a method for producing a medicament for the treatment of a leukemia, comprising: (a) a first expression that is exogenous in an immune cell; and a second expression cassette, wherein the first expression cassette is used to express a first CAR that targets CD19 and the second expression cassette is used to express a second CAR that targets BCMA; or (b) expressing in the immune cell a bispecific CAR according to the second aspect. The present invention provides methods for enhancing the in vivo survival of immune cells against tumor cells capable of clonal expansion, or enhancing the killing ability of immune cells against tumor cells capable of clonal expansion.

[0072]

[0121] In another preferred embodiment, the immune cells constructed by the above method are As described in the sixth and seventh aspects of the present invention.

[0122] In another preferred embodiment, the first expression cassette and the second expression cassette are The first expression cassette and the second expression cassette have the same meaning as those in the seventh aspect of the present invention.

[0073]

[0123] In another preferred embodiment, in vivo refers to autologous in vivo, or includes allogeneic in vivo.

[0124] In a seventeenth aspect of the present invention, an exogenous first expression in an engineered immune cell

[0010] Methods are provided for enhancing the in vivo survival or killing ability of BCMA-targeting engineered immune cells against tumor cells capable of clonal expansion, comprising expressing a first expression cassette, wherein the first expression cassette is used to express a first CAR that targets CD19.

[0074]

[0125] In another preferred embodiment, the first expression cassette is The terms "first expression cassette" and "second expression cassette" have the same meaning as those in the above.

[0126] In another preferred embodiment, the engineered immune cells that target BCMA are 1 is an immune cell expressing a CAR according to the first aspect of the invention.

[0075]

[0127] In another preferred embodiment, in vivo refers to autologous in vivo, or includes allogeneic in vivo.

[0128] In an eighteenth aspect of the present invention, there is provided a use of a first expression cassette, The expression cassette is used to express a first CAR that targets CD19, or is used to enhance the in vivo viability or killing ability of BCMA-targeting engineered immune cells against tumor cells capable of clonal expansion, or is used to prepare a kit, wherein the kit is used to enhance the in vivo viability or killing ability of BCMA-targeting engineered immune cells against tumor cells capable of clonal expansion.

[0076]

[0129] In another preferred embodiment, in vivo refers to autologous in vivo, or includes allogeneic in vivo.

[0130] Within the scope of the present invention, each of the above-mentioned technical features of the present invention and the following (e.g., It should be understood that each of the various technical features specifically described in the embodiments may be combined with each other to form new or preferred technical solutions, which will not be repeated in this specification due to limited space. [Brief explanation of the drawings]

[0077] [Figure 1]

[0131] FIG. 1 shows a schematic diagram of a construct containing a CAR and a cell suicide element according to the present invention, where the bispecific CAR and suicide switch element are linked via 2A. [Figure 2]

[0132] FIG. 2 shows the results of flow cytometric analysis of CAR-BB and CAR-S1 expression on the surface of Jurkat cells and primary T cells, respectively, according to the present invention. [Figure 3]

[0133] Figure 3 shows the killing effects of CAR-BB and CAR-S1 on Hela cells and BCMA-overexpressing cells (Hela-BCMA) (RTCA method), respectively, and the killing effects of CAR-April and CAR-S1 on Hela cells and BCMA-overexpressing cells (Hela-BCMA) (RTCA method), respectively, according to the present invention. [Figure 4]

[0134] FIG. 4 shows BCMA expression in target cells used in the present invention. [Figure 5]

[0135] FIG. 5 shows the results of in vitro killing experiments (luciferase method) of various batches of CAR-BB and CAR-S1 against MM.1s cells and RPMI-8226 cells, respectively, according to the present invention. [Figure 6]

[0136] FIG. 6 shows the release of cytokine IFNr during the killing process of CAR-BB and CAR-S1 against Hela cells and BCMA-overexpressing cells (Hela-BCMA), respectively, according to the present invention. [Figure 7]

[0137] FIG. 7 shows the tumor elimination ability of CAR-BB and CAR-S1 after intravenous reinfusion in immunodeficient mice modeled subcutaneously with RPMI-8226 according to the present invention. [Figure 8]

[0138] FIG. 8 shows the expression of CD19-CAR and BCMA-CAR in bispecific CAR-T cells according to the present invention. [Figure 9]

[0139] Figure 9 shows analysis of CD19-CAR and BCMA-CAR expression and EGFRt expression in safety-switched bispecific CAR-T cells according to the present invention. [Figure 10]

[0140] FIG. 10 is a comparison of killing of Hela cells and antigen-overexpressing Hela cell lines Hela-BCMA, Hela-CD19, and Hela-BCMA-CD19 by different batches of CAR-19, CAR-BCMA, and bispecific CAR-T according to the invention. [Figure 11]

[0141] FIG. 11 shows the results of in vitro killing experiments (luciferase method) of different batches of CAR-19, CAR-BCMA and bispecific CAR-T against MM.1s cells, RPMI-8226 cells and Nalm6 cells according to the present invention. [Figure 12]

[0142] FIG. 12 shows cytokine release during the killing process of CAR-BCMA and bispecific CAR-T cells against BCMA-overexpressing Hela cells (Hela-BCMA) according to the present invention. [Figure 13]

[0143] Figure 13 shows the analysis of the expression of CD107a molecules on the surface of dual CAR-T cells after co-culture with MM.1s or Raji tumor target cells. [Figure 14]

[0144] FIG. 14 shows the tumor elimination capacity of CAR-S1, bispecific CAR-S2, and CAR-S4 after intravenous reinfusion in immunodeficient mice modeled subcutaneously with RPMI-8226 according to the present invention. [Figure 15]

[0145] FIG. 15 shows the tumor elimination capacity of different doses of bispecific CAR-S2 and CAR-S4 cells after intravenous re-infusion in immunodeficient mice modeled intravenously with MM.1s-luc, according to the present invention. [Figure 16]

[0146] Figure 16 shows the inhibitory ability of different CAR-T cells on the clonal formation of CD34-negative monocytes in the bone marrow of MM patients. [Figure 17]

[0147] Figure 17 shows the in vivo tumor elimination ability of various CAR-T cells in NOG mice modeled with Nalm6-Luc cells. [Figure 18]

[0148] Figure 18 shows the expression of the CAR and safety switch of CAR-T cells on the surface of T cells. [Figure 19]

[0149] Figure 19 shows the killing effect (luciferase assay) of various CAR-T cells on Nalm6 or RMPI8226 cells. Compared with single CAR-T cells, dual CAR pairs have a stronger ability to kill target cells. [Figure 20] Figure 20 shows that various CAR-T cells do not have the ability to kill negative target cells (K562, Raji-KO19, Nalm6-KO19, and CCRF). [Figure 21]

[0150] Figure 21 shows the in vivo tumor elimination ability of various CAR-T cells against NOG cells modeled with Raji-luc. Compared with single CAR-T cells, dual CAR pairs have a stronger ability to kill target cells. [Figure 22]

[0151] Figure 22 shows the results of an in vitro killing experiment (luciferase method) of CAR-S1 against Raji lymphoma cells, where Figure 22A shows the expression of BCMA antigen on the surface of Raji lymphoma target cells, and Figure 22B shows the killing of Raji lymphoma target cells by CAR-S1 cells at various E:T ratios. DETAILED DESCRIPTION OF THE INVENTION

[0078]

[0152] After extensive and thorough research, the inventors have developed a novel manipulation targeting BCMA. For the first time, we have constructed immune cells containing BB scFv and the BCMA-binding domain of the CAR, which is an S-derived scFv. Experiments have shown that compared with CAR-T cells constructed using BB scFv and the BCMA-binding domain of April, the CAR-T cells constructed in this invention have a higher killing effect and tumor-eliminating ability. The present invention also uses S scFv and CD19 to construct dual CAR-T cells that can kill both BCMA-positive and CD19-positive CAR-T cells. Use scFv.

[0079]

[0153] Specifically, the CAR-T cells of the present invention utilize scFvs of various BCMA antibodies. By comparing them, we unexpectedly found that CAR-T cells constructed with the S-derived scFv had a higher ability to kill BCMA-overexpressing cells and BCMA-positive tumor target cells than those constructed with the BB scFv and the BCMA-binding domain from April. An in vivo mouse animal model also showed that CAR-T cells constructed with the S-derived scFv had a higher tumor-eliminating ability than BB-derived CAR-Ts. CAR-T cells constructed using several other BCMA-targeting scFvs commonly used in the art do not exhibit ideal in vitro and in vivo functions.

