Engineered immune cell targeting bcma and use thereof

Abstract

The present invention provides an engineered immune cell targeting BCMA and use thereof. In particular, the present invention provides a 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 as shown in SEQ ID NO: 9 and an antibody light chain variable region as shown in SEQ ID NO: 10. The present invention also provides a CAR-T cell comprising the CAR, a double CAR and CAR T cell comprising the S-derived scFv, and related use thereof. Compared to CAR-T cells constructed using other scFvs, the constructed CAR-T cell of the present invention has a better killing effect and tumor elimination capability.

Description

Engineered immune cells targeting BCMA and their applications Technical Field

[0001] This invention relates to the field of immunotherapy, and more specifically to an engineered immune cell that targets BCMA and its uses. Background Technology

[0002] Multiple myeloma (MM) is a malignant plasma cell tumor whose tumor cells originate from plasma cells in the bone marrow. Plasma cells are B lymphocytes that have reached their final functional stage. Multiple myeloma is essentially incurable, characterized by high morbidity and mortality. In 2017, the United States had 30,000 newly diagnosed cases of multiple myeloma, with an estimated 12,000 facing death. Current common treatments for multiple myeloma include cytotoxic drugs, protease inhibitors (such as bortezomib), lenalidomide, monoclonal antibodies, and corticosteroids. However, these treatments are only partially effective, do not provide lasting remission, and have a high relapse rate. Therefore, improvements in multiple myeloma treatment are crucial.

[0003] Therefore, there is an urgent need in the field for an effective and safe treatment for multiple myeloma with a low recurrence rate.

[0004] Summary of the Invention

[0005] The purpose of this invention is to provide an engineered immune cell that targets BCMA and its uses.

[0006] Another object of the present invention is to provide an engineered immune cell that simultaneously targets CD19 and BCMA and its uses.

[0007] In a first aspect of the invention, a chimeric antigen receptor (CAR) or TCR is provided, wherein the antigen-binding domain (scFv) of the CAR or TCR includes 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.

[0008] In another preferred embodiment, the scFv further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0009] In another preferred embodiment, the scFv is shown in equation A or equation B as follows:

[0010] V H -V L , (A); V L -V H (B)

[0011] In the formula, V H V is the variable region of the antibody heavy chain;L "-" represents the variable region of the antibody light chain; "-" represents a linking peptide or peptide bond.

[0012] In another preferred embodiment, the V H and V L The linking peptides are 1-4 consecutive sequences as shown in SEQ ID NO:7 (GGGGS), preferably 1-4, more preferably 3-4.

[0013] In another preferred embodiment, the structure of the CAR is shown in Equation I:

[0014] L-scFv-H-TM-C-CD3ζ (I)

[0015] In the formula,

[0016] Each "-" independently represents a linking peptide or peptide bond;

[0017] L represents the absence of a signal peptide sequence;

[0018] H represents the area with no hinge or no connection.

[0019] TM represents a transmembrane domain;

[0020] C is a co-stimulatory signaling molecule;

[0021] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0022] In a second aspect of the invention, a bispecific CAR or TCR is provided, the bispecific CAR or TCR targeting BCMA and a first target.

[0023] The antigen-binding domain (scFv) targeting BCMA in the bispecific CAR includes 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.

[0024] Furthermore, the first target is selected from the following group:

[0025] CD138、Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD 28. CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD1 23. 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, 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, N Y-ESO-1, PSMA, RANK, RORl, 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 receptorα, Folate receptorβ, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof. In another preferred embodiment, the bispecific CAR or TCR includes an antigen-binding domain targeting CD19.

[0026] In another preferred embodiment, the first target is CD19, and the antigen-binding domain (scFv) targeting CD19 in the bispecific CAR includes the antibody heavy chain variable region shown in SEQ ID NO:11 and the antibody light chain variable region shown in SEQ ID NO:12.

[0027] In another preferred embodiment, the first target is CD19, and the antigen-binding domain (scFv) targeting CD19 in the bispecific CAR includes the antibody heavy chain variable region shown in any of SEQ ID NO:21-30 and the antibody light chain variable region shown in any of SEQ ID NO:31-36.

[0028] The specific sequence is shown below.

[0029] The CD19 antibody heavy chain variable region (H9) shown in SEQ ID NO:21

[0030]

[0031] The CD19 antibody heavy chain variable region (H1) shown in SEQ ID NO:22

[0032]

[0033] The CD19 antibody heavy chain variable region (H8) shown in SEQ ID NO:23

[0034]

[0035] The CD19 antibody heavy chain variable region (H10) shown in SEQ ID NO:24

[0036]

[0037] The CD19 antibody heavy chain variable region (H2) shown in SEQ ID NO:25

[0038]

[0039] The CD19 antibody heavy chain variable region (H3) shown in SEQ ID NO:26

[0040]

[0041] The CD19 antibody heavy chain variable region (H4) shown in SEQ ID NO:27

[0042]

[0043] The CD19 antibody heavy chain variable region (H5) shown in SEQ ID NO:28

[0044]

[0045] The CD19 antibody heavy chain variable region (H6) shown in SEQ ID NO:29

[0046]

[0047] The CD19 antibody heavy chain variable region (H7) shown in SEQ ID NO:30

[0048]

[0049] The CD19 antibody light chain variable region (L5) shown in SEQ ID NO:31

[0050]

[0051] The CD19 antibody light chain variable region (L1) shown in SEQ ID NO:32

[0052]

[0053] The CD19 antibody light chain variable region (L6) shown in SEQ ID NO:33

[0054]

[0055] The CD19 antibody light chain variable region (L2) shown in SEQ ID NO:34

[0056]

[0057] The CD19 antibody light chain variable region (L3) shown in SEQ ID NO:35

[0058]

[0059] The CD19 antibody light chain variable region (L4) shown in SEQ ID NO:36

[0060]

[0061] In another preferred embodiment, the bispecific CAR simultaneously includes an antigen-binding domain targeting the first target and an antigen-binding domain targeting the BCMA.

[0062] In another preferred embodiment, the structure of the bispecific CAR is shown in Formula II:

[0063] L-scFv1-I-scFv2-H-TM-C-CD3ζ (II)

[0064] In the formula,

[0065] Each "-" independently represents a linking peptide or peptide bond;

[0066] L represents the absence of a signal peptide sequence;

[0067] I represents a flexible joint;

[0068] H represents the area with no hinge or no connection.

[0069] TM represents a transmembrane domain;

[0070] C is a co-stimulatory signaling molecule;

[0071] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ;

[0072] Of the two, scFv1 and scFv2, one is an antigen-binding domain that targets the first target, and the other is an antigen-binding domain that targets BCMA.

[0073] In another preferred embodiment, scFv1 and scFv2 can be independent, connected in series, or in a loop structure.

[0074] In another preferred embodiment, scFv1 is an antigen-binding domain targeting a first target, and scFv2 is an antigen-binding domain targeting BCMA.

[0075] In another preferred embodiment, scFv1 is an antigen-binding domain targeting BCMA, and scFv2 is an antigen-binding domain targeting a first target.

[0076] In another preferred embodiment, the sequence of the flexible joint I comprises 1-6, preferably 3-5 consecutive sequences as shown in SEQ ID NO:7 (GGGGS).

[0077] In another preferred embodiment, the flexible connector I has a sequence as shown in SEQ ID NO:17, 18 or 19.

[0078] In another preferred embodiment, the structure of the antigen-binding domain targeting the first target is shown in formula C or formula D as follows:

[0079] V L1 -V H1 (C); V H1 -V L1 (D)

[0080] Among them, V L1 For the variable region of the light chain of the antibody against the first target; V H1 This represents the variable region of the heavy chain of the antibody against the first target; "-" indicates a linking peptide or peptide bond.

[0081] In another preferred embodiment, the structure of the CD19-targeting antigen-binding domain is shown in formula C or formula D as follows:

[0082] V L1 -VH1 (C); V H1 -V L1 (D)

[0083] Among them, V L1 For the variable region of the light chain of the anti-CD19 antibody; V H1 This is the variable region of the heavy chain of the anti-CD19 antibody; "-" indicates a linking peptide or peptide bond.

[0084] In another preferred embodiment, the antigen-binding domain targeting CD19 includes the heavy chain variable region and the light chain variable region of the monoclonal antibody FMC63.

[0085] In another preferred embodiment, the variable region of the anti-CD19 antibody heavy chain has an amino acid sequence as shown in SEQ ID NO:11.

[0086] In another preferred embodiment, the variable region of the anti-CD19 antibody light chain has an amino acid sequence as shown in SEQ ID NO:12.

[0087] In another preferred embodiment, the structure of the antigen-binding domain targeting BCMA is shown in formula A or formula B as follows:

[0088] V H -V L , (A); V L -V H (B)

[0089] In the formula, V H V is the variable region of the antibody heavy chain; L "-" represents the variable region of the antibody light chain; "-" represents a linking peptide or peptide bond.

[0090] In another preferred embodiment, the scFv1 includes an antibody heavy chain variable region as shown in SEQ ID NO:11 and an antibody light chain variable region as shown in SEQ ID NO:12; and the scFv2 includes an antibody heavy chain variable region as shown in SEQ ID NO:9 and an antibody light chain variable region as shown in SEQ ID NO:10.

