Double-target engineered immune cell as well as preparation and application thereof

Abstract

The invention provides a double-target engineered immune cell as well as preparation and application thereof. According to the present invention, the chimeric antigen receptor simultaneously targeting TROP2 and CD133 is expressed in the immune cell, such that the killing range of the CAR-T cell on the tumor cell is wide, the tumor stem cell is removed, and the risk of the immune escape and the recurrence of the tumor is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of tumor immunity and cell therapy, and specifically relates to a dual-target engineered immune cell and its preparation and application. Background Art

[0002] The success of chimeric antigen receptor T cell (CAR-T) therapy in early clinical trials of hematological tumors has revolutionized anticancer therapy and provided a new option for precision cancer treatment. CAR-T cells are genetically engineered T cells that express CAR molecules that can recognize specific tumor cell surface antigens and guide T cells to recognize and eliminate tumor cells. CAR molecules contain extracellular antigen binding domains, hinge domains, transmembrane domains, co-stimulatory domains, and CD3ζ intracellular domains. CAR-T cell technology has shown exciting efficacy in some lymphomas, but it can also relapse due to the proliferation of CD19-negative lymphoma cells. The target heterogeneity of malignant solid tumors is stronger than that of lymphomas, and target-negative tumor cells are more likely to escape immune system, often leading to tumor recurrence after treatment. However, the CAR-T cell therapy currently undergoing clinical trials is still mainly based on single-target CAR-T cells. Targeting two or more tumor targets at the same time is an important breakthrough in solving the problem of tumor immune escape in CAR-T cell therapy.

[0003] CD133 is a member of the Prominin family of five-transmembrane glycoproteins. Studies have shown that CD133 is a marker for the prognosis of various tumor treatments. It can interact with substances such as vascular endothelial growth factor, and is also involved in membrane and cytoplasmic proteins of cell functions. It can regulate tumor cell survival by affecting glucose uptake, autophagy reaction and ATP synthesis. CD133-positive cells have the ability to initiate tumor growth and can produce tumors with self-renewal and differentiation capabilities. CD133 is highly expressed in a variety of solid tumors such as colorectal cancer, liver cancer, lung cancer, pancreatic cancer, and breast cancer, and is a specific target for the treatment of solid tumors.

[0004] TROP2 is a cell surface glycoprotein encoded by the TACSTD2 gene. It promotes tumor cell growth, proliferation, metastasis and epithelial-mesenchymal transition by regulating calcium ion signaling pathways, cell cycle protein expression and reducing fibronectin adhesion, resulting in a significant shortening of the survival of tumor patients. TROP2-positive colon cancer cells have long-term tumorigenic ability. TROP2 is continuously overexpressed in a variety of malignant solid tumors such as colorectal cancer, liver cancer, lung cancer, esophageal cancer, pancreatic cancer, breast cancer, nasopharyngeal carcinoma, etc. Therefore, TROP2 has become an attractive therapeutic target in the treatment of solid tumors.

[0005] CD133 and TROP2 are also tumor stem cell markers. Studies have shown that tumor stem cells with high expression of CD133 or TROP2 can be isolated and identified from a variety of solid tumors. In a hypoxic microenvironment, CD133-positive stem cells can still maintain the stemness characteristics of tumor stem cells, which is one of the important factors leading to tumor recurrence and metastasis. TROP2 is also related to the cell regeneration process and has an important regulatory effect on tumor growth and metastasis. Therefore, CAR-T cells that simultaneously target CD133 and TROP2 can not only expand the ability of CAR-T cells to recognize tumor cells, but also eliminate tumor stem cells at the same time, effectively control tumor growth, and reduce the risk of recurrence. Summary of the invention

[0006] The purpose of the present invention is to provide an engineered immune cell (such as CAR-T cell) with higher efficiency and better therapeutic effect for malignant tumors (especially solid tumors).

[0007] Another object of the present invention is to provide an engineered immune cell (such as a CAR-T cell) that jointly expresses a chimeric antigen receptor that specifically binds TROP2 and CD133, and a preparation method and application thereof.

[0008] In a first aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell is a T cell, a TIL cell, or a NK cell, and the immune cell has the following characteristics:

[0009] The immune cells express chimeric antigen receptors (CARs), wherein the CARs target two surface markers of tumor cells, CD133 and TROP2.

[0010] Wherein, the CAR includes a first CAR targeting CD133 and a second CAR targeting TROP2;

[0011] Alternatively, the CAR comprises a dual-targeting CAR antigen binding domain that specifically binds to both CD133 and TROP2.

[0012] In another preferred embodiment, the antigen binding domain of the first CAR specifically binds to CD133.

[0013] In another preferred example, the antigen binding domain of the second CAR specifically binds to TROP2.

[0014] In another preferred example, the antigen binding domain of the dual-targeting CAR specifically binds to CD133 and TROP2.

[0015] In another preferred example, the antigen binding domain of the dual-targeting CAR includes a first antigen binding domain that specifically binds to CD133 and a second antigen binding domain that specifically binds to TROP2.

[0016] In another preferred embodiment, the T cells include αβT cells, γδT cells, NKT cells, MAIT cells, or a combination thereof.

[0017] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0018] (i) Chimeric antigen receptor T cells (CAR-T cells);

[0019] (ii) chimeric antigen receptor NK cells (CAR-NK cells);

[0020] (iii) Chimeric antigen receptor TIL cells (CAR-TIL cells).

[0021] In another preferred embodiment, the engineered immune cells are autologous or allogeneic.

[0022] In another preferred embodiment, the antigen binding domain of the CAR is a single-chain antibody (scFv) or a single-domain antibody (or nanobody) against CD133 and / or TROP2.

[0023] In another preferred embodiment, the CAR may be constitutively expressed or inducibly expressed;

[0024] In another preferred embodiment, the expression cassette of the inducibly expressed CAR contains a gene expression regulatory element upstream or downstream;

[0025] In another preferred embodiment, the expression regulatory element contains 1 to 10 copies of a hypoxia response element (HRE);

[0026] In another preferred embodiment, the nucleotide sequence of the HRE is shown as SEQ ID No: 19 or SEQ ID No: 20 or SEQ ID No: 21.

[0027] In another preferred embodiment, the "activation" refers to the binding of the CAR to the surface markers of tumor cells.

[0028] In another preferred embodiment, the "tumor surface marker" refers to a specific antigen on the surface of the tumor.

[0029] In another preferred embodiment, the chimeric antigen receptor CAR is located on the cell membrane of the engineered immune cell.

[0030] In another preferred embodiment, the chimeric antigen receptor CAR is located on the cell membrane of the CAR-T cell.

[0031] In another preferred embodiment, the TROP2 protein is localized on the cell membrane of the tumor cells.

[0032] In another preferred embodiment, the CD133 protein is localized on the cell membrane of the tumor cells.

[0033] In another preferred embodiment, the structure of the first CAR targeting CD133 is shown in Formula I:

[0034] L-CD133scFv-H-TM-C-CD3ζ-D(I)

[0035] In the formula,

[0036] L is none or a signal peptide sequence;

[0037] CD133scFv is an antigen binding domain that specifically binds to CD133;

[0038] H is none or hinge region;

[0039] TM is the transmembrane domain;

[0040] C is no or co-stimulatory signaling domain;

[0041] CD3ζ is absent or derived from the cytoplasmic signal transduction sequence of CD3ζ (including wild type, or mutant / modified form thereof);

[0042] D is the absence or degradation domain (including the wild type, or its mutant / modified form);

[0043] The "-" is a connecting peptide or a peptide bond.

[0044] In another preferred embodiment, the L is respectively selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or a combination thereof.

[0045] In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID No:3.

[0046] In another preferred embodiment, the H is selected from the hinge region of the following proteins: CD8, CD28, CD137, IgG1, IgG4, or mutants / modified forms thereof, or a combination thereof.