[0080] term

[0154] In order to better understand this disclosure, certain terms will first be defined. As used in this application, unless expressly stated otherwise herein, each of the following terms has the meaning given below. Other definitions are set forth throughout the specification.

[0081]

[0155] The term "about" refers to an acceptable range for a particular value or composition as determined by one of ordinary skill in the art. A specific example may refer to a value or composition that is within a reasonable error range, which depends in part on how the value or composition is measured or determined.

[0082]

[0156] The term "administration" refers to administration by intravenous, intramuscular, subcutaneous, intraperitoneal, or other route, such as via injection or infusion. It refers to the use of any of a variety of methods and delivery systems known to those skilled in the art for physically introducing the products of the invention into a subject, including intravenous, intraspinal or other parenteral routes of administration.

[0083]

[0157] The term "antibody" (Ab) refers to a molecule that specifically binds to an antigen and binds to a target molecule via disulfide bonds. The term "immunoglobulin" includes, but is not limited to, immunoglobulins comprising at least two heavy (H) chains and two light (L) chains interconnected by a nucleotide sequence, or antigen-binding portions thereof. Each H chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains a constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is From the amino terminus to the carboxyl terminus, it contains three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0084]

[0158] The amino acid names in this specification are internationally recognized English single letter equivalents. It should be understood that the corresponding three-letter abbreviations for amino acid names are Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V), respectively.

[0085] B cell maturation antigen (BCMA)

[0159] BCMA is expressed on the surface of mature B lymphocytes, i.e., plasmablasts and plasma cells. BCMA is a transmembrane protein expressed in multiple myeloma cells. Multiple myeloma is caused by the abnormal proliferation and infiltration of plasma cells into the bone marrow. Research has shown that BCMA-targeting CAR-T cells have been shown to specifically kill myeloma cells. However, some patients experience relapse after receiving BCMA-targeting CAR-T cell therapy. For these relapsed patients, it is necessary to find a target other than BCMA to continue treatment.

[0086] CD19

[0160] The CD19 molecule is a transmembrane protein on the surface of B cells. , which is closely related to B cell activation, signal transduction, and growth control. As shown in Figure 1, CD19 is expressed on the surface of nearly all B cells, and CD19-targeting CAR-T cells currently have remarkable efficacy in the treatment of leukemia and lymphoma. Generally, 99.95% of plasma cells do not express CD19 on their surface, and therefore, the possibility of using CD19 for the treatment of multiple myeloma is considered to be overlooked.

[0087] Chimeric antigen receptor (CAR)

[0161] The chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains a target-specific binding element (also called an antigen-binding domain). The intracellular domain contains a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to the portion of the intracellular domain that contains a costimulatory molecule. Costimulatory molecules are cell surface molecules required for an effective response of lymphocytes to antigens, but are not antigen receptors or their ligands.

[0088]

[0162] Between the extracellular domain of CAR and the transmembrane domain, or between the cytoplasmic domain of CAR A linker may be incorporated between the transmembrane domain and the extracellular or cytoplasmic domain. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain of a polypeptide chain to the extracellular or cytoplasmic domain. The linker may contain 0 to 300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0089]

[0163] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention is The main comprises an antigen-binding domain that targets BCMA (or BCMA and CD19). When the CAR of the present invention is expressed in a T cell, the CAR of the present invention can perform antigen recognition based on antigen-binding specificity. When the CAR of the present invention binds to its bound antigen, the CAR of the present invention affects tumor cells, causing them to stop growing, promote death, or otherwise affect tumor cells, thereby reducing or eliminating the tumor burden in the patient. The antigen-binding domain preferably binds to one of a costimulatory molecule and a zeta chain. Preferably, the antigen-binding domain is fused to the intracellular domain in combination with the 4-1BB signaling domain and the CD3ζ signaling domain.

[0090]

[0164] As used herein, "antigen-binding domain" and "single-chain antibody fragment" refer to All of these terms refer to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment having antigen-binding activity. An Fv antibody is the smallest antibody fragment containing an antibody heavy chain variable region and an antibody light chain variable region, but no constant region, and all of the antigen-binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH domain and the VL domain, allowing it to form the structure required for antigen binding. The antigen-binding domain is usually an scFv (single-chain variable fragment). The size of an scFv is generally one-sixth the size of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. In a preferred embodiment of the present invention, the antigen-binding domain comprises an antibody that specifically recognizes BCMA, and optionally, the antigen-binding domain further comprises an antibody (preferably a single-chain antibody) that specifically recognizes CD19.

[0091]

[0165] With respect to the hinge region and transmembrane region (transmembrane domain), CAR is The extracellular domain may be designed to include a transmembrane domain fused to it. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding of the domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.

[0092]

[0166] The intracellular domain in the CAR of the present invention is the signaling domain of 4-1BB. It contains the signaling domains of CD3ζ and CD3ζ.

[0167] Preferably, the CAR of the present invention also comprises a cell suicide element.

[0093]

[0168] Preferably, the BCMA-targeting scFv of the present invention is an S scFv. In the examples, the BB scFv and April chain are used as controls. Both the BB scFv and April chain are binding sequences commonly used in the art to target BCMA. The BB scFv is described in PCT Publication No. 2010104949 A3, and the April chain is described in Chinese Patent No. 105658671A.

[0094] Bispecific CAR targeting CD19 and BCMA

[0169] Multiple myeloma (MM) is a malignant plasma cell neoplasm. Tumor cells are distributed in the bone marrow. Multiple myeloma arises from plasma cells, which are cells that develop from B lymphocytes in their final functional stage. Multiple myeloma is a fundamentally incurable disease characterized by high morbidity and mortality. Statistics from 2017 indicate that there were 30,000 newly diagnosed multiple myeloma patients in the United States, of whom 12,000 will likely die. Currently, common treatments for multiple myeloma include cytotoxic drug therapy, protease inhibitors (e.g., bortezomib), lenalidomide, monoclonal antibodies, and corticosteroids. However, all current treatments are only partially effective, do not provide sustained relief, and carry a high risk of relapse. Therefore, improved treatments for multiple myeloma are considered particularly important.

[0095]

[0170] CD19 is a 95kJ molecule expressed on the membrane surface of pre-B cells and mature B cells. It is a glycoprotein with Da, which is closely related to the B cell Ca++ transmembrane signaling pathway and has a regulatory effect on B cell proliferation and differentiation. It is expressed on normal and cancerous B cells with good tissue expression specificity, making it a good target for antibody or CAR-T immunotherapy. However, during the course of immunotherapy, the CD19 epitope on B cells is often lost, leading to non-response to immunotherapy or relapse in patients.

[0096]

[0171] Bispecificity is when the same CAR specifically binds to two different antigens, It is capable of immunologically recognizing different antigens, meaning that the CAR is capable of generating an immune response when combined with any one of the antigens.

[0097]

[0172] In another preferred embodiment, a bispecific C4 antibody targeting CD19 and BCMA is AR is described in the second aspect of the present invention.

[0173] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention is The main components contain antigen-binding domains that target CD19 and BCMA, including anti-CD19 scFv and anti-BCMA scFv.

[0098]

[0174] In another preferred embodiment, the present invention provides antibodies against the CD19 antigen and the BCMA antigen. The present invention provides a bispecific chimeric antigen receptor (CAR) that targets both CD19 and BCMA. CAR structural components that target both CD19 and BCMA can include a signal peptide, an anti-CD19 scFv, an anti-BCMA scFv, a hinge region, a transmembrane region, and an intracellular T cell signaling region, where the CD19 scFv and the BCMA scFv are linked via a short peptide segment (G4S)xN. CAR structures that target both CD19 and BCMA are described in a second aspect of the present invention.

[0099]

[0175] In another preferred embodiment, the CD19 and BCMA bispecific CAR of the invention has a single structure and contains scFvs against CD19 and BCMA, where the CAR contains CD19 scFv and BCMA scFv, and the ordering of the CD19 scFv and BCMA scFv and hinge is a major influencer of its function.

[0100]

[0176] In another preferred embodiment, the present invention provides a method for optimizing the sequence of a BCMA scFv. BCMA scFv (S scFv) has high affinity and good specificity for BCMA and can specifically target the full-length antigen and extracellular domain of BCMA.

[0101]

[0177] In a preferred embodiment of the present invention, (G4S)x3 is a CD19 scFv and Used to bind BCMA ScFv, this CAR has the best activity and killing potential.