[0091] In another preferred embodiment, the scFv1 includes an antibody heavy chain variable region as shown in SEQ ID NO:9 and an antibody light chain variable region as shown in SEQ ID NO:10; and the scFv2 includes an antibody heavy chain variable region as shown in SEQ ID NO:11 and an antibody light chain variable region as shown in SEQ ID NO:12.

[0092] In another preferred embodiment, the scFv1 and / or scFv2 are murine, human, human-mice chimeric, or fully humanized single-chain antibody variable region fragments.

[0093] In another preferred embodiment, the structure of the bispecific CAR is shown in formula III or III':

[0094] LV L3 -scFv3-V H3 -H-TM-C-CD3ζ (III)

[0095] LV H3 -scFv3-V L3 -H1-TM-C-CD3ζ (III')

[0096] In the formula,

[0097] Each "-" independently represents a linking peptide or peptide bond;

[0098] Components L, H, TM, C, and CD3ζ are as described above;

[0099] scFv3 is an antigen-binding domain that targets BCMA, V H3 For the variable region of the heavy chain of the antibody against the first target, and V L3 The variable region of the light chain of the antibody against the first target; or scFv3 is the antigen-binding domain targeting the first target, V H3 It is the variable region of the heavy chain of the anti-BCMA antibody, and V L3 This is the variable region of the light chain of the anti-BCMA antibody.

[0100] In another preferred embodiment, the scFv3 includes 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.

[0101] In another preferred embodiment, the V H3 It has an antibody heavy chain variable region as shown in SEQ ID NO:9, and V L3 It has the antibody light chain variable region as shown in SEQ ID NO:10.

[0102] In another preferred embodiment, the scFv3 comprises an antibody heavy chain variable region as shown in SEQ ID NO:11 and an antibody light chain variable region as shown in SEQ ID NO:12; and the V H3 It has an antibody heavy chain variable region as shown in SEQ ID NO:9, and V L3 It has the antibody light chain variable region as shown in SEQ ID NO:10.

[0103] In another preferred embodiment, the scFv3 comprises an antibody heavy chain variable region as shown in SEQ ID NO:9 and an antibody light chain variable region as shown in SEQ ID NO:10; and the V H3 It has the antibody heavy chain variable region as shown in SEQ ID NO:11, and V L3 It has the antibody light chain variable region as shown in SEQ ID NO:12.

[0104] In another preferred embodiment, the structure of the CAR is shown in Figure 1.

[0105] 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.

[0106] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0107] In another preferred embodiment, the L has an amino acid sequence as shown in SEQ ID NO:16 or 1.

[0108] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or combinations thereof. In yet another preferred embodiment, each H is an independent hinge region derived from CD8.

[0109] In another preferred embodiment, H has an amino acid sequence as shown in SEQ ID NO:8.

[0110] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof. In another preferred embodiment, each of the TMs is independently a transmembrane region derived from CD8 or CD28. In another preferred embodiment, the CD8-derived transmembrane region has the amino acid sequence shown in SEQ ID NO:7.

[0111] In another preferred embodiment, the transmembrane region derived from CD28 has an amino acid sequence as shown in SEQ ID NO:6.

[0112] In another preferred embodiment, C is a co-stimulatory signaling molecule 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 combinations thereof. In another preferred embodiment, C is a co-stimulatory signaling molecule derived from CD28 and / or 4-1BB.

[0113] In another preferred embodiment, the 4-1BB-derived co-stimulatory signaling molecule has an amino acid sequence as shown in SEQ ID NO:5.

[0114] In another preferred embodiment, the CD28-derived co-stimulatory signaling molecule has an amino acid sequence as shown in SEQ ID NO:4.

[0115] In another preferred embodiment, the CD3ζ has an amino acid sequence as shown in SEQ ID NO:3.

[0116] In another preferred embodiment, the CAR (preferably C-terminus or N-terminus) further includes a cell suicide element.

[0117] In another preferred embodiment, the cell suicide element is connected to the L or CD3ζ of the CAR or the bispecific CAR via T2A.

[0118] In a third aspect of the invention, a nucleic acid molecule is provided, said nucleic acid molecule encoding the CAR or TCR described in the first aspect of the invention or the bispecific CAR or TCR described in the second aspect of the invention.

[0119] In a fourth aspect of the invention, a carrier is provided, said carrier containing the nucleic acid molecule described in the third aspect of the invention.

[0120] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0121] In another preferred embodiment, the vector is a lentiviral vector.

[0122] In a fifth aspect of the invention, a host cell is provided, said host cell containing the vector described in the fourth aspect of the invention, or having an exogenous nucleic acid molecule described in the third aspect of the invention integrated into the chromosome, or expressing the CAR or TCR described in the first aspect of the invention or the bispecific CAR or TCR described in the second aspect of the invention.

[0123] In a sixth aspect of the invention, an engineered immune cell is provided, said immune cell containing the vector described in the fourth aspect of the invention, or having an exogenous nucleic acid molecule described in the third aspect of the invention integrated into the chromosome, or expressing the CAR or TCR described in the first aspect of the invention or the bispecific CAR or TCR described in the second aspect of the invention.

[0124] In another preferred embodiment, the immune cells have one or more characteristics selected from the group consisting of:

[0125] (a) The PD-1 gene expression of the immune cells was silenced;

[0126] (b) The immune cells are T cells, and the TCR gene expression of the T cells is silenced; and

[0127] (c) The immune cells express exogenous cell suicide elements;

[0128] (d) The immune cells express or secrete PD-1 antibody, PD-L1 antibody, CD47 antibody, Tim3 antibody, Lag3 antibody, Tigit antibody, OX40 antibody, ICOS antibody, IL7, CXCL19, IL21, IL15, IL2, IL18, or combinations thereof; and

[0129] (e) The cytokine-related signaling pathways of the immune cells are enhanced, wherein the cytokines are selected from the group consisting of IL7, CXCL19, IL21, IL15, IL2, IL18, or combinations thereof.

[0130] In another preferred embodiment, the engineered immune cells are selected from the group consisting of:

[0131] (i) Chimeric antigen receptor T cells (CAR-T cells); or

[0132] (ii) Chimeric antigen receptor NK cells (CAR-NK cells).

[0133] In another preferred embodiment, the immune cells express exogenous cell suicide elements.

[0134] In another preferred embodiment, the CAR is co-expressed with the cell suicide element in the immune cells.

[0135] In another preferred embodiment, the CAR is connected to the cell suicide element via a self-cleaving element.

[0136] In another preferred embodiment, the cell suicide element is located at the N-terminus or C-terminus of the CAR.

[0137] In another preferred embodiment, the self-cutting element comprises a 2A sequence or an IRES sequence, preferably P2A and T2A.

[0138] In another preferred embodiment, the cell suicide element is selected from the group consisting of HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or combinations thereof.

[0139] In another preferred embodiment, the structure of the cell suicide element is shown in Formula IV:

[0140] L2-DF(IV)

[0141] In the formula,

[0142] Each "-" independently represents a linking peptide or peptide bond;

[0143] L2 is an optional signal peptide sequence;

[0144] D is a suicide switch element;

[0145] F represents a transmembrane element.

[0146] In another preferred embodiment, the signal peptide is a signal peptide derived from GM-CSFR.

[0147] In another preferred embodiment, the cell suicide element is selected from the group consisting of: truncated epidermal growth factor receptor (EGFRt), truncated CD19 (CD19t) gene, induced caspase 9 gene (iCasp9), HSV-TK, ΔCD20, mTMPK, or combinations thereof.

[0148] In another preferred embodiment, the cell suicide element is EGFRt.

[0149] In another preferred embodiment, the engineered immune cells are used for autologous immunotherapy and / or allogeneic immunotherapy.

[0150] In another preferred embodiment, the engineered immune cells can kill tumor cells with clonal proliferation capabilities.

[0151] In another preferred embodiment, immune cells expressing the bispecific CAR of the second aspect have a longer in vivo survival time compared to immune cells expressing the CAR of the first aspect of the invention.

[0152] In another preferred embodiment, the body includes either the autologous body or the allogeneic body.

[0153] In a seventh aspect of the invention, an engineered immune cell is provided, the immune cell comprising an exogenous first expression cassette and a second expression cassette, wherein the first expression cassette is used to express a first CAR or a first exogenous TCR targeting a first target, and the second expression cassette is used to express a second CAR or a second exogenous TCR targeting BCMA.

[0154] Alternatively, the immune cells may express the first CAR or first exogenous TCR targeting the first target and the second CAR or second exogenous TCR targeting BCMA;

[0155] The antigen-binding domain (scFv) targeting BCMA in the second CAR or the second exogenous TCR includes 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.

[0156] Furthermore, the first target is selected from the following group:

[0157] CD138、Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD 57. 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, 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, N Y-ESO-1, PSMA, RANK, RORl, 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 receptorα, Folate receptorβ, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

[0158] In another preferred embodiment, the first target is CD19, and the antigen-binding domain (scFv) targeting CD19 in the first CAR includes the antibody heavy chain variable region shown in SEQ ID NO:11 and the antibody light chain variable region shown in SEQ ID NO:12.

[0159] In another preferred embodiment, the second CAR is the CAR described in the first aspect of the present invention.