[0047] In another preferred example, the amino acid sequence of H is shown as SEQ ID No: 10 or SEQ ID No: 11 or SEQ ID No: 17.

[0048] In another preferred embodiment, the TM is selected from the transmembrane region of a protein in the following group: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314, or a mutant / modified form thereof, or a combination thereof.

[0049] In another preferred embodiment, the TM is the transmembrane region of CD8.

[0050] In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID No:12.

[0051] In another preferred embodiment, the C is selected from the co-stimulatory domain of the protein in the following group: 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, OX40L, or its mutant / modified form, or a combination thereof.

[0052] In another preferred embodiment, the C is a co-stimulatory domain derived from 4-1BB.

[0053] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID No:13.

[0054] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID No:14.

[0055] In another preferred embodiment, the D is an oxygen-dependent degradation domain (ODD) or a partial sequence thereof;

[0056] In another preferred embodiment, the amino acid sequence of ODD is shown in SEQ ID No:18.

[0057] In another preferred embodiment, the CD133scFv is a single-chain variable region sequence of an antibody targeting a tumor antigen, or a partial sequence thereof.

[0058] In another preferred embodiment, the CD133scFv is a nanobody variable region sequence targeting a tumor antigen, or a partial sequence thereof.

[0059] In another preferred embodiment, the amino acid sequence of the CD133scFv is as shown in SEQ ID NO: 4 or 5.

[0060] In another preferred embodiment, in addition to the first CAR shown in Formula I, the CAR cell further contains a second CAR for targeting TROP2, and the structure of the second CAR is shown in Formula II:

[0061] L-TROP2scFv-H-TM-C-CD3ζ-D(II)

[0062] In the formula,

[0063] L is none or a signal peptide sequence;

[0064] TROP2scFv is an antigen-binding domain that specifically binds to TROP2;

[0065] H is none or hinge region;

[0066] TM is the transmembrane domain;

[0067] C is without or co-stimulatory domain;

[0068] CD3ζ is absent or derived from the cytoplasmic signal transduction sequence of CD3ζ (including wild type, or mutant / modified form thereof);

[0069] D is the absence or degradation domain (including the wild type, or its mutant / modified form);

[0070] The "-" is a connecting peptide or a peptide bond.

[0071] In another preferred embodiment, the L is respectively selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or a combination thereof.

[0072] In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID No:3.

[0073] In another preferred embodiment, the H is selected from the hinge region of the following proteins: CD8, CD28, CD137, IgG1, IgG4, or mutants / modified forms thereof, or a combination thereof.

[0074] In another preferred example, the amino acid sequence of H is shown as SEQ ID No: 10 or SEQ ID No: 11 or SEQ ID No: 17.

[0075] In another preferred embodiment, the TM is selected from the transmembrane region of a protein in the following group: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314, or a mutant / modified form thereof, or a combination thereof.

[0076] In another preferred embodiment, the TM is the transmembrane region of CD8.

[0077] In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID No:12.

[0078] In another preferred embodiment, the C is selected from the co-stimulatory domain of the protein in the following group: 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, OX40L, or its mutant / modified form, or a combination thereof.

[0079] In another preferred embodiment, C is a co-stimulatory domain derived from 4-1BB.

[0080] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID No:13.

[0081] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID No:14.

[0082] In another preferred embodiment, the D is an oxygen-dependent degradation domain (ODD) or a partial sequence thereof;

[0083] In another preferred embodiment, the amino acid sequence of ODD is shown in SEQ ID No:18.

[0084] In another preferred embodiment, the TROP2scFv is a single-chain variable region sequence of an antibody targeting a tumor antigen, or a partial sequence thereof.

[0085] In another preferred embodiment, the TROP2scFv is a nanobody variable region sequence targeting a tumor antigen, or a partial sequence thereof.

[0086] In another preferred embodiment, the amino acid sequence of the TROP2scFv is shown in SEQ ID NO: 6 or 7.

[0087] In another preferred embodiment, the dual-targeting CAR (such as combining the first CAR shown in Formula I and the second CAR shown in Formula II into one) has a structure as shown in Formula IIIa or IIIb:

[0088] L-CD133scFv-TROP2scFv-H-TM-C-CD3ζ-D(IIIa)

[0089] L-TROP2scFv-CD133scFv-H-TM-C-CD3ζ-D(IIIb)

[0090] In the formula,

[0091] L is none or a signal peptide sequence;

[0092] CD133scFv is the first antigen binding domain that specifically binds to CD133;

[0093] TROP2scFv is the second antigen binding domain that specifically binds to TROP2;

[0094] H is none or hinge region;

[0095] TM is the transmembrane domain;

[0096] C is without or co-stimulatory domain;

[0097] CD3ζ is absent or derived from the cytoplasmic signal transduction sequence of CD3ζ (including wild type, or mutant / modified form thereof);

[0098] D is the absence or degradation domain (including the wild type, or its mutant / modified form);

[0099] The "-" is a connecting peptide or a peptide bond.

[0100] In another preferred embodiment, the dual-targeting CAR has a structure as shown in Formula IVa or IVb:

[0101] L-CD133scFvP1-TROP2scFv-CD133scFvP2-H-TM-C-CD3ζ-D(IVa)

[0102] L-TROP2scFvP1-CD133scFv-TROP2scFvP2-H-TM-C-CD3ζ-D(IVb)

[0103] in,

[0104] CD133scFvP1 is a VH that specifically binds to the first antigen binding domain of CD133, and CD133scFvP2 is a VL that specifically binds to the first antigen binding domain of CD133; or CD133scFvP1 is a VL that specifically binds to the first antigen binding domain of CD133, and CD133scFvP2 is a VH that specifically binds to the first antigen binding domain of CD133;

[0105] In another preferred embodiment, the amino acid sequence of the CD133 antigen binding domain VH is shown in SEQ ID NO: 22 or 23;

[0106] In another preferred embodiment, the amino acid sequence of the CD133 antigen binding domain VL is shown in SEQ ID NO: 24 or 25;

[0107] TROP2scFvP1 is a VH that specifically binds to the second antigen-binding domain of TROP2, and TROP2scFvP2 is a VL that specifically binds to the second antigen-binding domain of TROP2; or TROP2scFvP1 is a VH that specifically binds to the second antigen-binding domain of TROP2, and TROP2scFvP2 is a VH that specifically binds to the second antigen-binding domain of TROP2;

[0108] In another preferred embodiment, the amino acid sequence of the TROP2 antigen binding domain VH is shown in SEQ ID NO: 26 or 27;

[0109] In another preferred embodiment, the amino acid sequence of the TROP2 antigen binding domain VL is shown in SEQ ID NO: 28 or 29;

[0110] Elements L, CD133scFv, TROP2scFv, H, TM, C, CD3ζ, and D are as defined above.

[0111] In another preferred embodiment, the CD133scFv and TROP2scFv are each independently a single-chain antibody (scFv) or a single-domain antibody (VHH).

[0112] In another preferred example, TROP2scFv and CD133scFv, CD133scFvP1, TROP2scFv and CD133scFvP2, TROP2scFvP1, CD133scFv and TROP2scFvP2, VH and VL of TROP2scFv, and / or VH and VL of CD133scFv are each independently connected via a flexible linker.

[0113] In another preferred embodiment, the length of the flexible linker is 4-30 amino acids, preferably 5-25 amino acids.

[0114] In another preferred embodiment, the L is respectively selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or a combination thereof.

[0115] In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID No:3.

[0116] In another preferred embodiment, the H is selected from the hinge region of the following proteins: CD8, CD28, CD137, IgG1, IgG4, or mutants / modified forms thereof, or a combination thereof.

[0117] In another preferred example, the amino acid sequence of H is shown as SEQ ID No: 10 or SEQ ID No: 11 or SEQ ID No: 17.