[0102]

[0178] Bispecific CARs targeting CD19 and BCMA can be used in the present invention. Compared with CARs targeting a single antigen, its affinity is significantly enhanced, and the activity of immune cells is significantly increased, resulting in a synergistic effect. Furthermore, due to the uneven expression levels of CD19 and BCMA on tumor cells, dual-targeting CAR-Ts have a broader therapeutic range. CAR immune cells targeting both CD19 and BCMA can reduce the possibility of antigen escape caused by the downregulation or lack of a single surface antigen. Furthermore, CD19 and BCMA bispecific CAR-Ts significantly outperform single CAR-Ts in the inactivation of CD34-negative monocytes in the bone marrow of patients with myeloma. The CD19 and BCMA bispecific CAR-Ts have the ability to inhibit tumor precursor cells significantly better than single CAR-Ts. Finally, the addition of the CD19 antigen may increase the continued viability of CD19 and BCMA bispecific CAR-Ts.

[0103] Chimeric antigen receptor T cells (CAR-T cells)

[0179] As used herein, the terms "CAR-T cells," "CAR-T," and "the present invention" are used interchangeably. "CAR-T cells of the invention" includes CAR-T cells encompassed in the third aspect of the invention.

[0104]

[0180] CAR-T cells have the following advantages over other T-cell-based therapies: (1) The action process of CAR-T cells is not restricted by MHC; (2) considering the fact that many tumor cells express the same tumor antigen, once the CAR gene directed to a specific antigen is constructed, it can be widely used; (3) CARs can use both tumor protein antigens and glycolipid non-protein antigens, expanding the range of tumor antigen targets; (4) by using the patient's own cells, the risk of rejection can be reduced; and (5) CAR-T cells have immunological memory function and can survive in the body for a long period of time.

[0105] Chimeric antigen receptor NK cells (CAR-NK cells)

[0181] As used herein, "CAR-NK cells," "CAR-NK," and All references to "CAR-NK cells of the present invention" refer to CAR-NK cells encompassed in the third aspect of the present invention. The CAR-NK cells of the present invention can be used to treat tumors with high BCMA expression, such as multiple myeloma.

[0106]

[0182] Natural killer (NK) cells act as a non-antigen specific pathway to protect against viruses. NK cells are the major type of immune effector cell that defends the body against infection and tumor cell infiltration. Engineered (genetically modified) NK cells can acquire new functions, including the ability to specifically recognize tumor antigens and enhanced antitumor cytotoxicity.

[0107]

[0183] Compared to autologous CAR-T cells, CAR-NK cells offer the following advantages: Additionally, CAR-NK cells have the following advantages: (1) they directly kill tumor cells by releasing perforin and granzymes without having a lethal effect on normal cells in the body; (2) they release small amounts of cytokines, thereby reducing the risk of cytokine storms; and (3) they are very easily expanded and developed in vitro into an "off-the-shelf" product. Furthermore, CAR-NK cells are similar to CAR-T cell therapy.

[0108] Suicide gene switch

[0184] Further investigation into adverse reactions of CAR-T cells, such as non-tumor targeting and cytokine release syndrome In order to control the proliferation of CAR-T cells, all CAR-T cells in the present invention have a suicide gene switch that can effectively eliminate CAR-T cells in the body under the action of exogenous drugs, thereby preventing unknown or uncontrollable long-term toxicity and ensuring patient safety.

[0109]

[0185] The suicide switch used in the present invention is the herpes simplex virus thymidine The kinase may be HSV-TK, inducible caspase 9 (iCasp9), CD20, mutant human thymidylate kinase (mTMPK), etc. In comparison, HSV-TK, iCasp9, and CD20 have the same elimination ability for CAR cells, but iCasp9 and CD20 have a faster elimination rate, and HSV-TK has a slower elimination rate.

[0110]

[0186] The iCasp9 suicide switch contains the FKBP12-F36V domain and is involved in the transcription of FK The FKBP12-F36V domain can be linked via a flexible linker to caspase-9, which does not contain a recruitment domain. FKBP12-F36V contains an FKBP domain with a phenylalanine substituted for valine at amino acid residue position 36. FKBP12-F36V can bind dimerization ligands, such as the otherwise inactive small molecule AP1903, with high selectivity and sub-nM affinity. Small molecules, when added, can promote dimerization. Small molecules can promote the activation of the suicide switch, thereby inducing cell apoptosis, but small molecules are ineffective against normal cells that do not possess the suicide switch.

[0111]

[0187] Inducible safety switch caspase 9 (iCasp9) is an FK506-binding protein We used human caspase 9 fused to a protein (FKBP), which can be induced to form dimers with a chemical inducer (AP1903 / Rimiducid, Bellicum Pharmaceuticals), causing apoptosis in cells expressing the fusion protein.

[0112]

[0188] CD19 and BCMA are highly expressed on tumor cells, but BCMA is also expressed on normal B cells, therefore the engineered immune cells of the present invention can attack normal B cells in vivo.

[0113]

[0189] How to control the safety of CAR cells is an urgent issue that needs to be resolved. This is an urgent issue. Adding a safety switch to CAR cells is the safest way to terminate CAR cell activity. The inducible iCasp9 safety switch controls the elimination of CAR cells after they cause severe toxicity (CRS / neurotoxicity) or after the patient reaches long-term, sustained remission.

[0114] vector

[0190] Nucleic acid sequences encoding the desired molecules can be, for example, cloned from cells that express the genes. The gene may be obtained using recombinant methods known in the art, such as by screening libraries, by obtaining the gene from a vector known to contain the gene, or by using standard techniques to directly isolate the gene from cells and tissues that contain it. Optionally, the gene of interest can be produced synthetically.

[0115]

[0191] The present invention also provides a vector into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and transmission of the transgene in daughter cells. Lentiviral vectors have an advantage over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. Lentiviral vectors also have the advantage of low immunogenicity.

[0116]

[0192] Generally, the expression cassette or nucleic acid sequence of the present invention is typically operable with a promoter. and incorporated into an expression vector. This vector is suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation termination elements, initiation sequences, and promoters that can be used to control the expression of the desired nucleic acid sequence.

[0117]

[0193] The expression constructs of the present invention also can be used in standard genetic applications for nucleic acid immunization and gene therapy. Gene delivery protocols can be utilized. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entireties. In another embodiment, the present invention provides a gene therapy vector.

[0118]

[0194] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be They may be cloned into such vectors, including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0119]

[0195] Additionally, the expression vector can be provided to the cell in the form of a viral vector. Vector technology is known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks in virology and molecular biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction sites, and one or more selectable markers (see, e.g., WO 01 / 96584, WO 01 / 29058, and U.S. Pat. No. 6,326,193).

[0120]

[0196] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells, either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0121]

[0197] Additional promoter elements, such as enhancers, can regulate the frequency at which transcription is initiated. Generally, these elements are located in the region 30 to 110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. Spacing between promoter elements can often be flexible to maintain promoter function when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, spacing between promoter elements can be increased by 50 bp before activity begins to decrease. Depending on the promoter, individual elements can act cooperatively or independently to initiate transcription.

[0122]

[0198] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter. The promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any operably linked polynucleotide sequence. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLv promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0123]

[0199] To assess the expression of a CAR polypeptide or a portion thereof, The expression vector may also contain one or both of a selectable marker gene and a reporter gene to facilitate identification and selection of expressing cells from a population of cells desired to be transfected or infected with the viral vector. In other embodiments, the selectable marker may be carried on a single piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences such that they can be expressed in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0124]

[0200] Reporter genes are useful for identifying potentially transfected cells. It is also used to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue, and may encode a polypeptide whose expression can be clearly indicated by some easily detectable property, such as enzymatic activity. After the DNA is introduced into the recipient cells, the expression of the reporter gene is measured at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (see, for example, Ui-Tei et al., 2000). (FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct with at least five flanking regions that exhibits the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to evaluate the ability of reagents to modulate promoter-driven transcription.

[0125]

[0201] Methods for introducing genes into cells and expressing the genes in cells are well known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0126]

[0202] As a physical method for introducing polynucleotides into host cells, calcium phosphate Examples of suitable methods for introducing polynucleotides into host cells include ELISA, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0127]

[0203] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA vectors. These include the use of viral vectors and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0128]

[0204] Chemical means for introducing polynucleotides into host cells include macromolecular complexes. colloidal dispersion systems such as bodies, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in ro and in vivo is a liposome (eg, an artificial membrane vesicle).