[0160] In another preferred embodiment, the first CAR and the second CAR are located on the cell membrane of the immune cell.

[0161] In another preferred embodiment, the immune cells express a first CAR targeting CD19 and a second CAR targeting BCMA on their cell membranes.

[0162] In another preferred embodiment, the first expression box and the second expression box are located on the same or different carriers.

[0163] In another preferred embodiment, the first expression box and the second expression box are located on the same carrier.

[0164] In another preferred embodiment, the structure of the first CAR is shown in equation V:

[0165] L-scFv1'-H-TM-C-CD3ζ (V)

[0166] In the formula,

[0167] Each "-" independently represents a linking peptide or peptide bond;

[0168] Components L, H, TM, C, and CD3ζ are as described above;

[0169] scFv1' is an antigen-binding domain that targets CD19.

[0170] In another preferred embodiment, the first CAR and the second CAR are linked by a 2A peptide.

[0171] In another preferred embodiment, the sequence of the 2A peptide is shown in SEQ ID NO:2.

[0172] In another preferred embodiment, the immune cells also include a cell suicide element.

[0173] In another preferred embodiment, the cell suicide element is connected (or tandemly) with the bispecific CAR via T2A.

[0174] In another preferred embodiment, the cell suicide element is connected to the first CAR and / or the second CAR via a T2A.

[0175] In another preferred embodiment, the PD1 gene expression of the immune cells is silenced.

[0176] In another preferred embodiment, "PD-1 gene expression is silenced" means that the PD-1 gene is not expressed or is expressed at low levels.

[0177] In another preferred embodiment, "low expression" refers to the ratio of the expression level G1 of the PD-1 gene on immune cells to the expression level G0 of the PD-1 gene on normal immune cells, i.e., G1 / G0≤0.5, preferably G1 / G0≤0.3, more preferably ≤0.2, even more preferably ≤0.1, and most preferably 0.

[0178] In another preferred embodiment, "low expression" refers to the ratio of the expression level G1 of the PD-1 gene in the CAR-T cells to the expression level G0 of the PD-1 gene in normal T cells, i.e., G1 / G0≤0.5, preferably G1 / G0≤0.3, more preferably ≤0.2, even more preferably ≤0.1, and most preferably 0.

[0179] In an eighth aspect of the invention, a formulation is provided comprising a CAR or TCR as described in the first or second aspect of the invention, or engineered immune cells as described in the sixth or seventh aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0180] In another preferred embodiment, the formulation is a liquid formulation.

[0181] In another preferred embodiment, the dosage form of the preparation is an injection.

[0182] In another preferred embodiment, the concentration of the engineered immune cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, preferably 1×10⁻⁶ 4 -1×10 7 Cells / ml

[0183] In another preferred embodiment, the CAR comprises a bispecific CAR.

[0184] In a ninth aspect of the invention, the use of CAR or TCR as described in the first or second aspect of the invention, or engineered immune cells as described in the sixth or seventh aspect of the invention, is provided for the preparation of medicaments or formulations for the prevention and / or treatment of cancer or tumors.

[0185] In another preferred embodiment, the tumor is a hematologic tumor.

[0186] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

[0187] In another preferred embodiment, the cancer or tumor is multiple myeloma.

[0188] In another preferred embodiment, the cancer or tumor is lymphoma.

[0189] In another preferred embodiment, the lymphoma is selected from the group consisting of: Hodgkin lymphoma (HL), 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 lymphoma (BL), and complex B-cell non-Hodgkin lymphoma.

[0190] In another preferred embodiment, the cancer or tumor includes recurrent cancer or tumor.

[0191] In another preferred embodiment, the drug or preparation treats cancer or tumors by killing tumor cells with clonal proliferation capabilities.

[0192] In another preferred embodiment, the tumor cells with clonal proliferation capacity include clonogenic cells, tumor cell precursor cells, and tumor progenitor cells.

[0193] In a tenth aspect of the present invention, a method for preparing engineered immune cells expressing the CAR or TCR described in the first or second aspect of the present invention is provided, comprising the following steps: transducing the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention into the immune cells, thereby obtaining the engineered immune cells.

[0194] In another preferred embodiment, the immune cells are T cells or NK cells.

[0195] In an eleventh aspect of the present invention, a method for preparing engineered immune cells is provided, comprising the following steps:

[0196] (1) Provide an immune cell to be modified; and

[0197] (2) Introducing a first expression cassette for expressing a first CAR targeting a first target into the immune cells; and

[0198] (3) The second expression cassette for expressing the second CAR targeting BCMA is introduced into the immune cells to obtain the engineered immune cells.

[0199] The antigen-binding domain (scFv) targeting BCMA in the second CAR includes 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.

[0200] Furthermore, the first target is selected from the following group:

[0201] CD138、Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD 28. CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD1 23. 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, 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, N Y-ESO-1, PSMA, RANK, RORl, 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 receptorα, Folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

[0202] In another preferred embodiment, step (2) may be performed before, after, simultaneously with, or alternately with step (3).

[0203] In another preferred embodiment, if the immune cells to be modified in step (1) have already expressed the first CAR or the second CAR, then step (2) or step (3) can be omitted.

[0204] In a twelfth aspect of the invention, a kit is provided for preparing engineered immune cells as described in the sixth or seventh aspect of the invention, and the kit contains a container and a nucleic acid molecule as described in the third aspect of the invention, or a carrier as described in the fourth aspect of the invention, located within the container.

[0205] In a thirteenth aspect of the invention, a kit is provided for preparing engineered immune cells as described in the sixth or seventh aspect of the invention, and the kit includes a container, and the following are located within the container:

[0206] (1) A first nucleic acid sequence, the first nucleic acid sequence containing a first expression cassette, the first expression cassette being used to express a first CAR targeting a first target; and

[0207] (2) A second nucleic acid sequence, the second nucleic acid sequence containing a second expression cassette, the second expression cassette being used to express the second CAR targeting BCMA;

[0208] The antigen-binding domain (scFv) targeting BCMA in the second CAR includes 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.

[0209] Furthermore, the first target is selected from the following group:

[0210] CD138、Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD 28. CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD1 23. 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, 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, N Y-ESO-1, PSMA, RANK, RORl, 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 receptorα, Folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

[0211] In another preferred embodiment, the first and second nucleic acid sequences are located in the same or different containers.

[0212] In another preferred embodiment, the first and second nucleic acid sequences are located in the same expression vector.

[0213] In a fourteenth aspect of the invention, the use of engineered immune cells as described in the sixth or seventh aspect of the invention is provided for the prevention and / or treatment of cancer or tumors.

[0214] In another preferred embodiment, the cancer or tumor is multiple myeloma.

[0215] In a fifteenth aspect of the invention, a method for treating a disease is provided, comprising administering an appropriate amount of the cells described in the sixth or seventh aspect of the invention, or the preparation described in the fifth aspect of the invention, to a subject requiring treatment.

[0216] In another preferred embodiment, the disease is cancer or a tumor.

[0217] In a sixteenth aspect of the invention, a method is provided to enhance the in vivo survival ability of immune cells or to enhance the killing ability of immune cells against tumor cells with clonal proliferation capacity, comprising (a) simultaneously expressing an exogenous first expression cassette and a second expression cassette in the immune cells, wherein the first expression cassette is used to express a first CAR targeting CD19 and the second expression cassette is used to express a second CAR targeting BCMA; or (b) expressing the bispecific CAR of the second aspect in the immune cells.

[0218] In another preferred embodiment, the immune cells constructed by the method are as described in the sixth and seventh aspects of the present invention.

[0219] In another preferred embodiment, the first expression box and the second expression box have the same meaning as the first expression box and the second expression box in the seventh aspect of the present invention.

[0220] In another preferred embodiment, the body includes either the autologous body or the allogeneic body.

[0221] In a seventeenth aspect of the invention, a method is provided to enhance the in vivo survival or killing ability of engineered immune cells targeting BCMA against tumor cells with clonal proliferation capacity, comprising expressing an exogenous first expression cassette in the engineered immune cells, the first expression cassette being used to express a first CAR targeting CD19.

[0222] In another preferred embodiment, the first expression box has the same meaning as the first expression box and the second expression box in the seventh aspect of the present invention.

[0223] In another preferred embodiment, the engineered immune cells targeting BCMA are immune cells expressing the CAR described in the first aspect of the present invention.

[0224] In another preferred embodiment, the body includes either the autologous body or the allogeneic body.

[0225] In an eighteenth aspect of the invention, a first expression cassette is provided for expressing a first CAR targeting CD19, for enhancing the in vivo survival of engineered immune cells targeting BCMA or their ability to kill tumor cells with clonal proliferation capacity, or for preparing a kit for enhancing the in vivo survival of engineered immune cells targeting BCMA or their ability to kill tumor cells with clonal proliferation capacity.

[0226] In another preferred embodiment, the body includes either the autologous body or the allogeneic body.

[0227] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0228] Figure 1 shows a schematic diagram of the structure containing the CAR and cell suicide element of the present invention. The bispecific CAR and the suicide switch element are connected via 2A.

[0229] Figure 2 shows the flow cytometry analysis results of CAR-BB and CAR-S1 expression on the surface of Jurkat cells and primary T cells, respectively, in this invention.