[0118] In another preferred embodiment, the TM is selected from the transmembrane region of a protein in the following group: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314, or a mutant / modified form thereof, or a combination thereof.

[0119] In another preferred embodiment, the TM is the transmembrane region of CD8.

[0120] In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID No:12.

[0121] In another preferred embodiment, the C is selected from the co-stimulatory domain of the protein in the following group: 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, OX40L, or its mutant / modified form, or a combination thereof.

[0122] In another preferred embodiment, C is a co-stimulatory domain derived from 4-1BB.

[0123] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID No:13.

[0124] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID No:14.

[0125] In another preferred embodiment, the D is an oxygen-dependent degradation domain (ODD) or a partial sequence thereof;

[0126] In another preferred embodiment, the amino acid sequence of ODD is shown in SEQ ID No:18.

[0127] In another preferred embodiment, the CD133scFv and TROP2scFv are single-chain variable region sequences of antibodies targeting tumor antigens, or partial sequences thereof.

[0128] In another preferred embodiment, the CD133scFv and TROP2scFv are nanoantibody variable region sequences targeting tumor antigens, or partial sequences thereof.

[0129] In another preferred embodiment, the amino acid sequence of the CD133scFv is as shown in SEQ ID NO: 4 or 5.

[0130] In another preferred embodiment, the amino acid sequence of the TROP2scFv is shown in SEQ ID NO: 6 or 7.

[0131] In a second aspect of the present invention, there is provided a method for preparing the engineered immune cell according to the first aspect of the present invention, comprising the following steps:

[0132] (A) providing an immune cell to be modified; and

[0133] (B) Transforming the immune cells so that the immune cells express CAR molecules, thereby obtaining the engineered immune cells as described in claim 1, wherein the CAR targets surface markers of tumor cells, wherein the CAR targets two surface markers of tumor cells, TROP2 and CD133.

[0134] In another preferred embodiment, in step (B), an expression cassette expressing the dual-targeting CAR that simultaneously targets CD133 and TROP2 (as shown in Formulas IIIa, IIIb, IVa and IVb in claim 1) is introduced into the immune cells.

[0135] In another preferred embodiment, step (B) comprises: (B1) introducing a first expression cassette (as shown in Formula I of claim 1) expressing the first CAR targeting CD133 into the immune cell; (B2) introducing a second expression cassette (as shown in Formula II of claim 1) of the second CAR targeting TROP2 into the immune cell; wherein steps (B1) and (B2) can be performed in any order.

[0136] In another preferred embodiment, the step (B1) can be performed before, after, simultaneously with, or alternately with step (B2).

[0137] In another preferred embodiment, steps (B1) and (B2) are performed simultaneously or alternately.

[0138] In another preferred embodiment, the antigen binding domain of the CAR specifically binds to the surface markers TROP2 and CD133.

[0139] In another preferred embodiment, a method for preparing the CAR-T cells according to the first aspect of the present invention is provided, comprising the following steps:

[0140] (A) providing a T cell to be modified;

[0141] (B) Transforming the T cells so that the T cells express the CAR molecules, thereby obtaining the engineered immune cells as described in the first aspect of the present invention.

[0142] In another preferred embodiment, in step (B), an expression cassette expressing the dual-targeting CAR that simultaneously targets CD133 and TROP2 (as shown in Formula IIIa, IVa and IVb in the first aspect of the present invention) is introduced into the T cells.

[0143] In another preferred embodiment, in step (B), it includes: introducing a first expression cassette expressing the first CAR targeting CD133 (as shown in Formula I in the first aspect of the present invention) into the T cell; and introducing a first expression cassette expressing the second CAR targeting TROP2 (as shown in Formula II in the first aspect of the present invention) into the T cell; wherein the introduction steps can be performed in any order or simultaneously or alternately.

[0144] In another preferred embodiment, for any two expression cassettes in the first and second expression cassettes (taking the first expression cassette and the second expression cassette as an example), their transcription directions are in the same direction (→→), in opposite directions (→←) or in opposite directions (←→).

[0145] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors.

[0146] In another preferred embodiment, the first expression cassette and the second expression cassette are located in the same vector.

[0147] In another preferred embodiment, when two of the first CAR molecule targeting CD133 and the second CAR molecule targeting TROP2 are expressed in series, a connecting peptide is also included between the two proteins.

[0148] In another preferred example, the coding sequences of the two CAR molecules are connected by an internal ribosome entry site (IRES), a coding sequence of a 2A connecting peptide, or a coding sequence of a flexible connecting peptide.

[0149] In another preferred embodiment, the connecting peptide is selected from the following group: a 2A connecting peptide, or a flexible connecting peptide.

[0150] In another preferred embodiment, the 2A connecting peptide is P2A or T2A.

[0151] In another preferred embodiment, the flexible connecting peptide is a connecting peptide composed of one or more G4S, such as 4×GGGGS.

[0152] In another preferred embodiment, the connecting peptide is P2A, T2A, F2A, or a mutant / modified form thereof, or a combination thereof.

[0153] In another preferred example, the amino acid sequence of the connecting peptide is shown in SEQ ID:15.

[0154] In another preferred example, the nucleotide sequence of the IRES is shown as SEQ ID No:16.

[0155] In another preferred embodiment, the vector is a viral vector, and preferably the viral vector contains the first and second expression cassettes in tandem.

[0156] In another preferred embodiment, the vector is selected from the following group: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or a combination thereof.

[0157] In another preferred embodiment, the vector is a pCDH series lentiviral vector.

[0158] In the third aspect of the present invention, a preparation is provided, which contains the engineered immune cells as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0159] In another preferred embodiment, the preparation contains the CAR-T cells described in the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0160] In another preferred embodiment, the preparation is a liquid preparation.

[0161] In another preferred embodiment, the dosage form of the preparation includes injection.

[0162] In another preferred embodiment, the concentration of the engineered immune cells (such as CAR-T cells) in the preparation is 1×10 3 -1×10 8cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0163] In a fourth aspect of the present invention, there is provided a use of the engineered immune cells as described in the first aspect of the present invention for preparing a drug or preparation for preventing and / or treating cancer.

[0164] In another preferred embodiment, there is provided a use of the CAR-T cells as described in the first aspect of the present invention for preparing a drug or preparation for preventing and / or treating cancer or tumors.

[0165] In another preferred embodiment, the preparation contains CAR-T cells and a pharmaceutically acceptable carrier, diluent or excipient.

[0166] In another preferred embodiment, the tumor comprises a solid tumor.

[0167] In another preferred embodiment, the tumor is selected from the following group: colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer, breast cancer, liver cancer, ovarian cancer, and nasopharyngeal cancer.

[0168] In another preferred embodiment, the tumor is a tumor with high expression, medium expression or low expression of TROP2 and / or CD133.

[0169] In a fifth aspect of the present invention, a kit for preparing the engineered immune cells according to the first aspect of the present invention is provided, the kit comprising a container, and:

[0170] (1) a first nucleic acid sequence comprising a first expression cassette for expressing the first CAR (as shown in Formula I in the first aspect of the present invention), wherein the antigen binding domain of the CAR specifically binds to CD133; and

[0171] (2) a second nucleic acid sequence, the second nucleic acid sequence comprising a second expression cassette for expressing the second CAR (as shown in Formula II in the first aspect of the present invention), wherein the antigen binding domain of the CAR specifically binds to TROP2;

[0172] Alternatively, the kit comprises a container, and located within the container:

[0173] (3) a third nucleic acid sequence, wherein the third nucleic acid sequence contains a third expression cassette for expressing the dual-targeting CAR (as shown in Formulas IIIa, IIIb, IVa and IVb in the first aspect of the present invention), wherein the antigen binding domain of the dual-targeting CAR specifically binds to CD133 and TROP2.

