Engineered immune cell, preparation therefor and use thereof

Abstract

An engineered immune cell, preparation therefor and a use thereof. A TROP2-targeted chimeric antigen receptor is expressed in immune cells, resulting in an unexpected killing effect of engineered immune cells on tumor cells.

Description

An engineered immune cell and its preparation and application Technical Field

[0001] The present invention belongs to the field of tumor immunity and cell therapy, and specifically relates to an 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 for hematologic malignancies has revolutionized cancer treatment and provided a new option for precision medicine. CAR-T cells are genetically engineered T cells that express a CAR molecule that recognizes specific tumor cell surface antigens, guiding the T cells to recognize and eliminate the tumor cells. The CAR molecule consists of an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain. CAR-T cell technology has shown promising efficacy in some lymphomas, but relapse can also occur due to the proliferation of CD19-negative lymphoma cells. Malignant solid tumors exhibit greater target heterogeneity than lymphomas, making target-negative tumor cells more susceptible to immune evasion, often leading to relapse after treatment. However, CAR-T cell therapies currently in clinical trials primarily target single-target CAR-T cells. Simultaneously targeting two or more tumor targets is a key breakthrough in addressing the problem of tumor immune evasion in CAR-T cell therapy.

[0003] TROP2 is a cell surface glycoprotein encoded by the TACSTD2 gene (amino acid sequence shown in SEQ ID No: 1). 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 tumor patient survival. 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, bile duct cancer, lung cancer, esophageal cancer, pancreatic cancer, and breast cancer. Therefore, TROP2 has become an attractive therapeutic target in the treatment of solid tumors.

[0004] TROP2 is also a marker for cancer stem cells. Studies have shown that cancer stem cells with high TROP2 expression can be isolated and identified from various solid tumors. TROP2 is associated with cell regeneration and plays a crucial role in regulating tumor growth and metastasis.

[0005] Therefore, there is an urgent need in this field to develop engineered immune cells with excellent performance targeting TROP2, so that they can have better recognition ability of tumor cells and can also eliminate tumor stem cells at the same time, effectively control tumor growth and reduce the risk of recurrence.

[0006] Summary of the Invention

[0007] 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).

[0008] Another object of the present invention is to provide an engineered immune cell (such as a CAR-T cell) expressing a chimeric antigen receptor that specifically binds to TROP2, and a preparation method and application thereof.

[0009] The first aspect of the present invention provides a chimeric antigen receptor (CAR) protein, wherein the CAR targets TROP2 and has a structure as shown in Formula Ia or Ib: L-TROP2scFv-H-TM-C1-C2-CD3ζ-D (Ia) L-TROP2scFv-H-TM-CD3ζ-C1-C2-D (Ib)

[0010] Where,

[0011] L is none or a signal peptide sequence;

[0012] TROP2scFv is a single-chain antibody (scFv) targeting TROP2; H is the hinge region;

[0013] TM is the transmembrane domain;

[0014] C1 is none or the first costimulatory domain;

[0015] C2 is no or a second costimulatory domain;

[0016] CD3ζ is absent or derived from a cytoplasmic signaling sequence of CD3ζ (including wild type, mutants, or modified forms thereof);

[0017] D is the absence or degradation domain (including wild type, or mutant / modified form thereof);

[0018] The "-" is a connecting peptide or peptide bond.

[0019] In another preferred embodiment, the L is respectively selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα, TCRβ or their mutants / modified forms, or a combination thereof.

[0020] In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID No: 2.

[0021] In another preferred embodiment, the TROP2scFv has the following structure of Formula IIA or IIB: VH-VL (IIA); VL-VH (IIB);

[0022] Wherein, VH is the heavy chain variable region of TROP2scFv, and VL is the light chain variable region of TROP2scFv;

[0023] “-” indicates a connecting peptide or none.