[0230] Figure 3 shows the killing results of CAR-BB and CAR-S1 on HeLa cells and BCMA-overexpressing cells (HeLa-BCMA) in this invention (RTCA method), and the killing results of CAR-April and CAR-S1 on HeLa cells and BCMA-overexpressing cells (HeLa-BCMA) in this invention (RTCA method).

[0231] Figure 4 shows the BCMA expression in the target cells used in this invention.

[0232] Figure 5 shows the in vitro killing results (Luciferase method) of different batches of CAR-BB and CAR-S1 on MM.1s cells and RPMI-8226 cells, respectively.

[0233] Figure 6 shows the release of IFNr cytokines during the killing of HeLa cells and BCMA-overexpressing cells (HeLa-BCMA) by CAR-BB and CAR-S1, respectively, in this invention.

[0234] Figure 7 shows the tumor elimination ability of CAR-BB and CAR-S1 after intravenous reinfusion in the subcutaneous RPMI-8226 model of immunodeficient mice in this invention.

[0235] Figure 8 shows the expression of CD19-CAR and BCMA-CAR in dual-specific CAR-T cells in this invention.

[0236] Figure 9 shows the expression of CD19-CAR and BCMA-CAR and the expression of EGFRt in dual-specific CAR-T cells with added safety switches in this invention.

[0237] Figure 10 shows a comparison of the killing effects of different batches of CAR-19, CAR-BCMA, and bispecific CAR-T on HeLa and HeLa-overexpressing antigen cell lines HeLa-BCMA, HeLa-CD19, and HeLa-BCMA-CD19 in this invention.

[0238] Figure 11 shows the in vitro killing results (Luciferase method) of different batches of CAR-CD19, CAR-BCMA and bispecific CAR-T on MM.1s cells, RPMI-8226 cells and Nalm6 cells.

[0239] Figure 12 shows the release of cytokines during the killing of HeLa-overexpressing BCMA cells (HeLa-BCMA) by CAR-BCMA and bispecific CAR-T cells in this invention.

[0240] Figure 13 Analysis of CD107a molecule expression on the surface of dual CAR-T cells after co-culturing with MM.1s or Raji tumor target cells.

[0241] Figure 14 shows the tumor elimination ability of CAR-S1 and bispecific CAR-S2 and CAR-S4 after intravenous reinfusion in the RPMI-8226 subcutaneous model of immunodeficient mice in this invention.

[0242] Figure 15 shows the tumor elimination capacity of different doses of the bispecific CAR-S2 and CAR-S4 cells of the present invention after intravenous infusion into an immunodeficient mouse MM.1s-luc intravenous model.

[0243] Figure 16 shows the ability of different CAR-T cells to inhibit the clonogenic formation of CD34-negative monocytes in the bone marrow of MM patients.

[0244] Figure 17 shows the in vivo tumor clearance ability of different CAR-T cells in Nalm6-Luc cell-induced NOG mice.

[0245] Figure 18 shows the expression of CAR and safety switch on the surface of T cells in CAR-T cells.

[0246] Figure 19 shows the killing effect of different CAR-T cells on Nalm6 or RMPI8226 cells (Luciferase assay). Dual CAR pairs have a stronger ability to kill target cells compared to single CAR-T cells. Figure 20 shows that different CAR-T cells do not have the ability to kill negative target cells (K562, Raji-KO19, Nalm6-KO19, CCRF).

[0247] Figure 21. In vivo tumor elimination capacity of different CAR-T cells against Raji-Luc modeled NOG cells. Dual CAR pairs exhibit stronger target cell killing ability compared to single CAR-T cells.

[0248] Figure 22 shows the results of the in vitro killing experiment of CAR-S1 on Raji lymphoma cells (Luciferase method). Among them, Figure 22A shows the expression of BCMA antigen on the surface of Raji lymphoma target cells, and Figure 22B shows the killing effect of CAR-S1 cells on Raji lymphoma target cells at different E:T ratios. Detailed Implementation

[0249] Through extensive and in-depth research, the inventors have for the first time constructed a novel engineered immune cell targeting BCMA, whose antigen-binding domain in the CAR is derived from S-type scFv. Experiments show that, compared with CAR-T cells constructed using BB scFv and April-derived BCMA-binding domains, the CAR-T cells constructed in this invention exhibit higher killing efficacy and tumor clearance capacity. This invention also utilizes S-type scFv and CD19 scFv to construct dual CAR-T cells, which can simultaneously kill BCMA- and CD19-positive CAR-T cells.

[0250] Specifically, this invention utilizes scFvs containing different BCMA antibodies to construct CAR-T cells and compares them. Unexpectedly, it was found that scFvs derived from S exhibit a higher ability to kill BCMA-overexpressing cells and BCMA-positive tumor target cells compared to scFvs derived from BB and CAR-T cells constructed from BCMA-binding domains derived from April. In an in vivo mouse model, CAR-T cells also demonstrated higher tumor-clearing ability than BB-derived CAR-T cells. CAR-T cells constructed using other commonly used BCMA-targeting scFvs did not exhibit ideal in vitro and in vivo functions.

[0251] the term

[0252] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0253] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0254] The term “giving” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0255] The term "antibody" (Ab) should include, but is not limited to, immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or their antigen-binding portions. Each H chain contains a heavy chain variable region (abbreviated 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 VL) and a light chain constant region. The light chain constant region contains one 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 contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0256] It should be understood that the amino acid names in this article adopt the internationally accepted single-letter identifiers, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), I1e (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0257] B cell maturation antigen (BCMA)

[0258] BCMA is a transmembrane protein expressed on the surface of mature B lymphocytes, specifically plasmablasts and plasma cells. Multiple myeloma is caused by the abnormal proliferation and invasion of the bone marrow by plasma cells. Studies have shown that BCMA is expressed on multiple myeloma cells. CAR-T cell therapy targeting BCMA has been shown to specifically kill myeloma cells. However, some patients still experience relapse after receiving BCMA-targeted CAR-T cell therapy. For these relapsed patients, it is necessary to find a target different from BCMA to continue treatment.

[0259] CD19

[0260] CD19 is a transmembrane protein on the surface of B cells, closely related to B cell activation, signal transduction, and growth regulation. As shown in Figure 1, CD19 is expressed on the surface of almost all B cells, and CAR-T cells targeting CD19 have shown significant efficacy in the treatment of leukemia and lymphoma. It is generally believed that 99.95% of plasma cells do not express CD19, thus the potential of CD19 for the treatment of multiple myeloma has been overlooked.

[0261] Chimeric antigen receptor (CAR)

[0262] The chimeric antigen receptor (CAR) of this invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than an antigen receptor or its ligands.

[0263] A linker may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of the polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0264] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting BCMA (or BCMA and CD19). When expressed in T cells, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to stop growing, be induced to die, or otherwise be affected, leading to a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with an intracellular domain derived from one or more of the co-stimulatory molecules and the ζ chain. Preferably, the antigen-binding domain is fused with an intracellular domain combining a 4-1BB signaling domain and a CD3ζ signaling domain.

[0265] As used herein, "antigen-binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen-binding activity. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant regions, and are the smallest antibody fragments possessing all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The antigen-binding domain is typically scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably a single amino acid chain sequence encoded by a single nucleotide chain. As a preferred embodiment of the invention, the antigen-binding domain contains an antibody that specifically recognizes BCMA; optionally, the antigen-binding domain also contains an antibody that specifically recognizes CD19, preferably a single-chain antibody.

[0266] For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one implementation, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected, or modified by amino acid substitution, to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0267] The intracellular domains in the CAR of this invention include the 4-1BB signal transduction domain and the CD3ζ signal transduction domain.

[0268] Preferably, the CAR of the present invention also includes a cell suicide element.

[0269] Preferably, the BCMA-targeting scFv of the present invention is an S scFv, and the BB scFv and April chain in the embodiments are used as controls. Both BB scFv and April chain are commonly used BCMA-targeting binding sequences in the art. BB scFv is described in PCT application WO 2010104949 A3, and April chain is described in CN105658671A.

[0270] Bispecific CAR targeting CD19 and BCMA

[0271] Multiple myeloma (MM) is a malignant plasma cell tumor whose tumor cells originate from plasma cells in the bone marrow. Plasma cells are B lymphocytes that have reached their final functional stage. Multiple myeloma is essentially incurable, characterized by high morbidity and mortality. In 2017, the United States had 30,000 newly diagnosed cases of multiple myeloma, with an estimated 12,000 facing death. Current common treatments for multiple myeloma include cytotoxic drugs, protease inhibitors (such as bortezomib), lenalidomide, monoclonal antibodies, and corticosteroids. However, these treatments are only partially effective, do not provide lasting remission, and have a high relapse rate. Therefore, improvements in multiple myeloma treatment are crucial.

[0272] CD19 is a 95kDa glycoprotein expressed on the surface of pre-B cells and mature B cells. It is closely related to the transmembrane signaling pathway of Ca++ in B cells and plays a regulatory role in B cell proliferation and differentiation. CD19 is mainly expressed in normal B cells and cancerous B cells, exhibiting high tissue specificity, making it a promising target for antibody or CAR-T immunotherapy. However, during immunotherapy, the loss of the CD19 epitope on B cells frequently occurs, leading to unresponsiveness or relapse in patients.