[0174] In another preferred embodiment, the engineered immune cells are engineered T cells.

[0175] In another preferred embodiment, the first and second nucleic acid sequences are independent or linked.

[0176] In another preferred embodiment, the first and second nucleic acid sequences are located in the same or different containers.

[0177] In another preferred embodiment, the first and second nucleic acid sequences are located on the same or different vectors.

[0178] In another preferred embodiment, the first and second nucleic acid sequences are located in the same vector.

[0179] In another preferred embodiment, when two of the first and second nucleic acid sequences are located in the same vector, a coding sequence of a connecting peptide or an internal ribosome entry site (IRES) is also included between two adjacent expression cassettes.

[0180] In another preferred embodiment, the connecting peptide is P2A, T2A, F2A, or a mutant / modified form thereof, or a combination thereof.

[0181] In another preferred embodiment, the vector is a viral vector, and preferably the viral vector contains the first, second and / or third nucleic acid sequences in tandem form.

[0182] In another preferred embodiment, the dual-targeting CAR has a structure as shown in Formula IIIa or IIIb:

[0183] L-CD133scFv-TROP2scFv-H-TM-C-CD3ζ-D(IIIa)

[0184] L-TROP2scFv-CD133scFv-H-TM-C-CD3ζ-D(IIIb)

[0185] Among them, elements L, CD133scFv, TROP2scFv, H, TM, C, CD3ζ, and D are defined as above.

[0186] In another preferred embodiment, the dual-targeting CAR has a structure as shown in Formula IVa or IVb:

[0187] L-CD133scFvP1-TROP2scFv-CD133scFvP2-H-TM-C-CD3ζ-D(IVa)

[0188] L-TROP2scFvP1-CD133scFv-TROP2scFvP2-H-TM-C-CD3ζ-D(IVb)

[0189] in,

[0190] CD133scFvP1 is a VH that specifically binds to the first antigen binding domain of CD133, and CD133scFvP2 is a VL that specifically binds to the first antigen binding domain of CD133; or CD133scFvP1 is a VL that specifically binds to the first antigen binding domain of CD133, and CD133scFvP2 is a VH that specifically binds to the first antigen binding domain of CD133;

[0191] TROP2scFvP1 is a VH that specifically binds to the second antigen-binding domain of TROP2, and TROP2scFvP2 is a VL that specifically binds to the second antigen-binding domain of TROP2; or TROP2scFvP1 is a VH that specifically binds to the second antigen-binding domain of TROP2, and TROP2scFvP2 is a VH that specifically binds to the second antigen-binding domain of TROP2;

[0192] Elements L, CD133scFv, TROP2scFv, H, TM, C, CD3ζ, and D are as defined above.

[0193] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0194] Figure 1 The structure of the CAR molecule is shown.

[0195] Figure 2 The comparison of the killing effects of single-target and dual-target CAR-T cells targeting CD133 and TROP2 on colorectal cancer cells is shown.

[0196] Figure 3 The comparison of the killing effects of single-target and dual-target CAR-T cells targeting CD133 and NKG2D on colorectal cancer cells is shown.

[0197] Figure 4 The comparison of the killing effects of single-target and dual-target CAR-T cells targeting TROP2 and NKG2D on colorectal cancer cells is shown.

[0198] Figure 5 The results showed that the dual-target CAR-T cells targeting CD133 and TROP2 prepared with a single lentiviral vector had a killing effect on colorectal cancer cells.

[0199] Figure 6The results showed that CAR-T cells targeting CD133 and TROP2 have a killing effect on esophageal cancer cells and pancreatic cancer.

[0200] Figure 7 The in vivo efficacy of dual-target CAR-T cells targeting CD133 and TROP2 was shown. DETAILED DESCRIPTION

[0201] After extensive and in-depth research and a large number of screenings, the inventors have provided for the first time engineered immune cells that simultaneously target TROP2 and CD133, and their preparation and application. Specifically, the present invention co-expresses chimeric antigen receptors targeting TROP2 and CD133 in CAR-T cells at the same time. By simultaneously targeting TROP2 and CD133, CAR-T cells have a wider range of killing tumor cells, while clearing tumor stem cells and reducing the risk of tumor immune escape and recurrence. The present invention was completed on this basis.

[0202] The present invention takes CAR-T cells as an example, and representatively describes the engineered immune cells of the present invention in detail. The engineered immune cells of the present invention are not limited to the CAR-T cells described in the context, and the engineered immune cells of the present invention have the same or similar technical features and beneficial effects as the CAR-T cells described in the context. Specifically, when immune cells express chimeric antigen receptor CAR, NK cells and TIL cells are equivalent to T cells (or T cells can be replaced by NK cells or TIL cells).

[0203] the term

[0204] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0205] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0206] The terms "administer" or "administration" refer to introducing the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0207] Antibody

[0208] As used herein, the term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises 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 antigens.

[0209] "Antibodies" also include single-domain antibodies (also called nanobodies). Single-domain antibodies are antibodies that are naturally missing light chains and exist in the peripheral blood of animals such as alpacas, camels, and sharks. The antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. The VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to the target antigen.

[0210] Antigen binding domain

[0211] As used herein, "antigen binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments, or VHH fragments of single-domain antibodies that have antigen binding activity. Fv antibodies contain the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with 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 usually scFv (single-chain variable fragment). A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain.

[0212] In the present invention, the scFv comprises an antibody or an active fragment thereof that specifically recognizes CD133 and TROP2 antigens expressed by tumors.

[0213] In addition, the immune cells of the present invention may also contain additional antibodies that specifically recognize antigens highly expressed in tumors, preferably single-chain antibodies or Fv antibodies.

[0214] Chimeric Antigen Receptor (CAR)

[0215] As used herein, chimeric immune antigen receptor (CAR) includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain. The extracellular domain includes an optional signal peptide and a target-specific binding domain (also referred to as an antigen binding domain). The intracellular domain includes a co-stimulatory domain and a CD3ζ chain portion. When CAR is expressed in T cells, the extracellular segment can recognize a specific antigen, and then the signal is transduced through the intracellular domain, causing cell activation proliferation, cytolytic toxicity, and secretion of cytokines such as IL-2 and IFN-γ, etc., affecting tumor cells, causing tumor cells to not grow, be prompted to die or otherwise be affected, and causing the patient's tumor load to shrink or be eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a co-stimulatory molecule and a CD3ζ chain. Preferably, the antigen binding domain is fused with the intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0216] In one embodiment, the CAR of the present invention targets CD133 and TROP2, and can specifically bind to CD133 and TROP2 on the surface of tumor cells.

[0217] In one embodiment, the engineered immune cell of the present invention is a dual-targeting CAR engineered immune cell, which contains the first CAR targeting CD133 and the second CAR targeting TROP2.

[0218] In the present invention, the two CARs contained in the dual-targeting CAR engineered immune cells can be activated through the "ANDGATE" activation mode.

[0219] In the activation mode of the "AND GATE", the intracellular structure of one CAR contains a 41BB element but does not contain a CD3ζ element, while the intracellular structure of another CAR does not contain a 41BB element but contains a CD3ζ element. For example, one CAR has a "CD133scFv-41BB" structure, while another CAR has a "TROP2scFv-CD3ζ" structure, and vice versa. The dual CAR in the "ANDGATE" activation mode requires two CARs to be activated at the same time to kill, and has a higher safety.

[0220] In addition, in the present invention, the two CARs contained in the dual CAR engineered immune cells can also be activated by the "ORGATE" activation mode.

[0221] In the activation mode of the "OR GATE", the intracellular structures of both CARs contain 41BB elements and CD3ζ elements. One CAR has a "CD133scFv-41BB-CD3ζ" structure, while the other CAR has a "TROP2scFv-41BB-CD3ζ" structure. The dual CAR in the "OR GATE" activation mode only needs one of the CARs to be activated to kill, and has a wider killing range.