[0129]

[0205] When a non-viral delivery system is used, an exemplary delivery tool is a liposome. The use of lipid formulations is considered to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid can be bound to a lipid. The lipid-bound nucleic acid can be encapsulated in the aqueous interior of a liposome, dispersed in the lipid bilayer of a liposome, bound to a liposome via a linking molecule bound to both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained in a lipid as a suspension, contained in a micelle, complexed with a micelle, or otherwise associated with a lipid. The lipid, lipid / DNA, or lipid / expression vector associated with the composition is not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a "disintegrated" structure. They can also simply be dispersed in a solution, sometimes forming aggregates of uneven size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include lipid droplets that occur naturally in the cytoplasm and compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0130]

[0206] In a preferred embodiment of the invention, the vector is a lentiviral vector. . formulation

[0207] The present invention relates to a CAR-T cell according to the first aspect of the invention, and a pharmaceutically acceptable carrier. In one embodiment, the formulation is a lipid formulation. Preferably, the formulation is for injection. Preferably, the concentration of CAR-T cells in the formulation is 1×10 3 ~1×10 8 cells / ml, more preferably 1 x 10 4 pieces~1×10 7 cells / ml.

[0131]

[0208] In one embodiment, the formulation is a saline solution such as neutral buffered saline, sulfate buffered saline, etc. The formulations of the present invention may contain buffers, carbohydrates such as glucose, mannose, sucrose, dextran, mannitol, etc., proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0132] therapeutic use

[0209] The present invention relates to a lentiviral vector (LV) encoding an expression cassette of the present invention. Therapeutic applications include using cells (e.g., T cells) transduced with BCMA and / or CD19. The transduced T cells can target the tumor cell markers BCMA and / or CD19, synergistically activating T cells, resulting in a T cell immune response that significantly improves their killing efficiency against tumor cells.

[0133]

[0210] Thus, the present invention also provides a method for the treatment of a target cell population or tissue in a mammal. A method of stimulating a T cell-mediated immune response is provided, the method comprising the step of administering a CAR-T cell of the invention to a mammal.

[0134]

[0211] In one embodiment, the present invention provides a method for determining whether a patient's autologous T cells (or allogeneic donor-derived T cells) are This involves a type of cell therapy in which cells are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected back into the same patient. In this method, the cells are injected into the same patient, who has graft-versus-host disease. In the rare event that an antigen is recognized by T cells in a non-MHC-restricted manner, a single CAR-T cell can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, creating long-term persistence that can lead to sustained tumor control.

[0135]

[0212] In one embodiment, the CAR-T cells of the present invention are stable in vivo T cells. They can undergo cell expansion and persist for long periods of time. Furthermore, CAR-mediated immune responses can be part of adoptive immunotherapy procedures, in which CAR-modified T cells induce immune responses specific to the antigen-binding domains in the CAR. For example, CAR-T cells directed against BCMA and / or CD19 induce immune responses specific to cells expressing BCMA and / or CD19.

[0136]

[0213] The data disclosed herein specifically support the use of anti-BCMA and / or CD19 Although lentiviral vectors comprising scFv, hinge and transmembrane regions, and 4-1BB / CD28 and CD3ζ signaling domains are disclosed, the invention should be construed to include any variation in the number of each of the construct components.

[0137]

[0214] Treatable cancers include those that are non-vascularized or substantially vascularized. Cancers include non-solid tumors, as well as vascularized tumors. Cancers can include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or solid tumors. Cancer types treated with the CARs of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant diseases such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0138]

[0215] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematopoietic) cancers include acute myeloid leukemia, leukemia, and leukemia. Leukemias including leukemia (such as myeloid lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, granulomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and spondylodysplasia.

[0139]

[0216] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Tumors can be benign or malignant. Various types of solid tumors can be designated after their cell type is formed (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

[0140]

[0217] The CAR-modified T cells of the present invention can also be used in ex vivo immunization and and / or can be used as a type of vaccine for in vivo therapy. Preferably, the mammal is a human.

[0141]

[0218] For ex vivo immunization, at least one of the following is required to inoculate the cells into a mammalian host: Prior to administration to a subject, in vitro procedures include: 1) expanding the cells; ii) introducing a nucleic acid encoding a CAR into the cells; and / or iii) cryopreserving the cells.

[0142]

[0219] Ex vivo procedures are well known in the art and are described more fully below. Briefly, cells are isolated from a mammal, preferably a human. , genetically modified (i.e., in (transduced or transfected in vitro). The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic effect. The mammalian recipient can be human, and the CAR-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0143]

[0220] In addition to using cell-based vaccines for ex vivo immunization The present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0144]

[0221] The present invention provides a method for treating tumors, the method comprising administering a therapeutically effective amount of the present invention. The method includes administering the CAR-modified T cells of the present invention to a patient in need of treatment.

[0222] The CAR-modified T cells of the present invention may be administered alone or in combination with diluent and / or IL-2, I The compositions of the present invention may be administered as a pharmaceutical composition in combination with other components, such as L-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention comprise a target cell population as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer solution, such as neutral buffered saline or sulfate buffered saline; a carbohydrate, such as glucose, mannose, sucrose, or dextran, mannitol; a protein; an amino acid, such as a polypeptide or glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

[0145]

[0223] The pharmaceutical composition of the present invention is suitable for the disease to be treated (or prevented). The amount and frequency of administration can be determined by factors such as the patient's disorder and the type and severity of the patient's disease, but an appropriate dosage can be determined by clinical trials.

[0146]

[0224] "Immunologically effective amount," "antitumor effective amount," "tumor suppression effective amount," or "therapeutic When referring to "amounts," the exact amount of the composition of the invention to be administered may be determined by a physician, who will take into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disorder. Generally, pharmaceutical compositions comprising the T cells described herein are administered in amounts of 10 4 ~10 pieces 9 At a dose of 10 cells / kg body weight, preferably 10 5 ~10 pieces 6 It may be noted that T cell compositions may be administered in doses of cells / kg body weight (including all integer values ​​within these ranges). The T cell compositions may also be administered multiple times at these doses. The cells may be administered by using injection techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0147]

[0225] The composition may be administered to a subject by spraying, injection, swallowing, infusion, implantation or transfer. The administration of T cells can be performed in any convenient manner, including by transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscular, intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the invention are preferably administered by intravenous injection. The T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[0148]

[0226] In certain embodiments of the invention, the present invention provides a method for expanding T cells to therapeutic levels. Cells activated and expanded using the methods described herein or other methods known in the art can be administered to a patient in combination with (e.g., prior to, concurrently with, or following) any number of relevant forms of therapy, including, but not limited to, treatment with the following agents: antiviral therapy cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab therapy for MS patients, or efalizumab therapy for psoriasis patients, or other therapies for PML patients. In further embodiments, the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention can be administered to a patient in combination with (e.g., prior to, concurrently with, or following) a bone marrow transplant using chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), and cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment of high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, after the transplant, the subject receives an infusion of the expanded immune cells of the present invention. In further embodiments, the expanded cells are administered after or before surgery.

[0149]

[0227] The dosage of the above treatments administered to a patient will depend on the exact nature of the disorder being treated and the Dosage rates administered to humans can be carried out according to art-accepted practices. Generally, 1 x 10 per treatment or series of treatments. 6 pieces~1×10 10 The modified T cells (e.g., CAR-T20 cells) according to the present invention can be administered to the patient, for example, by intravenous reinfusion.

[0150]

[0228] The main advantages of the present invention include:

[0229] (a) CAR-T cells containing S scFv constructed according to the present invention are and has higher in vivo and in vitro tumor-killing and functional activity than April CAR-T.

[0151]

[0230] (b) The bispecific CAR-T constructed according to the present invention comprises BCMA; Capable of recognizing two or more targets.

[0231] The present invention will now be further described in conjunction with the following specific examples, which are provided by way of example only. It should be understood that they are merely used to describe the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions are usually performed according to conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated on a weight basis. [Example]

[0152] Example 1 Isolation of PBMCs from Donor Blood and Expansion of T Cells

[0232] Mononuclear cells were isolated from donor blood and then cultured in Histopaque-1077 ( T cells (EasySep Human T Cell Enrichment Kit, Stemcell Technologies) were enriched by density gradient centrifugation using a Sigma-Aldrich® T cell culture medium. T cells were activated using coupled anti-CD3 / anti-CD28 magnetic beads, cultured, and expanded in X-vivo15 (300 IU / ml rhIL2) medium. All cells were then cultured at 37°C in a constant temperature incubator at 5% CO2.