[0273] Bispecificity means that the same CAR can specifically bind to and be recognized by the immune system for two different antigens. The CAR can generate an immune response by binding to either antigen.

[0274] In another preferred embodiment, the bispecific CAR targeting CD19 and BCMA is as described in the second aspect of the invention.

[0275] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting CD19 and BCMA, including an anti-CD19 scFv and an anti-BCMA scFv.

[0276] In another preferred embodiment, the present invention provides a bispecific chimeric antigen receptor targeting both CD19 and BCMA antigens. The CAR structural components simultaneously targeting CD19 and BCMA may 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, wherein the CD19 scFv and the BCMA scFv are linked by a short peptide (G4S)xN. The CAR structure simultaneously targeting CD19 and BCMA is as described in the second aspect of the present invention.

[0277] In another preferred embodiment, the CD19 and BCMA bispecific CAR of the present invention is a single structure comprising scFvs that resist both CD19 and BCMA. The CAR comprises CD19 scFvs and BCMA scFvs, and the ordering and hinge of the CD19 scFvs and BCMA scFvs are the main factors influencing its function.

[0278] In another preferred embodiment, the present invention optimizes the sequence of the BCMA scFv, wherein the BCMA scFv (S scFv) has high affinity for BCMA and good specificity, and can specifically target the full-length BCMA antigen and extracellular region.

[0279] In a preferred embodiment of the invention, (G4S)x3 is used to connect CD19scFv and BCMAscFv, at which point the activity and lethality of the CAR are optimal.

[0280] This invention utilizes a CAR that specifically targets CD19 and BCMA, exhibiting significantly enhanced affinity and increased immune cell activity compared to CARs targeting a single antigen, demonstrating a synergistic effect. Furthermore, due to the heterogeneous expression levels of CD19 and BCMA in tumor cells, the dual-targeting CAR-T therapy has a broader therapeutic scope. Simultaneously targeting CD19 and BCMA with CAR-immune cells can reduce the possibility of antigen escape caused by downregulation or absence of a single surface antigen. Additionally, the CD19 and BCMA bispecific CAR-T has a significantly superior ability to inhibit the in vitro colony formation of CD34-negative monocytes in the bone marrow of myeloma patients compared to single CAR-T cells, indicating a significantly better ability to suppress tumor progenitor cells than single CAR-T cells. Finally, the addition of the CD19 antigen can increase the sustained survival of the CD19 and BCMA bispecific CAR-T.

[0281] Chimeric antigen receptor T cells (CAR-T cells)

[0282] As used herein, the terms “CAR-T cell”, “CAR-T”, and “CAR-T cell of the present invention” include the CAR-T cells included in the third aspect of the present invention.

[0283] CAR-T cells have the following advantages over other T-cell-based therapies: (1) The action of CAR-T cells is not restricted by MHC; (2) Given that many tumor cells express the same tumor antigens, once the CAR gene targeting a certain tumor antigen is constructed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, thus expanding the target range of tumor antigens; (4) Using the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0284] Chimeric antigen receptor NK cells (CAR-NK cells)

[0285] As used herein, the terms "CAR-NK cell," "CAR-NK," and "CAR-NK cell of the present invention" all refer to the CAR-NK cell included in the third aspect of the present invention. The CAR-NK cell of the present invention can be used to treat tumors with high BCMA expression, such as multiple myeloma.

[0286] Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infections and tumor cell invasion through non-antigen-specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

[0287] Compared with autologous CAR-T cells, CAR-NK cells have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzymes, without killing normal cells in the body; (2) they release very little cytokine, thus reducing the risk of cytokine storm; and (3) they are very easy to expand in vitro and develop into "ready-made" products. In addition, they are similar to CAR-T cell therapy.

[0288] Suicide Gene Switch

[0289] To further control adverse effects such as non-tumor targeting of CAR-T cells and cytokine release syndrome, the CAR-T cells in this invention all carry suicide gene switches. Under the action of exogenous drugs, they can effectively eliminate CAR-T cells in the body and block unknown or uncontrollable long-term toxicity, thereby ensuring the safety of patients.

[0290] The suicide switch used in this invention can be the herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (iCasp9), CD20, mutated human thymidylate kinase (mTMPK), etc. Comparatively, HSV-TK, iCasp9, and CD20 have equivalent clearance abilities against CAR-cells, but iCasp9 and CD20 clear cells more rapidly, while HSV-TK clears cells more slowly.

[0291] The iCasp9 suicide switch contains the FKBP12-F36V domain, which connects to cysteine-aspartic protease 9 via a flexible linker. The latter lacks a recruitment domain. FKBP12-F36V contains an FKBP domain where phenylalanine replaces valine at the 36th amino acid residue. It exhibits high selectivity and sub-nanomolar affinity, enabling it to bind dimerizing ligands such as other inert small molecules like AP1903. Upon addition of these small molecules, dimerization is induced, thereby inducing apoptosis, while having no effect on normal cells lacking the suicide switch.

[0292] The inducible safety switch caspase9 (iCasp9) uses human caspase9 fused with FK506 binding protein (FKBP), which can be induced to form a dimer with a chemical inducer (AP1903 / Rimiducid, Bellicum Pharmaceutical), leading to apoptosis in cells expressing the fusion protein.

[0293] Although CD19 and BCMA are highly expressed in tumor cells, they are also expressed in normal B cells. The engineered immune cells of this invention will attack normal B cells in vivo.

[0294] Controlling the safety of CAR cells has always been a pressing issue. Adding a safety switch to CAR cells is the safest way to terminate their activity. Inducible iCasp9 safety switches can control CAR cell clearance in cases of severe toxicity (CRS / neurotoxicity) or after a patient achieves long-term remission.

[0295] carrier

[0296] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be synthesized.

[0297] This invention also provides vectors in which the expression cassette of this invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0298] In short, the expression cassette or nucleic acid sequence of this invention is typically operatively linked to a promoter and incorporated into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0299] The expression constructs of the present invention can also be used with standard gene delivery protocols for nucleic acid immunotherapy and gene therapy. 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 entirety. In another embodiment, the present invention provides a gene therapy vector.

[0300] This nucleic acid can be cloned into many types of vectors. For example, it can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0301] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0302] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. 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, lentiviral vectors are used.

[0303] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. These are typically located in a 30–110 bp region upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible to maintain promoter function when an element is inverted or moved relative to another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to initiate transcription.

[0304] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively 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, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0305] To assess the expression of CAR peptides or portions thereof, the expression vector introduced into cells may also contain either or both of an optional marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. Alternatively, the optional marker may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes such as neo.

[0306] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is either absent from or expressed by the recipient organism or tissue, and that encodes a polypeptide whose expression is clearly indicated by readily detectable properties such as enzyme activity. After DNA has been introduced into the recipient cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein (e.g., 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. Typically, a construct with at least five flanking regions exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated into reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0307] Methods for introducing genes into cells and expressing genes into cells are known in the art. Within the scope of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0308] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and so on. Methods for producing cells comprising 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). Calcium phosphate transfection is a preferred method for introducing polynucleotides into host cells.

[0309] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA 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 may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0310] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and 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 both in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0311] In the case of using a non-viral delivery system, an exemplary delivery tool is a liposome. Consider using a lipid formulation to introduce nucleic acid into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, conjugated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any specific structure in solution. For example, they may be present in a bilayer structure, as micelles, or have a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0312] In a preferred embodiment of the present invention, the carrier is a lentivirus carrier.

[0313] preparation

[0314] This invention provides a formulation containing CAR-T cells as described in the first aspect of this invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAR-T cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, more optimal 1×104 -1×10 7 Cells / ml

[0315] In one embodiment, the formulation may include buffer solutions such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides 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.

[0316] Therapeutic applications

[0317] This invention includes therapeutic applications using cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of this invention. The transduced T cells can target tumor cell markers BCMA and / or CD19, synergistically activating T cells and evoking a T cell immune response, thereby significantly enhancing their efficiency in killing tumor cells.

[0318] Therefore, the present invention also provides a method for stimulating a T-cell-mediated immune response against a target cell population or tissue of a mammal, comprising the step of administering the CAR-T cells of the present invention to a mammal.

[0319] In one embodiment, the present invention includes a type of cell therapy in which the patient's own T cells (or those from a heterologous donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This method results in an extremely low probability of graft-versus-host disease, and the antigen is recognized by the T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-lasting, durable antibodies that lead to sustained tumor control.

[0320] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and sustain for an extended period of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, anti-BCMA and / or CD19 CAR-T cells elicit a specific immune response against cells expressing BCMA and / or CD19.

[0321] Although the data disclosed herein specifically disclose lentiviral vectors including anti-BCMA and / or CD19scFv, hinge and transmembrane regions, and 4-1BB / CD28 and CD3ζ signaling domains, the invention should be construed as including any number of variations in each of the construct components.

[0322] Treatable cancers include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematologic malignancies, such as leukemia and lymphoma) or may include solid tumors. Types of cancers treatable with the CAR of this invention include, but are not limited to, carcinomas, germ cell tumors, and sarcomas, and certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0323] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-borne) cancers include leukemia, including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal dysplasia.