[0222] Chimeric Antigen Receptor T Cells (CAR-T Cells)

[0223] As used herein, the terms "CAR-T cells", "CAR-T", and "CAR-T cells of the present invention" all refer to the CAR-T cells described in the first aspect of the present invention. The CAR-T cells of the present invention can be used to treat tumors expressing CD133 and / or TROP2, such as colorectal cancer, ovarian cancer, pancreatic cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, nasopharyngeal cancer, etc.

[0224] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigens, once the CAR gene construction targeting a certain tumor antigen is completed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) The use of the patient's own cells reduces the risk of rejection; and (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0225] Chimeric Antigen Receptor NK Cells (CAR-NK Cells)

[0226] As used herein, the terms "CAR-NK cells", "CAR-NK", and "CAR-NK cells of the present invention" all refer to the CAR-NK cells described in the first aspect of the present invention. The CAR-NK cells of the present invention can be used to treat tumors expressing CD133 and / or TROP2, such as colorectal cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, breast cancer, liver cancer, nasopharyngeal carcinoma, etc.

[0227] Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infection 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 have enhanced anti-tumor cytotoxic effects.

[0228] Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are very easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

[0229] Expression cassette

[0230] As used herein, "expression cassette" or "expression cassette of the present invention" include the first expression cassette for expressing the first CAR targeting CD133, the second expression cassette for expressing the second CAR targeting TROP2, or the third expression cassette for expressing the dual-targeting CAR targeting CD133 and TROP2. Typically, the expression cassette includes the coding sequence of the CAR operably linked to the promoter.

[0231] In the present invention, the first expression cassette comprises a nucleic acid sequence encoding the first CAR targeting CD133; the second expression cassette comprises a nucleic acid sequence encoding the second CAR targeting TROP2. The third expression cassette comprises a nucleic acid sequence encoding the dual-targeting CAR targeting CD133 and TROP2.

[0232] In the present invention, the CAR can be constitutively expressed or inducibly expressed.

[0233] In the case of induced expression, when the CAR-T cell is activated by the corresponding inducer or induction condition, the expression cassette expresses the corresponding CAR; thus, when the CAR-T cell of the present invention is not exposed to the corresponding inducer or induction condition, the expression cassette does not express the corresponding CAR.

[0234] In one embodiment, the first expression cassette, the second expression cassette and / or the third expression cassette further include a promoter and / or a terminator, respectively. The promoters of the second expression cassette and the third expression cassette may be constitutive or inducible promoters.

[0235] Carrier

[0236] The present invention also provides a vector containing the expression cassette of the present invention. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow transgenes to be stably integrated into the cell genome for a long time and replicate with the replication of the daughter cell genome. Lentivirus vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells and have the advantage of low immunogenicity.

[0237] Usually, expression cassette or nucleic acid sequence of the present invention can be connected to the promoter downstream by routine operation, and incorporated into expression vector.This vector can be integrated into the eukaryotic cell genome and replicated therewith.Typical cloning vectors include transcription and translation terminators, initial sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0238] The expression vectors of the present invention can also be used in standard gene delivery protocols for nucleic acid immunization 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, 5,589,466, which are incorporated herein by reference in their entirety.

[0239] The expression cassette or nucleic acid sequence can be cloned into many types of vectors. For example, the expression cassette or nucleic acid sequence can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, etc.

[0240] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally, suitable vectors include at least one replication origin, promoter sequence, convenient restriction enzyme site and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193) that work in organisms.

[0241] Many virus-based systems have been developed and used for gene transduction of mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In one embodiment, a lentiviral vector is used. Many DNA viral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art.

[0242] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these elements are located in the 30-110bp region upstream of the start site, although recently many promoters have been shown to also contain functional elements downstream of the start site. The intervals between promoter elements are often flexible, so that when an element is inverted or moved relative to another element, the promoter function is maintained. In the thymidine kinase (tk) promoter, the intervals between promoter elements can be increased by 50bp, and activity begins to decline. Depending on the promoter, it is shown that a single element can work cooperatively or independently to start transcription.

[0243] An example of a suitable promoter is a cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. 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 simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus (Epstein-Barr virus, EBV) immediate early promoter, Rous sarcoma virus promoter, and human gene promoter, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be a part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence connected to the inducible promoter when desired, or turn off expression when not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0244] The expression vector introduced into the cell may also comprise any one or both of a selectable marker gene or a reporter gene, so that the expression cell can be identified and selected from a transfected or infected cell population by a viral vector. In other respects, selectable markers may be carried on a single section of DNA and used for cotransfection procedures. Both selectable marker genes and reporter genes may be flanked by suitable regulatory sequences so that they can be expressed in host cells. Useful selectable marker genes include, for example, antibiotic resistance genes, such as neomycin, etc.

[0245] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, for example, mammals (such as human T cells), bacteria, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0246] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, cationic complex transfection, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001). Preferred methods for introducing polynucleotides into host cells are liposome transfection and cationic complex polyethyleneimine transfection.

[0247] Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0248] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0249] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid formulations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipid, mixed with lipid, combined with lipid, included in lipid as a suspension, included in micelle or compounded with micelle, or otherwise associated with lipid. The lipid, lipid / DNA or lipid / expression vector associated with the composition are not limited to any specific structure in the solution. They can also be simply dispersed in the solution, and may form an aggregate of size or shape inhomogeneity. Lipid is a lipid substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0250] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0251] It should be understood that in the present invention, in addition to using lentivirus for transduction, CAR targeting CD133 and TROP2 can also be expressed in immune cells such as T cells by directly transfecting mRNA or plasmids, or by expressing artificial transcription factors.

[0252] preparation

[0253] The present invention provides a composition containing the engineered immune cells (such as CAR-T cells) described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0254] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0255] Therapeutic applications

[0256] The present invention provides therapeutic applications of cells (e.g., T cells) transduced by a vector (e.g., a lentiviral vector) of the expression cassette of the present invention. The transduced T cells can target the surface markers CD133 and / or TROP2 of tumor cells, synergistically and significantly improving their killing efficiency of tumor cells.

[0257] Therefore, the present invention also provides a method for stimulating an immune response mediated by T cells targeting a tumor cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cells of the present invention to a mammal.

[0258] In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient. This approach makes the probability of graft-versus-host reaction extremely low, and the antigen is recognized by the T cell in an MHC-free manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

[0259] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and can last for months to years. In addition, the CAR-mediated immune response can be part of the adoptive immunotherapy step. Among them, the CAR-T cells can produce a specific immune response to tumor cells expressing the corresponding antigens. For example, the CAR-T cells of the present invention cause a specific immune response to tumor cells expressing CD133 and / or TROP2.

[0260] Treatable cancers include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors. The types of cancers treated with the CAR of the present invention include, but are not limited to, colorectal cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer, breast cancer, nasopharyngeal cancer, etc.

[0261] Generally, cells activated and expanded as described herein can be used to treat and prevent diseases such as tumors. Therefore, the present invention provides a method for treating cancer, which comprises administering a therapeutically effective amount of CAR-T cells of the present invention to a subject in need thereof.

[0262] The CAR-T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell populations. In short, the pharmaceutical composition of the present invention may include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0263] The pharmaceutical composition 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, or can be determined by clinical trials.

[0264] When "therapeutically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. The pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 7 The T cell composition can also be administered multiple times at these doses. The cells can be administered using well-known infusion techniques in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0265] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The composition of the T cell can be directly injected into the tumor, lymph node or infection site.

[0266] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, at the same time, or after), including but not limited to treatment with the following agents: agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants, such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, at the same time, or after). For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered prior to or after surgery.