[0153] Example 2 Cell Culture and Construction

[0233] BCMA-expressing cell lines MM.1s and RPMI8226, MM.1s-ffl uc cells, RPMI8226-ffluc cells, and Hela cells expressing BCMA, CD19, or both BCMA and CD19 were all cultured in RPMI 1640 medium, and 293T (human renal epithelial cell line, ATCC® CRL-3216) were cultured in DM. The cells were cultured in EM medium, all of which was supplemented with 10% (v / v) fetal bovine serum, 100 U / ml penicillin and streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate.

[0154]

[0234] Among them, those expressing BCMA, CD19, or both BCMA and CD19 Hela cells are stably transfected cell lines obtained by transferring BCMA and / or CD19 antigens via lentiviral vectors and capable of specifically expressing BCMA and / or CD19 protein molecules. MM.1s-ffluc and RPMI8226-ffluc cells are stably transfected cell lines obtained by screening after infection with firefly luciferase lentivirus.

[0155] Example 3: CAR structural design and transduction

[0235] Single CAR targeting BCMA and CAR targeting both BCMA and CD19 A dual CAR was designed and constructed, and its structural schematic is shown in Figure 1. Among them, the CAR, CD19 CAR, and suicide switch-EGFRt element were linked via the 2A peptide. Specifically, the CAR structure involved in the present invention is shown in Figure 1, and its name and composition are shown in Table 1.

[0156]

[0236]

[0157] [Table 1]

[0158]

[0237] The specific sequences of each element involved in CAR described in Figure 1 and Table 1 are as follows: This is: S scFv (S scFv) heavy chain QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYIHWVRQAPGQGLEWIGYINPNSGYTNYAQKFQGRATMTADKSINTAYVELSRLRSDDTAVYFCTRYMWERVTGFFDFWGQGTMVTVSS (SEQ ID NO: 9) S scFv (S scFv) light chain DIQMTQSPSSVSASVGDRVTITCLASEDISDDLAWYQQKPGKAPKVLVYTTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQTYKFPPTFGGGTKVEIKR (SEQ ID NO: 10) BB scFv heavy chain DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK (SEQ ID NO: 13) BB scFv light chain QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAA (SEQ ID NO: 14) April chain SVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKLSGGGSDP (SEQ ID NO: 15) CD8 signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO: 16) (G4S)3 linker peptide GGGGSGGGGSGGGGS (SEQ ID NO: 17) (G4S)5 linker peptide GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18) 218 linker peptide GSTSGSGKPGSGEGSTKG (SEQ ID NO: 19) CD8 hinge region TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 8) or KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKP (SEQ ID NO: 37) or SGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 38) CD8 transmembrane region IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 7) CD28 transmembrane domain FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 6) 41BB signal transduction region KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 5) CD28 signaling domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY RS (SEQ ID NO: 4) CD3z signaling region RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 3) or RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 39) 2A peptide GSGATNFSLLKQAGDVEENP (SEQ ID NO: 2) FMC63 scFv (CD19 scFv) heavy chain EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 11) FMC63 scFv (CD19 scFv) light chain DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 12) GM-CSF signal peptide MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 1) EGFRt sequence RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO: 20)

[0238] The CAR gene in Table 1 was cloned into the FUW lentiviral vector backbone. A complete lentiviral expression vector that can be used to infect T cells was constructed using the BCMA CAR gene as an example for detailed description. The BCMA CAR gene was placed under the action of the EF1α (EF-1α) promoter to form Fuw-EF1α-BCMA CAR. Three plasmids, namely, Fuw-EF1α-BCMA CAR, lentiviral envelope plasmid pMD2.G (Addgene, plasmid #12259), and lentiviral packaging plasmid psPAX2 (Addgene, plasmid #12260), were transfected into 293T cells using Lipofectamine 3000 to prepare the complete lentiviral expression vector. The viral supernatant was collected at 48 and 72 hours and concentrated by ultracentrifugation. The concentrated virus could be used to infect T cells.

[0159]

[0239] Flow cytometry analysis revealed that lentiviruses expressing BCMA CAR It has been shown that viral vectors can be prepared from the constructed CAR gene.

[0160] Example 4 Preparation of CAR-T cells

[0240] The experimental method is as follows:

[0241] 4.1 Lentiviral infection

[0242] Two days after activating the isolated and purified primary T cells, they were incubated with the IgG1 construct described in Example 3. The cells were infected with the lentiviral vector using the prepared lentivirus, and then transferred to a cell culture flask and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0161]

[0243] 4.2 Detection of cell proliferation and CAR positivity

[0244] Three days after infection and before cryopreservation, BCMA-positive cells were identified using BCMA antigen. Samples were taken to detect the cell number and percentage, i.e., to detect the CAR-positive rate of T cells. Half of the medium was replaced every 2 to 3 days.

[0162]

[0245] As a result, the lentiviral vector constructed in Example 3 was used to Each of the RT cells was shown to be successfully constructed, and their designations are shown in Table 1.

[0246] Specifically, the results of constructing BCMA CAR-T cells are shown in Figure 2. AR expression can be detected in both CAR-BB and CAR-S1 CAR-T cells after viral transfection, and CAR expression can reach greater than 50%.

[0163] Example 5: In vitro killing of cells

[0247] In vitro killing experiments were performed using the CAR-S1 CAR obtained in Example 4. The study was conducted using CAR-T cells, CAR-BB CAR-T cells, and CAR-April CAR-T cells. RTCA was used to examine the killing of CAR-T cells against the target cell line, HeLa, which overexpresses BCMA.

[0164]

[0248] The results are shown in Figure 3 (tested using the RTCA method) and the NT control (transferase While the uninfected T cell control (uninfected T cell control) and medium control (blank control) had no killing against Hela-BCMA cells, CAR-S1 cells were able to carry out BCMA-specific killing function, and CAR-S1 cells performed better than CAR-BB cells in killing BCMA-positive Hela-BCMA cells.

[0165]

[0249] Luciferase-labeled tumor target cells were used to detect the killing activity. By transferring the luciferase gene into target cells, stably transfected cell lines MM.1s-Luc and RPMI8226-Luc were obtained after clonal screening. During the experiment, luciferase reacted with luciferin by adding luciferin substrate, producing fluorescence. By detecting the intensity of the fluorescence, the activity of luciferase could be measured, and subsequently the cell survival rate and the killing effect of CAR-T cells could be obtained.

[0166]

[0250] Figure 4 shows antigen expression on the surface of target cells. Figure 5 shows the same E:T ratio. These results show that NT cells have no killing function, CAR-S1 cells have a dose-dependent killing effect on MM.1s-Luc cells (MM.1S cells transfected with the luciferase gene) and RPMI8226-Luc cells (RPMI8226 cells transfected with the luciferase gene), and that CAR-S1 cells exhibited better killing ability than CAR-BB and CAR-April.

[0167]

[0251] Additionally, the applicant also provides various targeting BCMA antibodies commonly found in the art. When CAR-T cells were constructed and tested using the scFv, none of these CAR-T cells exhibited ideal killing function.

[0168]

[0252] In summary, CAR-T cells target cells (BCMA-overexpressing cells and BC After co-culture with MA-positive tumor cells, MM.1s-Luc and RPMI8226 cells, the target Target cells could be lysed by BCMA-targeting CAR-T cells, and CAR-S1 demonstrated higher killing ability than CAR-BB. Several other CAR-T cells constructed with BCMA-targeting scFvs commonly found in the art did not demonstrate ideal killing function.

[0169] Example 6 Detection of cytokine release

[0253] BCMA-targeting CAR T cells (CAR-S1 C) obtained in Example 4 AR-T cells and CAR-BB CAR-T cells were mixed with tumor cells (Hela, Hela-BCMA, Hela-CD19, or Hela-BCMA-CD19) and placed in RPMI medium. The cell density was 1 x 10 4 The CAR-T cells and tumor cells were cultured at 100 μl / ml. 100 μl each of the CAR-T cells and tumor cells was placed in a 96-well plate and co-cultured overnight. The supernatant was collected and centrifuged, and the released levels of cytokines such as IFN-γ were detected using an ELISA kit.