[0324] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0325] The CAR-modified T cells of the present invention can also be used as a type of vaccine for in vitro immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0326] For in vitro immunization, at least one of the following occurs in vitro before the cells are administered into a mammal: i) cell expansion, ii) introduction of nucleic acid encoding CAR into the cells, and / or iii) cryopreservation of the cells.

[0327] In vitro procedures are well known in the art and are discussed more fully below. Simply put, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) using a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0328] 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 antigens in a patient.

[0329] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need of the treatment.

[0330] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may comprise target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0331] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease—although the appropriate dosage can be determined by clinical trials.

[0332] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that a pharmaceutical composition including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6 The T-cell composition can be administered at a dose of cells per kg of body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0333] The application of the target composition can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0334] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may 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 composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded immune cells of the present invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0335] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 6 One to 1×10 10 The modified T cells of this invention (e.g., CAR-T20 cells) are administered to the patient via, for example, intravenous infusion.

[0336] The main advantages of this invention include:

[0337] (a) The CAR-T cells containing S scFv constructed in this invention have higher in vivo and in vitro tumor killing and functional activities than BB and April CAR-T.

[0338] (b) The bispecific CAR-T constructed in this invention can simultaneously recognize two or more targets, including BCMA.

[0339] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0340] Example 1: Isolation of PBMCs and Expansion of T Cells from Donor Blood

[0341] Mononuclear cells were isolated from donor blood, and T cells were enriched using a density gradient centrifugation method with Histopaque-1077 (Sigma-Aldrich) (EasySep Human T Cell Enrichment Kit, Stemcell Technologies). T cells were activated and expanded using magnetic beads conjugated with anti-CD3 / anti-CD28. X-vivo15 (300 IU / ml rhIL2) was used as the culture medium. All cells were cultured in a 37°C, 5% CO2 incubator.

[0342] Example 2 Cell Culture and Construction

[0343] Cell lines expressing BCMA, including MM.1s and RPMI8226, MM.1s-ffluc cells, RPMI8226-ffluc cells, HeLa cells expressing BCMA, CD19 alone, and BCMA / CD19 simultaneously, were all cultured in RPMI 1640 medium; 293T (human kidney epithelial cell line) were also included. CRL-3216 was cultured in DMEM medium. All media were supplemented with 10% (v / v) fetal bovine serum and 100 U / ml penicillin and streptomycin, 2 ml glutamine, and 1 mM sodium pyruvate.

[0344] Among them, HeLa cells expressing BCMA and CD19 alone and simultaneously expressing BCMA / CD19 are stable cell lines obtained by transfecting BCMA and CD19 antigens into a lentiviral vector. They can specifically express BCMA and / or CD19 protein molecules. MM.1s-ffluc cells and RPMI8226-ffluc cells are stable cell lines obtained by screening after infection with firefly luciferase lentivirus.

[0345] Example 3: CAR Structure Design and Transduction

[0346] The design and construction of a single CAR targeting BCMA and a dual CAR simultaneously targeting BCMA and CD19 are illustrated in Figure 1. The CAR, CD19 CAR, and the suicide switch – EGFRt element are connected via a 2A peptide. Specifically, the CAR structures involved in this invention are shown in Figure 1, and their nomenclature and composition are shown in Table 1.

[0347] Table 1. Structure of CAR

[0348] Structural Naming and Composition of CAR-T: S1 Single S scFv CAR-S1; S2 Parallel CD19 CAR + S CAR (Dual CAR) CAR-S2; S3 Parallel S CAR + CD19 CAR (Dual CAR) CAR-S3; S4 Loop Structure CD19 scFv + S scFv CAR-S4

[0349] S5 tandem CD19 scFv+S scFvCAR-S5BB single BB scFvCAR-BBApril single April chain CAR-April19 single CD19 scFvCAR-19S6 parallel CD19 CAR+S CAR+EGFRtCAR-S6S7 parallel S CAR+CD19 CAR+EGFRtCAR-S7

[0350] The specific sequence of the components involved in the CAR described in Figure 1 and Table 1 is as follows:

[0351] S scFv (S scFv) heavy chain

[0352]

[0353] S scFv (S scFv) light chain

[0354]

[0355] BB scFv heavy chain

[0356]

[0357] BB scFv light chain

[0358]

[0359] April Chain

[0360]

[0361] CD8 signal peptide

[0362]

[0363] (G4S)3-linked peptide

[0364]

[0365] (G4S)5-linked peptide

[0366]

[0367] 218 Linked Peptide

[0368]

[0369] CD8 hinge area

[0370]

[0371] orKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKP(SEQ ID NO:37)

[0372] or SGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:38)

[0373] CD8 transmembrane region

[0374]

[0375] CD28 transmembrane region

[0376]

[0377] 41BB signal area

[0378]

[0379] CD28 signal area

[0380]

[0381] CD3z signal area

[0382]

[0383] or

[0384]

[0385] 2A peptide

[0386]

[0387] FMC63 scFv (CD19 scFv) rechain

[0388]

[0389] FMC63 scFv (CD19 scFv) light chain

[0390]

[0391] GM-CSF signal peptide

[0392]

[0393] EGFRt sequence

[0394]

[0395] Each CAR gene in Table 1 was cloned into the FUW lentiviral vector backbone to construct a complete lentiviral expression vector that can be used to infect T cells. Specifically, taking the BCMA CAR gene as an example, the BCMA CAR gene was placed under the EF1α (EF-1α) promoter to form Fuw-EF1α-BCMA CAR. The three plasmids, Fuw-EF1α-BCMA CAR, lentiviral envelope plasmid pMD2.G (Addgene, Plasmid #12259), and lentiviral packaging plasmid psPAX2 (Addgene, Plasmid #12260), were transformed into 293T cells using Lipofectamine 3000 to prepare a complete lentiviral expression vector. Viral supernatants were collected at 48h and 72h and concentrated by ultracentrifugation. The concentrated virus can then be used to infect T cells.

[0396] Flow cytometry analysis showed that the constructed CAR gene could be used to prepare lentiviral vectors expressing BCMA CAR.

[0397] Example 4: Preparation of CAR-T cells

[0398] The experimental method is as follows:

[0399] 4.1 Lentiviral infection

[0400] Two days after activation, the isolated and purified primary T cells were infected with the lentiviral vector constructed in Example 3, transferred to cell culture flasks, and cultured in a 37°C, 5% CO2 incubator.

[0401] 4.2 Cell proliferation and CAR positivity rate detection

[0402] Samples were taken on day 3 after infection and before freezing to detect the number of cells and the percentage of BCMA-positive cells, i.e. the CAR positivity rate of T cells. Half of the culture medium was replaced every 2-3 days.

[0403] The results showed that various CAR-T cells were successfully constructed using the lentiviral vector constructed in Example 3, and their names are shown in Table 1.

[0404] Specifically, the construction results of BCMA CAR-T cells are shown in Figure 2. BCMA CAR expression can be detected in both virus-transfected CAR-BB and CAR-S1 CAR-T cells, with CAR expression reaching over 50%.

[0405] Example 5: In vitro cell killing

[0406] In vitro killing experiments were conducted on CAR-S1 CAR-T cells, CAR-BB CAR-T cells, and CAR-April CAR-T cells obtained in Example 4. The RTCA method was used to test the killing effect of CAR-T cells on HeLa cell lines overexpressing BCMA.

[0407] The results are shown in Figure 3 (RTCA assay). The NT control group (untransfected T cell control group) and the culture medium control group (blank control group) did not kill Hela-BCMA cells, while CAR-S1 cells could exert BCMA-specific killing function. Moreover, CAR-S1 cells showed better results than CAR-BB cells in killing BCMA-positive Hela-BCMA cells.

[0408] The cytotoxic ability of tumor target cells labeled with luciferase was detected. The luciferase gene was transferred into target cells, and stable cell lines MM.1s-Luc and RPMI8226-Luc were obtained after clone selection. During the experiment, a luciferin substrate was added, and the luciferase reacted with luciferin to produce fluorescence. The activity of luciferase was determined by detecting the intensity of the fluorescence, and the cell survival rate was measured to obtain the cytotoxic effect of each CAR-T cell.

[0409] Figure 4 shows the antigen expression on the surface of target cells. Figure 5 shows that at the same E:T ratio, NT cells did not show any killing function, while CAR-S1 cells had 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 CAR-S1 cells showed superior killing ability compared to CAR-BB and CAR-April.

[0410] In addition, the applicant constructed CAR-T cells using various BCMA-targeting scFvs commonly used in the field. However, these CAR-T cells did not exhibit the desired killing function after testing.

[0411] In summary, after co-culturing CAR-T cells with target cells (BCMA-overexpressing cells, BCMA-positive tumor cells, MM.1s-Luc cells, and RPMI8226 cells), the target cells could be lysed by BCMA-targeting CAR-T cells, and CAR-S1 cells exhibited higher killing ability than CAR-BB cells. Furthermore, some commonly used CAR-T cells constructed from scFv cells targeting BCMA did not demonstrate ideal killing function.