[0267] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Generally, 1×10 5 to 1×10 10 The modified T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0268] The main advantages of the present invention include:

[0269] (1) Compared with single-target CAR-immune cells, the dual-target CAR-immune cells of the present invention can improve tumor killing ability and prevent tumor immune escape and recurrence;

[0270] (2) Compared with single-target CAR-immune cells, the dual-target CAR-immune cells of the present invention can cover more tumor types and meet the needs of more different cancer patients;

[0271] (3) Compared with bispecific antibodies, the effect is sustained, no repeated administration is required, and there is memory.

[0272] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (Sambrook et al., New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0273] Unless otherwise stated, the reagents and materials used in the examples were commercially available.

[0274] Materials and Methods

[0275] Structural design of CAR molecules

[0276] The target gene structure of the lentiviral vector involved in this example is as follows Figure 1 shown.

[0277] (1) BW133-2 is composed of the following structures in series: human CD8 signal peptide [abbreviated as CD8(SP)], anti-human CD133 single-chain antibody 1 (abbreviated as CD133 scFv1), human IgG hinge region [abbreviated as IgG(Fc)], human CD8 transmembrane domain [abbreviated as CD8(TM)], human 4-1BB intracellular co-stimulatory domain [abbreviated as 4-1BB(ID)], and human CD3ζ intracellular signal transduction domain [abbreviated as CD3ζ(ID)];

[0278] (2) BW133-3 is composed of the following structures in series: CD8 (SP), anti-human CD133 single-chain antibody 2 (abbreviated as CD133scFv2), IgG (Fc), CD8 (TM), 4-1BB (ID), and CD3ζ (ID);

[0279] (3) BTRP003 is composed of the following structures in series: CD8 (SP), anti-human TROP2 single-chain antibody 1 (abbreviated as TROP2scFv1), human CD8 hinge region [abbreviated as CD8(hinge)], CD8 (TM), 4-1BB (ID), and CD3ζ (ID);

[0280] (4) BTRP101 is composed of the following structures in series: CD8 (SP), anti-human TROP2 single-chain antibody 2 (abbreviated as TROP2scFv2), CD8 (hinge), CD8 (TM), 4-1BB (ID), and CD3ζ (ID);

[0281] (5) The structure of BN001 is composed of the following structures in series: CD8 (SP), human NKG2D extracellular domain [abbreviated as NKG2D (ED)], CD8 (hinge), CD8 (TM), 4-1BB (ID), and CD3ζ (ID);

[0282] (6) BTRP133-3 is composed of the following structures in series: CD8 (SP), TROP2 scFv1, linker, CD133scFv1, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3ζ (ID);

[0283] (7) BTRP133-4 is composed of the following structures in series: CD8 (SP), CD133 scFv1, linker, TROP2scFv1, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3ζ (ID);

[0284] (8) BTRP133-13 is composed of the following structures in series: CD8 (SP), TROP2 scFv1, CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3ζ (ID), self-cleaving peptide (abbreviated as 2A), CD8 (SP), CD133 scFv1, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3ζ (ID);

[0285] (9) BTRP133-14 is composed of the following structures in series: CD8(SP), TROP2 scFv1, CD8(hinge), CD8(TM), 4-1BB(ID), CD3ζ(ID), 2A, CD8(SP), CD133 scFv1, IgG(Fc), CD8(TM), human CD28 intracellular co-stimulatory domain [abbreviated as CD28(ID)], CD3ζ(ID);

[0286] (10) BTRP133-15 is composed of the following structures in series: CD8 (SP), TROP2 scFv1, CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3ζ (ID), internal ribosome entry site (IRES), CD8 (SP), CD133 scFv1, IgG (Fc), CD8 (TM), CD28 (ID), CD3ζ (ID);

[0287] (11) BTRP133-16 is composed of the following structures in series: CD8 (SP), TROP2 scFv1, CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3ζ (ID), IRES, CD8 (SP), CD133 scFv1, IgG (Fc), CD8 (TM), CD28 (ID), CD3ζ (ID).

[0288] The amino acid or nucleotide sequences of the related structures are as follows:

[0289] CD133 (SEQ ID: 1):

[0290]

[0291] TROP2 (SEQ ID: 2):

[0292]

[0293] CD8(SP)(SEQ ID:3):MALPVTALLLPLALLLHAARPS

[0294] CD133 scFv1 (SEQ ID: 4):

[0295]

[0296] CD133 scFv2 (SEQ ID: 5):

[0297]

[0298] TROP2 scFv1 (SEQ ID: 6):

[0299]

[0300] TROP2 scFv2 (SEQ ID: 7):

[0301]

[0302] NKG2D (ED) (SEQ ID: 8):

[0303]

[0304] linker(SEQ ID:9):GGGGSGGGGSGGGGSGGGGS

[0305] IgG1(Fc) (SEQ ID:10):

[0306]

[0307] CD8(hinge)(SEQ ID:11):TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFA

[0308] CD8(TM)(SEQ ID:12):CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR

[0309] 4-1BB(ID)(SEQ ID:13):KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0310] CD3ζ(ID) (SEQ ID: 14):

[0311]

[0312]

[0313] 2A(SEQ ID:15):SGSGAPVKQTLNFDLLKLAGDVESNPGP

[0314] IRES (nucleotide sequence) (SEQ ID: 16):

[0315]

[0316] IgG4 (Fc) (SEQ ID: 17):

[0317]

[0318] ODD (SEQ ID: 18):

[0319]

[0320] HRE1 (nucleotide sequence) (SEQ ID: 19): GGCCCTACGTGCTGTCTCACACAGCCTGTCTGAC

[0321] HRE2 (nucleotide sequence) (SEQ ID: 20): CCACAGTGCATACGTGGGCTCCAACAGGTCCTCTT

[0322] HRE3 (nucleotide sequence) (SEQ ID: 21): GTCGTGCAGGACGTGACA

[0323] CD133 antigen binding structure and VH1 (SEQ ID: 22):

[0324]

[0325] CD133 antigen binding structure and VH2 (SEQ ID: 23):

[0326]

[0327] CD133 antigen binding structure and VL1 (SEQ ID: 24):

[0328]

[0329] CD133 antigen binding structure and VL2 (SEQ ID: 25):

[0330]

[0331] TROP2 antigen binding structure and VH1 (SEQ ID: 26):

[0332]

[0333] TROP2 antigen binding structure and VH2 (SEQ ID: 27):

[0334]

[0335] TROP2 antigen binding structure and VL1 (SEQ ID: 28):

[0336]

[0337] TROP2 antigen binding structure and VL2 (SEQ ID: 29):

[0338]

[0339] Example 1 Preparation of Lentivirus Vector

[0340] The whole gene synthesis of the above nucleotide sequences ( Figure 1), and then connected to the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning, so that it is expressed under the regulation of the human EF-1α promoter and Kozak sequence. The above-mentioned lentiviral vector expression plasmids were co-transfected into 293T cells with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G according to the relevant instructions using the transfection reagent Lipofectamine 3000. The virus supernatant was collected 48 hours after transfection to obtain the virus concentrate, which was named BW133-2, BW133-3, BTRP003, BTRP101, BN001, BTRP133-3, BTRP133-4, BTRP133-13, BTRP133-14, BTRP133-15 and BTRP133-16, and stored at -80°C for use. Finally, Jurkat cells were used as materials to detect the activity titer of the above-mentioned lentiviruses.

[0341] Example 2 Preparation of CAR-T cells

[0342] Peripheral blood mononuclear cells (PBMC) from healthy donors were resuscitated in AIM V medium and stimulated with anti-human CD3 / CD28 antibodies to culture T cells. T cells were transduced with lentivirus at an MOI of 5 IU / ml. On the 7th day after transduction, the expression of the corresponding CAR molecules was detected. On the 10th day after transduction, the cells were collected and stored in liquid nitrogen for later use. The obtained CAR-T cells continued to use the name of the lentivirus. The single-target CAR-T cells were BW133-2, BW133-3, BTRP003, BTRP101 and BN001, and the dual-target CAR-T cells were BTRP133-3, BTRP133-4, BTRP133-13, BTRP133-14, BTRP133-15 and BTRP133-16. T cells not transduced with lentivirus were named Ctrl T.