[0170]

[0254] The results are shown in Figure 6. After costimulation with Hela-BCMA target cells, C The secretion of the cytokine INF-γ by AR-S1 was significantly higher than that by CAR-BB, whereas no significant secretion was observed in the NT and medium groups.

[0171] Example 7 In vivo drug efficacy studies

[0255] Select 6- to 12-week-old NOG mice and inoculate them at 1 x 10 7 RPMI822 6 cells were subcutaneously injected. Two days later, tumor graft burden was measured. Ten days later, grouping was performed, and CAR-S1 CAR-T cells and CAR-BB CAR-T cells were injected one day after grouping. After CAR-T treatment, the tumor volume burden of the mice was assessed twice weekly.

[0172]

[0256] The results are shown in Figure 7. Compared to the control, the tumors in mice injected with CAR-S1 cells were Tumor burden was significantly suppressed, and CAR-S1 cells had a slightly greater antitumor effect than CAR-BB.

[0173] Example 8 Preparation of Dual CAR-T Cells

[0257] The experimental method is as follows:

[0258] This example describes CAR-T cells that target both BCMA and CD19. The CAR structures are shown in Figure 1 (CAR S2, CAR S3, CAR S4, CAR S5, CAR S6, and CAR S7). Among them, the BCMA CAR, CD19 CAR, and suicide switch-EGFRt elements were linked via the 2A peptide. Among them, the scFv in the BCMA CAR structure was composed of the heavy and light chains of S and BB scFvs. Here, the S scFv was composed of SEQ ID NOs: 9 and 10, the BB scFv was composed of SEQ ID NOs: 13 and 14, and the CD19 scFv was composed of SEQ ID NOs: 11 and 12. Furthermore, the scFvs can be replaced with a BCMA-binding region composed of a portion of the April sequence (SEQ ID NO: 15) to form a new CAR structure.

[0174]

[0259] The BCMA-CD19 CAR gene was cloned into the vector backbone and expressed in E The EF1α-BCMA-CD19-EGFRt CAR was placed under the action of the F1α (EF-1α) promoter to form the EF1α-BCMA-CD19-EGFRt CAR, and the EF1α-BCMA-CD19-EGFRt CAR and lentiviral envelope plasmid were transfected into 293T cells using Lipofectamine 3000 to prepare the complete lentiviral vector. The viral supernatant was collected at 48 and 72 hours and concentrated via ultracentrifugation, and the concentrated virus could be used to infect T cells.

[0175]

[0260] Lentiviral infection: Two days after activation of isolated and purified primary T cells, The cells were infected with lentiviral vectors using the lentivirus constructed as described above at an MOI (1–10), and then transferred to cell culture flasks and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0176]

[0261] Detection of cell proliferation and CAR positivity: Three days after infection, before cryopreservation, cell count and Samples were collected to detect BCMA / CD19 double-positive cells, i.e., to detect the CAR-positive rate of T cells. Half of the medium was replaced every 2 to 3 days.

[0177]

[0262] The results, as shown in Figure 1 and Table 1, demonstrate that BCMA-CD19 Using a CAR lentiviral vector, BCMA-CD19 CAR-T cells were shown to be successfully constructed.

[0178]

[0263] The results are shown in Figure 8. Expression of BCMA CAR and CD19 CAR was significantly increased in BCM Both the A antigen and the CD19 antigen can be used to detect on the surface of virus-infected T cells.

[0179]

[0264] Figure 9 shows that the expression of BCMA CAR, CD19 CAR, and EGFRt all It is shown that it can be detected on the surface of CAR-S6 and CAR-S7 cells. Example 9 Cell Killing in Vitro

[0265] An in vitro killing experiment was performed using the CAR-T cells obtained in Example 8. The experiment was carried out using a Hela cell line overexpressing BCMA and CD19 for RTCA, or luciferase-labeled tumor target cells for detection. Stably transfected cell lines (RPMI8226, MM.1s, and Nalm6) were obtained after clonal screening by transferring the luciferase gene into target cells. During the experiment, luciferase reacted with luciferin by adding a luciferin substrate, producing fluorescence. The activity of luciferase could be measured by detecting the intensity of the fluorescence, which could then be used to detect the cell survival rate and the killing effect of CAR-T cells.

[0180]

[0266] The results showed that CAR-T cells bind to target cells (CD19 / BCMA double positive, C After co-culture with CD19 / BCMA double-positive, CD19 single-positive, and BCMA single-positive cells, all target cells were shown to be lysed, demonstrating that BCMA-CD19 CAR-T has a killing effect against all CD19 / BCMA double-positive, CD19 single-positive, and BCMA single-positive cells.

[0181]

[0267] Specific results are shown in Figure 10. Bispecific CAR-T inhibited single-positive CD19-positive The bispecific CAR-T cells were able to significantly kill target cells (Hela-CD19) or single-positive BCMA-positive target cells (Hela-BCMA), and the bispecific CAR-T cells were also able to significantly kill CD19 / BCMA double-positive target cells, Hela-BCMA-CD19. Bispecific CAR-T cells with a combination of BCMA and CD19 were shown to have a killing effect against both single and dual target cells. Single CAR-T cells (CAR-19 or CAR-S1) only had a killing effect against one target antigen.

[0182]

[0268] Figure 11 shows that dual CAR-T inhibits BCMA single-positive tumor target cells MM.1s and This shows that the dual CAR-T can significantly kill RPMI8226 tumor cells. The dual CAR-T also significantly killed CD19-positive tumor target cells Raji and Nalm6. The dual CAR with a combination of BCMA and CD19 was shown to have a killing effect on both BCMA-positive and CD19-positive tumor target cells.

[0183] Example 10 Detection of cytokine release

[0269] BCMA-CD19 CAR-T cells (obtained in Example 8) were transfected with tumor cells ( Hela-BCMA) and placed in RPMI medium. The cell density was 1 × 10 4 The cells were prepared at 100 μl / ml. 100 μl of CAR-T cells and tumor cells were placed in a 96-well plate and co-cultured overnight. The supernatant was collected and centrifuged, and the supernatant was then harvested to detect cytokine release levels. The CBA method was used for detection.

[0184]

[0270] The results are shown in Figure 12. After stimulation with BCM-positive target cells, BCMA-C The CD19 CAR-T was able to secrete large amounts of cytokines, while the NT secreted only small amounts of cytokines. BCMA-CD19 CAR-T was shown to be able to be activated by BCMA.

[0185] Example 11 Upregulation of CD107 after stimulation

[0271] The CAR-T cells obtained in Example 8 were activated, and then the C107a expression The changes were analyzed by flow cytometry. Tumor cell lines expressing CD19 or BCMA were used in co-incubation activation experiments. After co-incubation, the cells were labeled with antibodies against CD3, CD8, and CD107a, followed by flow cytometry analysis.

[0186]

[0272] The results are shown in Table 13. Dual CAR-T cells were administered to BCMA-positive tumor target cells MM After co-culture with .1s and CD19-positive Raji, CD107a molecules on the surface of CAR-T cells were significantly upregulated.

[0187] Example 12: In vivo drug efficacy studies

[0273] Select 6- to 12-week-old NOG mice and inoculate them at 1 x 10 7 RPMI822 6 cells were subcutaneously injected. Two days later, tumor graft volume was measured. Ten days later, they were divided into groups with similar tumor burdens, and CAR-T cells were injected into each group one day after grouping. After CAR-T treatment, the tumor volume burden of the mice was assessed twice a week.

[0188]

[0274] From the results in Figure 14, CAR-S2 and CAR-S4 were It has been shown that the compounds can eliminate tumors in mice modeled subcutaneously with vesicles, demonstrating their remarkable antitumor efficacy.

[0189]

[0275] At the same time, select 6- to 12-week-old NOG mice and incubate them at 1 × 10 7 MM. 1s cells were intravenously injected. Tumor graft burden was detected, and mice were divided equally into groups according to tumor burden. CAR-T cells were injected one day after group division. After CAR-T treatment, the tumor volume burden of the mice was evaluated. Each mouse was intraperitoneally injected with 3 mg of d-luciferin (Perkin Elmer Life Sciences), and then photographed 4 minutes later with a 30-second exposure time using a Xenogen IVIS imaging system (Perkin Elmer Life Sciences). Bioluminescence signals were calculated according to the amount of photons emitted, and the amount of photons was normalized using the exposure time and surface area, and finally expressed as photons / s / cm. 2 / steradian (p / s / cm 2 / sr).