[0412] Example 6: Detection of Cytokine Release

[0413] The CAR T cells (CAR-S1 CAR-T cells and CAR-BB CAR-T cells) targeting BCMA obtained in Example 4 were mixed with tumor cells (HeLa, HeLa-BCMA, HeLa-CD19, HeLa-BCMA-CD19) and placed in RPMI medium, with each cell density prepared at 1 x 10⁻⁶. 4 CAR-T cells and tumor cells, 100 μL each, were placed in 96-well plates and co-cultured overnight. The supernatant was collected, centrifuged, and the release levels of cytokines such as IFN-γ were detected using an ELISA kit.

[0414] As shown in Figure 6, after CAR-S1 was co-stimulated by Hela-BCMA target cells, the secretion of cytokine INF-γ was significantly higher than that of CAR-BB, while no significant secretion was observed in the NT and Medium groups.

[0415] Example 7: In vivo efficacy study

[0416] Select 6-12 week old NOG mice and subcutaneously inject them with 1×10 7 RPMI8226 cells. Tumor graft burden was assessed two days later. Ten days later, the cells were divided into groups, and CAR-S1 CAR-T cells and CAR-BB CAR-T cells were injected one day after grouping. Tumor volume burden in mice was assessed twice a week after CAR-T treatment.

[0417] As shown in Figure 7, compared with the control group, the tumor burden of mice injected with CAR-S1 cells was significantly suppressed, and the anti-tumor effect was slightly higher than that of CAR-BB.

[0418] Example 8 Preparation of dual CAR-T cells

[0419] The experimental method is as follows:

[0420] This embodiment involves CAR-T cells that simultaneously target BCMA and CD19. The schematic diagram of the CAR structure is shown in Figure 1 (CAR S2, CAR S3, CAR S4, CAR S5, CAR S6, and CAR S7). The BCMA CAR, CD19 CAR, and the suicide switch – EGFRt element are linked via a 2A peptide. In the BCMA CAR structure, the scFv is composed of the heavy and light chains of the S and BB scFvs. The S scFv is composed of SEQ ID NO:9 and SEQ ID NO:10; the BB scFv is composed of SEQ ID NO:13 and SEQ ID NO:14; and the CD19 scFv is composed of SEQ ID NO:11 and SEQ ID NO:12. Furthermore, the scFv can be replaced by the BCMA-binding region composed of the April partial sequence (SEQ ID NO:15) to form a new CAR structure.

[0421] The BCMA-CD19CAR gene was cloned into a vector backbone and placed under the promoter of EF1α (EF-1α) to form EF1α-BCMA-CD19-EGFRt CAR. The EF1α-BCMA-CD19-EGFRt CAR and lentiviral envelope plasmid were transformed into 293T cells using Lipofectamine 3000 to prepare a complete lentiviral expression vector. Viral supernatants were collected at 48h and 72h and concentrated by ultrafiltration. The concentrated virus can then be used to infect T cells.

[0422] Lentiviral infection: Two days after activation, the isolated and purified primary T cells were infected with the lentivirus constructed above at an MOI of 1-10, transferred to cell culture flasks, and cultured in a 37°C, 5% CO2 incubator.

[0423] Cell proliferation and CAR positivity rate detection: Samples were taken on the 3rd day after infection and before freezing to detect the number of cells and the proportion of BCMA / CD19 double positive cells, that is, to detect the CAR positivity rate of T cells. Half of the culture medium was replaced every 2-3 days.

[0424] The results showed that BCMA-CD19 CAR-T cells were successfully constructed using the BCMA-CD19 CAR lentiviral vector, as shown in Figure 1 and Table 1.

[0425] As shown in Figure 8, the expression of BCMA CAR and CD19 CAR could be detected on the surface of T cells after viral transfection using both BCMA antigen and CD19 antigen.

[0426] Figure 9 shows that the expression of BCMA CAR, CD19 CAR and EGFRt can be detected simultaneously on the surface of CAR-S6 and CAR-S7 cells.

[0427] Example 9: In vitro cell killing

[0428] In vitro killing experiments were performed on the CAR-T cells obtained in Example 8. RTCA was performed using HeLa overexpressing cell lines expressing BCMA and CD19, or on tumor target cells labeled with luciferase. Stable cell lines (RPMI8226, MM.1s, and Nalm6) were obtained after clonal selection by transferring the luciferase gene into target cells. During the experiment, a luciferase substrate was added, and the luciferase reacted with luciferin to produce fluorescence. The activity of luciferase was determined by detecting the fluorescence intensity, and the cell survival rate was measured to obtain the killing effect of CAR-T cells.

[0429] The results showed that after co-culturing CAR-T cells with various target cells (CD19 / BCMA double positive, CD19 single positive, and BCMA single positive), the target cells all lysed, indicating that BCMA-CD19 CAR-T had a killing effect on CD19 / BCMA double positive, CD19 single positive, and BCMA single positive cells.

[0430] The specific results, as shown in Figure 10, indicate that bispecific CAR-T cells significantly killed both single-positive CD19-positive target cells (HeLa-CD19) and single-positive BCMA-positive target cells (HeLa-BCMA), and also significantly killed double-positive target cells (HeLa-BCMA-CD19). This demonstrates that bispecific CAR-T cells combining BCMA and CD19 have cytotoxic effects on both single-target and dual-target cells. In contrast, single-target CAR-T cells (CAR-19 or CAR-S1) can only exert a cytotoxic effect against one target antigen.

[0431] Figure 11 shows that the dual CAR-T therapy significantly killed BCMA-positive tumor target cells MM.1s and RPMI8226, and also significantly killed CD19-positive tumor target cells Raji and Nalm6. This indicates that the dual CAR-T therapy combining BCMA and CD19 has a killing effect on both BCMA- and CD19-positive tumor target cells.

[0432] Example 10 Detection of Cytokine Release

[0433] BCMA-CD19 CAR-T cells (obtained in Example 8) were mixed with tumor cells (HeLa-BCMA) and placed in RPMI medium, with each cell density set at 1 x 10⁻⁶. 4CAR-T cells and tumor cells, 100 μL each, were placed in a 96-well plate and co-cultured overnight. The supernatant was collected, centrifuged, and the cytokine release level was detected using the CBA method.

[0434] As shown in Figure 12, BCMA-CD19 CAR-T cells secreted a large amount of cytokines after stimulation by BCM-positive target cells, while NT cells secreted only a small amount of cytokines. This indicates that BCMA-CD19 CAR-T cells can be activated by BCMA.

[0435] Example 11: Upregulation of CD107 after stimulation

[0436] Flow cytometry analysis was performed on the changes in CD107a expression in the CAR-T cells obtained in Example 8 after activation. Co-incubation activation experiments were conducted using tumor cell lines expressing CD19 or BCMA. After co-incubation, the cells were labeled with antibodies for CD3, CD8, and CD107a, and then analyzed by flow cytometry.

[0437] As shown in Figure 13, after co-culturing dual CAR-T cells with BCMA-positive tumor target cells MM.1s and CD19-positive Raji, the CD107a molecule on the surface of CAR-T cells was significantly upregulated.

[0438] Example 12 In vivo efficacy study

[0439] Select 6-12 week old NOG mice and subcutaneously inject them with 1×10 7 RPMI8226 cells. Tumor graft burden was assessed two days later. Ten days later, the mice were divided into groups with similar tumor burdens. CAR-T cells were injected into each group one day after grouping. Tumor volume burden was assessed twice weekly after CAR-T treatment.

[0440] Figure 14 shows that CAR-S2 and CAR-S4 can eliminate tumors in mice with subcutaneous RPMI8226 cell modeling, indicating their significant anti-tumor efficacy.

[0441] Meanwhile, NOG mice aged 6-12 weeks were selected and intravenously injected with 1×10 7MM.1s cells. Tumor graft burden was assessed, and mice were grouped according to average tumor burden. One day after grouping, CAR-T cells were injected into each group. Tumor burden in mice was assessed after CAR-T treatment. Each mouse received an intraperitoneal injection of 3 mg d-luciferin (Perkin Elmer Life Sciences), and images were taken four minutes later using the Xenogen IVIS Imaging System (Perkin Elmer Life Sciences) with a 30-second exposure. The bioluminescent signal was calculated based on the emitted photon quantity, which was normalized using exposure time and surface area. The final photon quantity / s / cm² was then obtained. 3 / Spherical angle (p / s / cm) 2 / sr).

[0442] The results in Figure 15 show that, compared with the control group, the tumor burden of mice injected with dual CAR-T cells was significantly reduced until it disappeared, indicating that BCMA-CD19 CAR-T cells have a significant anti-tumor effect.

[0443] Example 13 Study on the killing of tumor-forming cells

[0444] Relapse in myeloma (MM) patients is a common clinical phenomenon. Clinically, most tumor cells can generally be eliminated, but the tumor cells with clonal proliferative capacity that lead to relapse often have relatively higher drug resistance. To investigate the killing ability of CAR-T cells against tumor cells with clonal proliferative capacity and to compare the advantages of bispecific CAR-T cells with mono-CAR-T cells, this study established a method for myeloma clonogenic assays and examined the inhibitory effect of CAR-T cells on clonogenicity.

[0445] MM tumor cells with proliferative capacity can grow in clonal proliferation medium, but it is necessary to remove the interference of CD34+ hematopoietic stem cells with proliferative capacity during the experiment and harvest cells mainly composed of tumor proliferating cells. This is an important issue that needs to be controlled in this experiment.