[0343] The lentivirus BW133-2, BW133-3, BTRP003, BTRP101, and BN001 were combined in pairs and co-transduced into T cells at an MOI of 5 IU / ml. On the 7th day after transduction, the expression of the corresponding CAR molecules was detected, and the cells were collected on the 10th day after transduction and frozen in liquid nitrogen for later use. The obtained dual-target CAR-T cells were named BW133-2&BTRP003, BW133-3&BTRP003, BW133-2&BTRP101, BW133-3&BTRP101, BN001&BTRP003, BN001&BTRP101, BN001&BW133-2, and BN001&BW133-3.

[0344] Example 3 CAR-T cell expression detection

[0345] Wash the cells of each group to be tested twice with FACS buffer, add human CD133 recombinant protein and human TROP2 protein with His-tag respectively, and incubate at 4°C for 1h. After washing once with FACS buffer, add anti-His-tag antibody and incubate at 4°C for 1h. After washing with FACS buffer, use cells stained with anti-His-tag antibody only as negative control, and use flow cytometry to detect the expression rate of CD133 CAR molecules and TROP2CAR molecules in each group of cells. For the expression of NKG2D CAR molecules, stain with anti-human NKG2D antibody and detect the corresponding expression rate by flow cytometry.

[0346] The analysis was performed using FlowJo software, and the expression rates of each CAR-T cell are shown in Table 1 .

[0347] Table 1 Detection of the expression rate of CAR molecules in each T cell

[0348]

[0349]

[0350] Example 4 Detection of target cells

[0351] The tumor cells used in this example are all reporter cell lines expressing luciferase and fluorescent protein, including colorectal cancer cell HT-29-Luc-mCherry (cultured in McCoy's 5a medium + 10% fetal bovine serum + 100U / ml penicillin + 100μg / ml streptomycin), esophageal cancer cell KYSE-520-Luc-mCherry (cultured in RPMI 1640 medium + 10% fetal bovine serum + 100U / ml penicillin + 100μg / ml streptomycin) and pancreatic cancer cell line Capan-2-Luc-mCherry (cultured in McCoy's 5a medium + 10% fetal bovine serum + 100U / ml penicillin + 100μg / ml streptomycin). The expression rates of CD133, TROP2, and NKG2D ligands of target cells were detected by flow cytometry. FlowJo software was used for analysis, and the results are shown in Table 2.

[0352] Table 2 Detection of target protein expression rate of each tumor cell

[0353]

[0354] Example 5 Combination screening of dual-target CAR molecules

[0355] First, the dual-target CAR-T cells (BW133-2&BTRP003, BW133-3&BTRP003, BW133-2&BTRP101, BW133-3&BTRP101, BN001&BTRP003, BN001&BTRP101, BN001&BW133-2, and BN001&BW133-3) prepared by dual virus co-transduction were used for experiments and compared with the killing effects of the corresponding single-target CAR-T cells to determine which target combination can effectively improve the killing effect of CAR-T cells. McCoy's 5a complete medium was used at 1×10 5 HT-29-Luc-mCherry cells were resuspended at a density of 100 μl / ml and inoculated in a 96-well plate at a volume of 100 μl per well. After being cultured overnight in the IncuCyteSX5 live cell imaging analysis system, single-target or dual-target CAR-T cells were added for co-culture at an effect-target ratio of 2:1. The mCherry fluorescence signal of the target cells in each group was recorded in real time, and the target cells were added again at the appropriate time to repeatedly stimulate the T cells. After the co-culture, the changes in the mCherry fluorescence area of ​​the target cells in each group were calculated by the IncuCyte software. The lower the signal value, the smaller the area occupied by the target cells in the group, and the better the T cell killing effect. By comparing the fluorescence signal values ​​detected at the last time point, it is possible to statistically determine whether there are significant differences in the killing effects of the CAR-T cells in each group.

[0356] For CAR-T cells targeting CD133 and TROP2, the killing effect is as follows Figure 2 As shown. After co-culture with target cells, the killing effects of BW133-2, BW133-3, BTRP003 and BTRP101 gradually weakened, but the dual-target CAR-T group (BW133-2&BTRP003, BW133-3&BTRP003, BW133-2&BTRP101, BW133-3&BTRP101) can still maintain the killing effect, and the killing effect is significantly better than the corresponding single-target CAR-T group (P<0.05). This result shows that simultaneous targeting of CD133 and TROP2 can effectively improve the killing effect of CAR-T cells on colorectal cancer cells.

[0357] For CAR-T cells targeting CD133 and NKG2D ligands, the killing effect is as Figure 3As shown. After co-culture with target cells, the killing effect of the dual-target CAR-T cell group (BW133-2&BN001 and BW133-3&BN001) was not completely superior to that of the single-target CAR-T cell group (BW133-2, BW133-3 and BN001). For example, although the killing effect of CAR-T cells in the BW133-2&BN001 group was significantly better than that of BW133-2, its killing effect was not significantly different from that of BN001 (P>0.05). Therefore, simultaneous targeting of CD133 and NKG2D cannot effectively improve the killing effect of CAR-T cells on colorectal cancer cells.

[0358] For CAR-T cells targeting TROP2 and NKG2D ligands, the killing effect is as Figure 4 As shown. After co-culture with target cells, the killing effect of the dual-target CAR-T cell group (BTRP003&BN001 and BTRP101&BN001) was not completely superior to that of the single-target CAR-T cell group (BTRP003, BTRP101 and BN001). For example, the CAR-T cells in the BTRP003&BN001 group had no significant difference from BN001 (P>0.05) and were even significantly weaker than BN001 (P<0.05). Therefore, simultaneous targeting of TROP2 and NKG2D cannot effectively improve the killing effect of CAR-T cells on colorectal cancer cells.

[0359] In summary, although CD133, TROP2, and NKG2D ligands have been reported to be highly expressed in a variety of solid tumor cells, when constructing dual-target CAR-T cells, not any combination of two targets can effectively enhance the killing effect of CAR-T cells on tumor cells. For colorectal cancer cells, when NKG2D ligand is used as one of the targets of dual-target CAR-T cells, the killing effect of CAR-T cells cannot be enhanced. Only by targeting CD133 and TROP2 at the same time can the killing effect of dual-target CAR-T cells be significantly enhanced.

[0360] Example 6 Structural screening of dual-target CAR molecules

[0361] The dual-target CAR-T cells in the above experiment were prepared by dual-virus co-transduction. To determine the feasibility of preparing dual-target CAR-T cells using a single lentiviral vector, HT-29-Luc-mCherry was used as the target cell, and experiments were conducted using BTRP133-3, BTRP133-4, BTRP133-13, BTRP133-14, BTRP133-15 and BTRP133-16 to compare the killing effects with those of the corresponding single-target CAR-T cells.

[0362] After co-culture with target cells, the killing effect of BTRP133-4 was comparable to that of single-target CAR-T cells BW133-2 and BTRP003, while the killing effect of BTRP133-3 was significantly stronger than that of other groups (P<0.05)( Figure 5 ), indicating that when the scFv targeting CD133 and TROP2 are connected in series on the same CAR molecule, the scFv targeting TROP2 is located at the N-terminus of the scFv targeting CD133, which is more conducive to improving the targeted killing effect of immune cells. In addition, the killing effects of BTRP133-13, BTRP133-14, BTRP133-15 and BTRP133-16 are weak (not shown), and this killing effect is also consistent with its lower expression level (Table 1). This result shows that compared with the structure expressing two CAR molecules separately, the structure of scFv targeting CD133 and TROP2 connected in series on the same CAR molecule is more conducive to improving the targeted killing effect of immune cells.