[0190]

[0276] The results in Figure 15 show that mice injected with dual CAR-T cells showed significantly improved survival compared to controls. The tumor burden was shown to be significantly reduced until it disappeared, indicating that the BCMA-CD19 CAR-T cells had a significant anti-tumor effect.

[0191] Example 13: Studies on the killing of tumor-forming cells

[0277] Relapse in patients with multiple myeloma (MM) is a common clinical phenomenon, and clinical findings include Generally, the majority of tumor cells can be eliminated, but clonal proliferation can occur, leading to tumor recurrence. Tumor cells capable of clonal proliferation tend to have relatively high drug resistance. To investigate the killing ability of CAR-T cells against tumor cells capable of clonal proliferation and to compare the advantages of bispecific CAR-T cells and single CAR-T cells, this study established a myeloma clonogenesis experiment method and investigated the inhibitory ability of CAR-T against clonogenesis.

[0192]

[0278] Proliferative MM tumor cells can be grown in clonal growth medium, but during experiments CD34+ hematopoietic stem cells capable of proliferation, which may interfere with the experiment, need to be removed. The important issue that needs to be controlled for this experiment is that the collected cells should be dominated by tumor-propagating cells.

[0193]

[0279] The specific experimental methods are as follows:

[0280] In the first step, bone marrow mononuclear cells are isolated and extracted using Ficoll; The phenotypes were analyzed by flow cytometry. In the second step, CD34+ cells were depleted using a CD34+ cell sorting kit. In the third step, killing experiments were performed on the resulting cells using various groups of CAR-T cells (double CAR-T and single CAR-T). After killing was completed, CAR-T cells were depleted using a T cell depletion sorting kit. In the fourth step, semi-solid cloning growth medium was used for clonal growth, and statistics, counting, and summary of results were performed after 1 to 2 weeks.

[0194]

[0281] The results are shown in Figure 16. CAR-S2 and CAR-S4 were significantly higher than CAR-19 and C CAR-S2 and CAR-S4 showed a greater ability to inhibit myeloma cell clonogenesis than single CARs, with a significant advantage over CAR-S1 in killing clonogenic or tumor cell precursor cells.

[0195] Example 14 In vivo vein modeling experiment of Nalm6

[0282] Select 6- to 12-week-old NOG mice and inoculate them at 1 x 10 7 Nalm6 cells Six days later, tumor graft burden was detected, and the mice were divided into groups according to tumor burden. CAR-T cells were injected one day after group division. After CAR-T treatment, the tumor burden of the mice was evaluated. Each mouse was injected with d-luciferin (Perkin Elmer). The cells were then intraperitoneally injected with 3 mg of luminescence imaging (Perkin Elmer Life Sciences) and subsequently photographed 4 minutes later with a 30-second exposure time using a Xenogen IVIS imaging system (Perkin Elmer Life Sciences). Bioluminescence signals were calculated according to the amount of photons emitted, which was normalized using the exposure time and surface area, and finally expressed as photons / s / cm. 2 / steradian (p / s / cm 2 / sr).

[0196]

[0283] The results are shown in Figure 17. Tumors in mice injected with CAR-S2 and CAR-S4 The amount was shown to be significantly reduced until it disappeared, and BCMA-CD19 CAR-T cells had a more pronounced anti-CD19-positive tumor effect than CAR-19.

[0197] Example 15: CAR-T cell safety switch experiment

[0284] CAR-T containing the EGFRt element was stained with an EGFR antibody and analyzed by flow cytometry. The cells were analyzed by cytometry and CAR expression was analyzed simultaneously.

[0198]

[0285] The results are shown in Figure 18. Safety switch expression was detected in CAR-T cells. . Example 16 Preparation of humanized CAR-T cells and detection of killing effect

[0286] Using the methods of Examples 3 and 4, humanized CAR-T cells (CAR-h We constructed humanized dual CAR-T cells (CAR-hS2 and CAR-hS4) and humanized dual CAR-T cells (CAR-hS1 and CAR-hS2). The structure of the humanized CAR-T cells was similar to that of CAR-19. The structure of AR-T cell CAR-hS2 is similar to that of CAR-S2, and the structure of CAR-hS4 is similar to that of CAR-S4, with the only difference being that a humanized CD19 scFv can be used to replace the mouse-derived scFv in the original structure. The humanized CD19 scFv comprises an antibody heavy chain variable region set forth in any one of SEQ ID NOs: 21 to 30 and an antibody light chain variable region set forth in any one of SEQ ID NOs: 31 to 36.

[0199]

[0287] Using the method of Example 9, humanized double CRA-T cells were cultured in vitro. The killing effect of

[0288] The in vitro killing effect is shown in Figures 19 and 20. Humanized CAR-T The cells and humanized dual CAR-T cells showed a significant killing effect on target cells without killing non-target cells.

[0200] Example 17: Study of in vivo drug efficacy of humanized CAR-T cells

[0289] Select 6- to 12-week-old NOG mice and inoculate them with 3 × 10 5 Raji cells The mice were subcutaneously injected. Six days later, the tumor graft burden was measured, and the mice were divided into groups with similar tumor burdens. The double CAR-T cells prepared as described above were injected into each group one day after the grouping. After CAR-T treatment, the tumor volume burden of the mice was evaluated. Each mouse was intraperitoneally injected with 3 mg of d-luciferin (Perkin Elmer Life Sciences), and then photographed 4 minutes later using a Xenogen IVIS imaging system (Perkin Elmer Life Sciences) with an exposure time of 30 seconds. The bioluminescence signal was calculated according to the amount of emitted photons, and the amount of photons was normalized using the exposure time and surface area, and finally expressed as photons / s / cm. 2 / steradian (p / s / cm 2 / sr).

[0201]

[0290] The results are shown in Figure 21. Humanized CAR-hS2 cells showed a higher R In mice modeled with aji cells, they had a stronger ability to eliminate tumors, demonstrating their remarkable antitumor efficacy.

[0202] Example 18 Killing of Raji Lymphoma Cells by CAR-T Cells

[0291] Luciferase-labeled Raji lymphoma target cells were used to detect killing activity. By transferring the luciferase gene into Raji target cells, a stably transfected cell line, Raji-Luc, was obtained after clonal screening. During the experiment, luciferase reacted with luciferin by adding luciferin substrate, producing fluorescence. The activity of luciferase could be measured by detecting the intensity of the fluorescence, and subsequently the cell survival rate and the killing effect of CAR-T cells could be determined.

[0203]

[0292] The results are shown in Figure 22. NT cells have no killing function, and CAR-S1 cells They had a dose-dependent killing effect on Raji-Luc cells (Raji cells transfected with the luciferase gene), demonstrating their potential value for indications such as lymphoma.

[0204]