[0024] In another preferred embodiment, the amino acid sequence of VL is shown in SEQ ID NO: 3, 4 or 22;

[0025] In another preferred embodiment, VL is a partial sequence of SEQ ID NO: 3, 4 or 22, or a sequence formed by substituting, deleting, modifying and / or adding at least one (e.g., 1-20, preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) amino acid residues of the sequence as shown in SEQ ID NO: 3, 4 or 22;

[0026] In another preferred embodiment, the amino acid sequence of VH is shown in SEQ ID NO: 5, 6 or 23;

[0027] In another preferred embodiment, VH is a partial sequence of SEQ ID NO: 5, 6 or 23, or a sequence formed by substituting, deleting, modifying and / or adding at least one (e.g., 1-20, preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) amino acid residues of the sequence as shown in SEQ ID NO: 5, 6 or 23;

[0028] In another preferred example, the amino acid sequence of the VL is shown in SEQ ID NO: 3, and the amino acid sequence of the VH is shown in SEQ ID NO: 5.

[0029] In another preferred example, the amino acid sequence of the VL is shown in SEQ ID NO: 4, and the amino acid sequence of the VH is shown in SEQ ID NO: 6.

[0030] In another preferred example, the amino acid sequence of the VL is shown in SEQ ID NO: 22, and the amino acid sequence of the VH is shown in SEQ ID NO: 23.

[0031] In another preferred embodiment, the H is selected from the hinge region of the following proteins: CD8, CD28, CD137, IgG1, IgG4, TCRα, TCRβ or their mutants / modified forms, or a combination thereof.

[0032] In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID No: 10 or SEQ ID No: 11 or SEQ ID No: 12.

[0033] In another preferred embodiment, the TM is selected from the transmembrane region of the following proteins: CD28, CD3ε, CD3ζ, CD3γ, CD3δ, TCRα, TCRβ, CD40, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314, or their mutants / modified forms, or their combinations.

[0034] In another preferred embodiment, the TM is the transmembrane region of CD8.

[0035] In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID No: 13.

[0036] In another preferred embodiment, the TM is the transmembrane region of CD28.

[0037] In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID No: 25.

[0038] In another preferred embodiment, C1 and C2 are selected from the costimulatory domains of proteins in the following group: CD3ε, CD3γ, CD3δ, 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 mutants / modified forms thereof, or combinations thereof.

[0039] In another preferred embodiment, C1 is a costimulatory domain derived from 4-1BB.

[0040] In another preferred embodiment, the amino acid sequence of C1 is shown in SEQ ID No: 14.

[0041] In another preferred embodiment, C2 is a costimulatory domain derived from CD28.

[0042] In another preferred embodiment, the amino acid sequence of C2 is shown in SEQ ID No: 15.

[0043] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID No: 16.

[0044] In another preferred embodiment, the D is an oxygen-dependent degradation domain (ODD) or a partial sequence thereof.

[0045] In another preferred embodiment, the amino acid sequence of ODD is shown in SEQ ID No: 17.

[0046] In another preferred embodiment, the connecting peptide consists of 1 to 5 GGGGS short peptides, and the amino acid sequence is shown in SEQ ID No: 18.

[0047] The second aspect of the present invention is an engineered immune cell, wherein the engineered immune cell is a T cell, a TIL cell, or a NK cell, and the immune cell

[0048] The immune cells express the CAR described in the first aspect of the present invention.

[0049] In another preferred embodiment, the amino acid sequence of the TROP2scFv is shown in SEQ ID NO: 8 or 9.

[0050] In another preferred embodiment, the amino acid sequence of the TROP2scFv is shown in SEQ ID NO:7.

[0051] In another preferred embodiment, the antigen binding domain of the CAR specifically binds to TROP2.

[0052] In another preferred embodiment, the T cells include αβT cells, γδT cells, NKT cells, MAIT cells, or a combination thereof.

[0053] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0054] (i) Chimeric antigen receptor T cells (CAR-T cells);

[0055] (ii) chimeric antigen receptor NK cells (CAR-NK cells);

[0056] (iii) Chimeric antigen receptor TIL cells (CAR-TIL cells).

[0057] In another preferred embodiment, the antigen binding domain of the CAR is a single-chain antibody against TROP2.

[0058] In another preferred embodiment, the CAR can be constitutively expressed or inducibly expressed;

[0059] In another preferred embodiment, the expression cassette of the inducibly expressed CAR contains a gene expression regulatory element upstream or downstream;

[0060] In another preferred embodiment, the expression control element contains 1 to 10 copies of a hypoxia response element (HRE);

[0061] 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.