[0446] The specific experimental method is as follows:

[0447] The first step involved isolating and extracting bone marrow mononuclear cells using Ficoll and performing flow cytometry phenotypic analysis. The second step involved removing CD34+ cells using a CD34+ cell sorting kit. The third step involved using different groups of CAR-T cells (dual CAR-T and single CAR-T) to perform a killing experiment. After the killing process, the CAR-T cells were removed using a T cell removal sorting kit. The fourth step involved clonal growth using semi-solid clonal proliferation medium. After 1-2 weeks, statistical analysis, counting, and result summarization were performed.

[0448] As shown in Figure 16, CAR-S2 and CAR-S4 have a more significant advantage in killing clonogenic cells or tumor cell precursor cells compared to CAR-19 and CAR-S1, indicating that they have a higher ability to inhibit myeloma cell clonogenicity than single CARs.

[0449] Example 14: Nalm6 In vivo vein modeling experiment

[0450] Select 6-12 week old NOG mice and inject them intravenously with 1×10 7 Nalm6 cells were used. Tumor graft burden was assessed 6 days later, and mice were grouped according to average tumor burden. One day after grouping, CAR-T cells were injected into each group. Tumor burden in mice was assessed after CAR-T treatment. Each mouse received an intraperitoneal injection of 3 mg d-luciferin (Perkin Elmer Life Sciences), and images were taken four minutes later using the Xenogen IVIS Imaging System (Perkin Elmer Life Sciences) with a 30-second exposure. The bioluminescent signal was calculated based on the emitted photon quantity, which was normalized using exposure time and surface area. The final photon quantity / s / cm² was then obtained. 2 / Spherical angle (p / s / cm) 2 / sr).

[0451] As shown in Figure 17, the tumor burden in mice injected with CAR-S2 and CAR-S4 was significantly reduced until it disappeared, and BCMA-CD19CAR-T cells had a more significant effect on anti-CD19 positive tumors compared with CAR-19.

[0452] Example 15: Safety Switch Experiment for CAR-T Cells

[0453] CAR-T cells containing EGFRt elements were analyzed by flow cytometry after staining with EGFR antibody, and CAR expression was also analyzed simultaneously.

[0454] As shown in Figure 18, the expression of the safety switch was detected in CAR-T cells.

[0455] Example 16 Preparation and Detection of Killing Effect of Humanized CAR-T Cells

[0456] Humanized CAR-T cells (CAR-h19) and humanized dual CAR-T cells (CAR-hS2 and CAR-hS4) were constructed using the methods described in Examples 3 and 4. The structure of the humanized CAR-T cells was similar to that of CAR-19, the structure of the humanized dual CAR-T cells CAR-hS2 was similar to that of CAR-S2, and the structure of CAR-hS4 was similar to that of CAR-S4. The only difference was that the mouse scFv in the original structure was replaced with a humanized CD19 scFv. The humanized CD19 scFv included the antibody heavy chain variable region shown in any of SEQ ID NO:21-30 and the antibody light chain variable region shown in any of SEQ ID NO:31-36.

[0457] The in vitro killing effect of humanized dual CAR-T cells was detected using the method described in Example 9.

[0458] The in vitro killing results are shown in Figures 19 and 20. Humanized CAR-T cells and humanized dual CAR-T cells showed significant killing effects on target cells without killing non-target cells.

[0459] Example 17: In vivo pharmacodynamic study of humanized CAR-T cells

[0460] NOG mice aged 6-12 weeks were selected and subcutaneously injected with 3×10 5 Raji cells were used. Six days later, tumor graft burden was assessed, and mice were divided into groups with comparable tumor burdens. One day after grouping, each group was injected with the prepared dual CAR-T cells. Tumor volume burden was evaluated after CAR-T treatment. Each mouse received an intraperitoneal injection of 3 mg d-luciferin (Perkin Elmer Life Sciences), and four minutes later, images were taken using the Xenogen IVIS Imaging System (Perkin Elmer Life Sciences) with a 30-second exposure. The bioluminescence signal was calculated based on the emitted photon quantity, which was normalized using exposure time and surface area to obtain the photon quantity / s / cm². 2 / Spherical angle (p / s / cm) 2 / sr).

[0461] As shown in Figure 21, humanized CAR-hS2 cells had a stronger ability to eliminate tumors in Raji cell-induced model mice than CAR-S2 cells, indicating their significant anti-tumor efficacy.

[0462] Example 18: Killing effect of CAR-T cells on Raji lymphoma cells

[0463] The cytotoxicity of CAR-T cells was assessed using luciferase-labeled Raji lymphoma target cells. The luciferase gene was transferred into Raji target cells, and a stable transfected cell line, Raji-Luc, was obtained after cloning and selection. During the experiment, a luciferin substrate was added, and the luciferase reacted with the luciferin to produce fluorescence. The activity of the luciferase was determined by detecting the intensity of the fluorescence, and the cell survival rate was measured to obtain the cytotoxic effect of each CAR-T cell.

[0464] As shown in Figure 22, NT cells did not exhibit killing function, while CAR-S1 cells showed dose-dependent killing effect on Raji-Luc cells (Raji cells transfected with the luciferase gene), indicating its potential application value in lymphoma indications.

[0465] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A chimeric antigen receptor (CAR), characterized in that, The antigen-binding domain (scFv) of the CAR includes 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.

2. The CAR as described in claim 1, characterized in that, The scFv is shown in either formula A or formula B: V H -V L ,(A);V L -V H ,(B) In the formula, V H V is the variable region of the antibody heavy chain; L " " represents the variable region of the antibody light chain; "-" represents a linking peptide or peptide bond.

3. A bispecific CAR, characterized in that, The bispecific CAR targets BCMA and the first target. The antigen-binding domain (scFv) targeting BCMA in the bispecific CAR includes 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. Furthermore, the first target is selected from the following group: CD138、Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD 28. CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD1 23. 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, 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, N Y-ESO-1, PSMA, RANK, RORl, 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 receptorα, Folate receptorβ, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

4. The bispecific CAR as described in claim 3, characterized in that, The first target is CD19, and the antigen-binding domain (scFv) targeting CD19 in the bispecific CAR includes the antibody heavy chain variable region shown in any of SEQ ID NO:11, 21-30, and the antibody light chain variable region shown in any of SEQ ID NO:12, 31-36.

5. The bispecific CAR as described in claim 3, characterized in that, The structure of the bispecific CAR is shown in Formula II below: L-scFv1-I-scFv2-H-TM-C-CD3ζ (II) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the absence of a signal peptide sequence; I represents a flexible joint; H represents the area with no hinge or no connection. TM represents a transmembrane domain; C is a co-stimulatory signaling molecule; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ; Of the two, scFv1 and scFv2, one is an antigen-binding domain that targets the first target, and the other is an antigen-binding domain that targets BCMA.

6. The bispecific CAR as described in claim 3, characterized in that, The structure of the bispecific CAR is shown in formula III or III' below: L-V L3 -scFv3-V H3 -H-TM-C-CD3ζ (III) L-V H3 -scFv3-V L3 -H1-TM-C-CD3ζ (III’) In the formula, Each "-" independently represents a linking peptide or peptide bond; Components L, H, TM, C, and CD3ζ are as described above; scFv3 is an antigen-binding domain that targets BCMA, V H3 For the variable region of the heavy chain of the antibody against the first target, and V L3 The variable region of the light chain of the antibody against the first target; Alternatively, scFv3 could be the antigen-binding domain targeting the first target, V H3 It is the variable region of the heavy chain of the anti-BCMA antibody, and V L3 This is the variable region of the light chain of the anti-BCMA antibody.

7. A nucleic acid molecule encoding the CAR of claim 1 or the bispecific CAR of claim 3.

8. A vector comprising the nucleic acid molecule of claim 7.

9. An engineered immune cell, said immune cell containing the vector of claim 8, or having an exogenous nucleic acid molecule of claim 7 integrated into a chromosome, or expressing the CAR of claim 1 or the bispecific CAR of claim 3.

10. An engineered immune cell, the immune cell comprising an exogenous first expression cassette and a second expression cassette, wherein the first expression cassette is used to express a first CAR targeting a first target, and the second expression cassette is used to express a second CAR targeting BCMA; Alternatively, the immune cells may express the first CAR targeting the first target and the second CAR targeting BCMA; in, The antigen-binding domain (scFv) targeting BCMA in the second CAR includes 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; Furthermore, the first target is selected from the following group: CD138、Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD 28. CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD1 23. 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, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewi s, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, NY-ESO-1, PSMA, RANK, RORl, 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 receptorα, Folate receptorβ, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

11. A formulation comprising the CAR of claim 1, the bispecific CAR of claim 3, or the engineered immune cell of claim 9 or 10, and a pharmaceutically acceptable carrier, diluent, or excipient.

12. Use of a CAR of claim 1, a bispecific CAR of claim 3, or an engineered immune cell of claim 9 or 10 for the preparation of a medicament or formulation for the prevention and / or treatment of cancer or tumors.

13. The use as described in claim 12, characterized in that, The drug or preparation described herein treats cancer or tumors by killing tumor cells with clonal proliferation capabilities.

14. The use as described in claim 13, characterized in that, The tumor cells with clonal proliferation capacity include clone-forming cells, tumor cell precursor cells, and tumor progenitor cells.