[0293] All documents mentioned in this application are expressly incorporated by reference in their entirety, as if each document were individually incorporated by reference. The present application is incorporated by reference as if it were incorporated herein by reference. Furthermore, after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and it should be understood that equivalents thereof also fall within the scope defined by the appended claims of this application. This specification includes the following disclosure of the invention. [Item 1] A chimeric antigen receptor (CAR) in which the antigen-binding domain (scFv) comprises the antibody heavy chain variable region shown in SEQ ID NO: 9 and the antibody light chain variable region shown in SEQ ID NO: 10. [Item 2] The scFv is represented by the following formula A or B: VH-VL, (A); VL-VH, (B) (wherein VH is an antibody heavy chain variable region; VL is an antibody light chain variable region; and "-" is a linker peptide or peptide bond). Item 1. The CAR according to item 1, wherein the CAR is as shown in [Item 3] A bispecific CAR targeting BCMA and a first target, wherein the BCMA-targeting antigen-binding domain (scFv) in the bispecific CAR comprises an antibody heavy chain variable region shown in SEQ ID NO: 9 and an antibody light chain variable region shown in SEQ ID NO: 10; the first target is CD138, kappa light chain, NKG2D-ligand, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133 , CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, a bispecific CAR selected from the group consisting of KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCR alpha, TCR beta, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, claudin 18.2, folate receptor alpha, folate receptor beta, GPC2, CD70, BAFF-R, TROP-2, or a combination thereof. [Item 4] The bispecific CAR according to Item 3, wherein the first target is CD19, and the antigen-binding domain (scFv) in the bispecific CAR that targets CD19 comprises an antibody heavy chain variable region shown in any one of SEQ ID NOs: 11 and 21 to 30, and an antibody light chain variable region shown in any one of SEQ ID NOs: 12 and 31 to 36. [Item 5] Formula II below: L-scFv1-I-scFv2-H-TM-C-CD3ζ (II) (In the formula, each "-" is independently a linker peptide or a peptide bond; L is absent or is a signal peptide sequence; I is a flexible linker; H is absent or is the hinge region; TM is the transmembrane domain; C is a costimulatory signal molecule, CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; One of scFv1 and scFv2 is an antigen-binding domain that targets the first target, and the other is an antigen-binding domain that targets BCMA. 4. The bispecific CAR of item 3, having a structure as shown in [Item 6] Formula III or III' below: L-VL3-scFv3-VH3-H-TM-C-CD3ζ (III) L-VH3-scFv3-VL3-H1-TM-C-CD3ζ (III') (In the formula, each "-" is independently a linker peptide or a peptide bond; The elements L, H, TM, C, and CDζ are as described above; scFv3 is an antigen-binding domain targeting said BCMA, VH3 is an antibody heavy chain variable region directed against said first target, and VL3 is an antibody light chain variable region directed against said first target; or scFv3 is an antigen-binding domain that targets the first target, VH3 is a heavy chain variable region of an antibody against BCMA, and VL3 is a light chain variable region of an antibody against BCMA. 4. The bispecific CAR of item 3, having a structure as shown in [Item 7] A nucleic acid molecule encoding the CAR described in Item 1 or the bispecific CAR described in Item 3. [Item 8] A vector comprising the nucleic acid molecule described in Item 7. [Item 9] An engineered immune cell that contains the vector described in Item 8 or the nucleic acid molecule described in Item 7 exogenously integrated into a chromosome, or that expresses the CAR described in Item 1 or the bispecific CAR described in Item 3. [Item 10] The immune cell comprises an exogenous first expression cassette and a second expression cassette, the first expression cassette is used to express a first CAR that targets a first target, and the second expression cassette is used to express a second CAR that targets BCMA; or an immune cell expressing the first CAR that targets the first target and the second CAR that targets the BCMA; the BCMA-targeting antigen-binding domain (scFv) of the second CAR comprises an antibody heavy chain variable region set forth in SEQ ID NO: 9 and an antibody light chain variable region set forth in SEQ ID NO: 10; The first target is selected from the group consisting of CD138, kappa light chain, NKG2D-ligand, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB, bB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TG FBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, TWEAK-R, LTPR, L IFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, claudin 18.2, folate receptor α, folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or a combination thereof selected from the group consisting of Engineered immune cells. [Item 11] A formulation comprising the CAR described in Item 1, the bispecific CAR described in Item 3, or the engineered immune cell described in Item 9 or Item 10, and a pharmaceutically acceptable carrier, diluent, or excipient. [Item 12] Use of the CAR described in Item 1, the bispecific CAR described in Item 3, or the engineered immune cell described in Item 9 or Item 10 in the preparation of a drug or formulation for preventing and / or treating cancer or tumors. [Item 13] The use of item 12, wherein the drug or preparation treats the cancer or tumor by killing tumor cells capable of clonal proliferation. [Item 14] The use described in Item 13, wherein the tumor cells capable of clonal proliferation include clonogenic cells, tumor cell precursor cells, and tumor progenitor cells.

Claims

1. 1. A polynucleotide encoding a bispecific chimeric antigen receptor (CAR) polypeptide, wherein the CAR polypeptide comprises: (i) a first heavy chain variable region (V) comprising the complementarity determining regions (CDRs) of a polypeptide sequence selected from the group consisting of SEQ ID NOs: 11 and 21-30; Ｈ，ＣＤ１９ ) and (ii) a first light chain variable region (V) comprising the CDRs of a polypeptide sequence selected from the group consisting of SEQ ID NOs: 12 and 31-36. Ｌ，ＣＤ１９ a first antigen-binding domain exhibiting specific binding to CD19, comprising: (i) a second heavy chain variable region (V) comprising the CDRs of the polypeptide sequence of SEQ ID NO: 9; Ｈ，ＢＣＭＡ ) and (ii) a second light chain variable region (V) comprising the CDRs of the polypeptide sequence of SEQ ID NO:

10. Ｌ，ＢＣＭＡ a second antigen-binding domain that exhibits specific binding to BCMA, comprising The second antigen-binding domain is V Ｈ，ＣＤ１９ and V Ｌ，ＣＤ１９ The polynucleotide is configured to be adjacent to

2. V Ｈ，ＢＣＭＡ comprises the polypeptide sequence of SEQ ID NO: 9, and V Ｌ，ＢＣＭＡ The polynucleotide of claim 1, comprising the polypeptide sequence of SEQ ID NO:

10.

3. V Ｈ，ＣＤ１９ comprises the polypeptide sequence of SEQ ID NO: 11, and V Ｌ，ＣＤ１９ The polynucleotide of claim 1, comprising the polypeptide sequence of SEQ ID NO:

12.

4. V Ｌ，ＣＤ１９ is located upstream of the N-terminus of the second antigen-binding domain, and V Ｈ，ＣＤ１９ is located downstream of the C-terminus of the second antigen-binding domain.

5. The bispecific CAR polypeptide has the formula I: L-V Ｌ，ＣＤ１９ -V Ｈ，ＢＣＭＡ -V Ｌ，ＢＣＭＡ -V Ｈ，ＣＤ１９ -H-TM-C ２ -C １ (I) wherein: - are each independently a linker peptide or a peptide bond; L is absent or is a signal domain; H is absent or is a hinge domain; TM is the transmembrane domain; C ２ is a costimulatory signal domain; C １ The polynucleotide of claim 1 , wherein:

6. L comprises the polypeptide sequence of SEQ ID NO: 16; V Ｌ，ＣＤ１９ comprises the polypeptide sequence of SEQ ID NO: 12; V Ｈ，ＢＣＭＡ comprises the polypeptide sequence of SEQ ID NO:9; V Ｌ，ＢＣＭＡ comprises the polypeptide sequence of SEQ ID NO: 10; V Ｈ，ＣＤ１９ comprises the polypeptide sequence of SEQ ID NO: 11; H comprises the polypeptide sequence of SEQ ID NO: 38; the TM comprises the polypeptide sequence of SEQ ID NO:7; C ２ comprises the polypeptide sequence of SEQ ID NO:5; and C １ The polynucleotide of claim 5, comprising the polypeptide sequence of SEQ ID NO:

3.

7. (i) V via a linker peptide comprising the polypeptide sequence of SEQ ID NO: 40 Ｌ，ＣＤ１９ and V Ｈ，ＢＣＭＡ is coupled; (ii) V via a linker peptide comprising the polypeptide sequence of SEQ ID NO: 19 Ｈ，ＢＣＭＡ and V Ｌ，ＢＣＭＡ couples; and (iii) V via a linker peptide comprising the polypeptide sequence of SEQ ID NO: 40 Ｌ，ＢＣＭＡ and V Ｈ，ＣＤ１９ The polynucleotide of claim 6, wherein:

8. 2. The polynucleotide of claim 1, wherein engineered immune cells expressing the bispecific CAR polypeptide are characterized by exhibiting greater cytotoxicity against target cells expressing BCMA compared to control engineered immune cells expressing a CAR polypeptide comprising a first antigen-binding domain and a second antigen-binding domain in tandem.

9. 2. The polynucleotide of claim 1, wherein engineered immune cells expressing the bispecific CAR polypeptide are characterized by exhibiting greater cytotoxicity against target cells expressing CD19 compared to control engineered immune cells expressing a CAR polypeptide comprising a first antigen-binding domain and a second antigen-binding domain in tandem.

10. A vector comprising the polynucleotide according to any one of claims 1 to 9.

11. The vector of claim 10, which is a lentiviral vector.

12. 12. The vector of claim 10 or 11, wherein the polynucleotide is operably linked to one or more regulatory elements suitable for expression in human T cells.

13. An engineered immune cell comprising the polynucleotide of any one of claims 1 to 12.

14. 14. The engineered immune cell of claim 13, which is an engineered T cell.

15. 15. A pharmaceutical composition comprising the engineered immune cells of claim 13 or 14 for use in a method for treating a subject in need thereof, the method comprising administering the composition to the subject.

16. 16. The pharmaceutical composition of claim 15, wherein the engineered immune cells are derived from the subject's autologous cells.

17. 16. The pharmaceutical composition of claim 15, wherein the engineered immune cells are allogeneic to the subject.

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