[0363] The results of the above structural screening showed that when the scFvs targeting CD133 and TROP2 are connected in series in the same CAR molecular structure and the scFv targeting TROP2 is located at the N-terminus of the scFv targeting CD133, dual-target CAR-T cells can be prepared through a single lentiviral vector, and the targeted killing effect of immune cells mediated by this structure is significantly stronger than that of single-target CAR-T cells, indicating that this approach is feasible.

[0364] Example 7: Expansion of indications for dual-target CAR-T cells

[0365] In addition to colorectal cancer, abnormal expression of CD133 and TROP2 can be detected in a variety of malignant solid tumors. Therefore, KYSE-520 esophageal cancer cells and Capan-2 pancreatic cancer cells were used as target cells to compare the killing effects of BTRP003, BW133-2 and BTRP133-3.

[0366] The results are as follows Figure 6 A and Figure 6 As shown in Figure B, BTRP133-3 can also achieve a significantly better killing effect than BTRP003 and BW133-2 for esophageal cancer cells and pancreatic cancer cells (P<0.05). The above results show that dual-target CAR-T cells targeting CD133 and TROP2 have better killing effects on a variety of solid tumors.

[0367] Example 8 Dual-target CAR-T cell in vivo tumor inhibition experiment

[0368] Using Ctrl T, BW133-2, BTRP003 and BTRP133-3 as effector cells and HT29 as target cells, the inhibitory effect of subcutaneous transplanted tumors in mice was tested. Fifteen 6-8 week old NOG mice were subcutaneously injected with 1×10 6 HT-29 cells. The average tumor volume is about 50mm 3 The mice were randomly divided into vehicle control group, Ctrl T control group, BW133-2 control group, BTRP003 control group, and BTRP133-3 experimental group, and the vehicle (100 μl / mouse) or the corresponding T cells (5×10 6 The day of injection of the test substance was recorded as treatment day 0. The tumor volume and mouse body weight were measured 2-3 times a week.

[0369] like Figure 7 As shown, in the HT-29 subcutaneous tumor model, the tumor volume detected in the BTRP133-3 experimental group was 91.3±76.1mm on day 31 after CAR-T cell infusion. 3 , which was significantly smaller than that of the BW133-2 control group (tumor volume was 1039.3±440.8mm 3 ) and BTRP003 control group (tumor volume was 375.7±100.6mm 3 )(P<0.05). The above data show that the inhibitory effect of dual-target CAR-T cells targeting CD133 and TROP2 on solid tumors in vivo is also significantly better than that of single-target CAR-T cells.

[0370] discuss

[0371] Although there are currently CAR-T cell or antibody technologies targeting CD133 or TROP2, they still fail to meet the needs of the huge patient population of malignant solid tumors. The existing technology still has a lot of room for improvement, and the shortcomings are as follows:

[0372] (1) Although single-target CAR-T cells of CD133 or TROP2 have certain in vivo efficacy, it is still difficult to completely eliminate tumor cells, especially to prevent immune escape and recurrence of tumors with low target expression rates;

[0373] (2) Dual-target CAR-T cells targeting CD133 or TROP2 can improve the therapeutic effect. However, in the existing technology, the combined targets are only significantly expressed in a few cancer types, so the scope of application is narrow (for example, GPC-3 is specifically expressed in liver cancer, so CD133 and GPC-3 dual-target CAR-T cells can only be used to treat liver cancer; TROP2×PD-L1 dual-target CAR-T cells must be in the form of third-generation CAR to treat gastric cancer, which poses a safety risk).

[0374] (3) Dual-target drugs targeting CD133 or TROP2 with a wide range of indications currently only have bispecific antibodies (such as CD133×EpCAM and TROP2×HER2 bispecific antibodies). During treatment, continuous administration is required, and the killing effect is mainly exerted through antibody-coupled drugs, which is not as effective as immune cell therapy.

[0375] Therefore, the purpose of the present invention is to obtain a multi-target engineered immune cell that can be used to treat a variety of solid tumors and solve the following problems:

[0376] (1) Although some proteins have been found in solid tumors that can be used as targets for immune cell therapy (such as CEA, EpCAM, HER2, CD133, TROP2, NKG2D ligand, etc.), not all combinations of targets can effectively enhance the killing effect of immune cells, nor can all combinations of targets expand the range of indications for immune cell therapy. To this end, this study screened and compared different combinations of therapeutic targets through a large number of experiments, and obtained a target combination that can enhance the killing effect of CAR-T cells and can be used for the treatment of a variety of solid tumors;

[0377] (2) There are many ways to prepare bispecific engineered immune cells. In some studies, the expression sequences of two CAR molecules can be transduced into immune cells simultaneously through two vectors, but the amount of lentivirus used in this method is higher, which is more likely to cause genetic mutations in immune cells, and there are safety risks in clinical use, and the actual production is more difficult. Some studies also transduce the expression sequences of two CAR molecules into immune cells simultaneously through one vector, but this method will increase the size of the target gene in the vector, which may reduce the expression level of the CAR molecule and affect the effectiveness. There are also studies that use design modifications to enable a CAR molecule to recognize two targets at the same time, but the CAR molecular structure needs to be screened. To this end, this study screened out an effective implementation form for CD133 and TROP2 dual-target immune cells through a large number of experiments to ensure that the dual-target engineered immune cells can be used in actual production.

[0378] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An engineered immune cell, characterized in that: The engineered immune cells are T cells, TIL cells, or NK cells, and the immune cells have the following characteristics: The immune cells express chimeric antigen receptors (CARs), wherein the CARs target two surface markers of tumor cells, CD133 and TROP2. Wherein, the CAR includes a first CAR targeting CD133 and a second CAR targeting TROP2; Alternatively, the CAR comprises a dual-targeting CAR antigen binding domain that specifically binds to both CD133 and TROP2.

2. A method for preparing the engineered immune cell according to claim 1, characterized in that: The following steps are involved: (A) providing an immune cell to be modified; and (B) Transforming the immune cells so that the immune cells express CAR molecules, thereby obtaining the engineered immune cells as described in claim 1, wherein the CAR targets surface markers of tumor cells, wherein the CAR targets two surface markers of tumor cells, TROP2 and CD133.

3. A preparation, characterized in that The preparation contains the engineered immune cell as claimed in claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

4. A use of the engineered immune cell according to claim 1, characterized in that: Used for preparing medicines or preparations for preventing and / or treating cancer.

5. A kit for preparing the engineered immune cell according to claim 1, characterized in that: The kit comprises a container, and located within the container: (1) a first nucleic acid sequence comprising a first expression cassette for expressing the first CAR (as shown in Formula I in claim 1), wherein the antigen binding domain of the CAR specifically binds to CD133; and (2) a second nucleic acid sequence, the second nucleic acid sequence comprising a second expression cassette for expressing the second CAR (as shown in Formula II of claim 1), wherein the antigen binding domain of the CAR specifically binds to TROP2; Alternatively, the kit comprises a container, and located within the container: (3) a third nucleic acid sequence, wherein the third nucleic acid sequence contains a third expression cassette for expressing the dual-targeting CAR (as shown in Formulas IIIa, IIIb, IVa and IVb of claim 1), wherein the antigen binding domain of the dual-targeting CAR specifically binds to CD133 and TROP2.

6. The kit according to claim 5, characterized in that The engineered immune cells are engineered T cells.

7. The kit according to claim 5, characterized in that The first and second nucleic acid sequences are independent or linked.

8. The kit according to claim 5, characterized in that The first and second nucleic acid sequences are located in the same or different containers.

9. The kit according to claim 5, characterized in that The first and second nucleic acid sequences are located on the same or different vectors.

10. The kit according to claim 5, characterized in that The first and second nucleic acid sequences are located in the same vector.

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