[0062] In another preferred embodiment, the "activation" refers to the binding of the CAR to a surface marker of a tumor cell.

[0063] In another preferred embodiment, the "tumor surface marker" refers to a specific antigen on the surface of the tumor.

[0064] In another preferred embodiment, the chimeric antigen receptor CAR is located on the cell membrane of the engineered immune cell.

[0065] In another preferred embodiment, the chimeric antigen receptor CAR is located on the cell membrane of the CAR-T cell.

[0066] In another preferred embodiment, the TROP2 protein is localized on the cell membrane of the tumor cells.

[0067] The third aspect of the present invention provides a method for preparing the engineered immune cells according to the second aspect of the present invention, comprising the following steps:

[0068] (A) providing an immune cell to be modified; and

[0069] (B) The immune cells are modified so that the immune cells express CAR molecules, thereby obtaining engineered immune cells as described in the second aspect of the present invention, wherein the CAR targets the surface marker of tumor cells, wherein the CAR targets the surface marker TROP2 of tumor cells, and the TROP2scFv of the CAR that specifically binds to TROP2 is the antigen binding domain.

[0070] In another preferred embodiment, the amino acid sequence of the TROP2scFv is shown in SEQ ID NO: 8 or 9.

[0071] In another preferred embodiment, in step (B), an expression cassette for expressing the TROP2-targeting CAR (as shown in Formulas Ia, Ib, Ic, and Id in the first aspect of the present invention) is introduced into the immune cells.

[0072] In another preferred example, the antigen binding domain of the CAR specifically binds to the surface marker TROP2.

[0073] In another preferred embodiment, a method for preparing the CAR-T cells according to the second aspect of the present invention is provided, comprising the following steps:

[0074] (A) providing a T cell to be modified;

[0075] (B) Transforming the T cells so that the T cells express the CAR molecules, thereby obtaining the engineered immune cells as described in the second aspect of the present invention.

[0076] In another preferred embodiment, in step (B), an expression cassette expressing the targeting CAR targeting TROP2 (as shown in Formula Ia, Ib, Ic, Id in the first aspect of the present invention) is introduced into the T cell.

[0077] In another preferred embodiment, the expression cassette is located on a vector.

[0078] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or a combination thereof.

[0079] In another preferred embodiment, the vector is a pCDH series lentiviral vector.

[0080] The fourth aspect of the present invention provides a preparation comprising the engineered immune cells as described in the second aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0081] In another preferred embodiment, the preparation contains the CAR-T cells of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0082] In another preferred embodiment, the preparation is a liquid preparation.

[0083] In another preferred embodiment, the dosage form of the preparation includes injection.

[0084] 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 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0085] The fifth aspect of the present invention provides a use of the engineered immune cells according to the second aspect of the present invention for preparing a drug or preparation for preventing and / or treating cancer.

[0086] In another preferred embodiment, there is provided a use of the CAR-T cells as described in the second aspect of the present invention for preparing a drug or preparation for preventing and / or treating cancer or tumors.

[0087] In another preferred embodiment, the preparation contains CAR-T cells and a pharmaceutically acceptable carrier, diluent or excipient.

[0088] In another preferred embodiment, the tumor comprises a solid tumor.

[0089] In another preferred embodiment, the tumor is selected from the group consisting of colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer, breast cancer, liver cancer, bile duct cancer, ovarian cancer or nasopharyngeal cancer.

[0090] In another preferred embodiment, the tumor is a tumor with high, medium or low expression of TROP2.

[0091] A sixth aspect of the present invention provides a kit for preparing an engineered immune cell as described in the second aspect of the present invention, the kit comprising a container, and a nucleic acid sequence located within the container. The nucleic acid sequence comprises an expression cassette for expressing the CAR (as shown in Formulas Ia, Ib, Ic, and Id in the first aspect of the present invention), wherein the antigen binding domain of the CAR specifically binds to TROP2.

[0092] In another preferred embodiment, the engineered immune cells are engineered T cells.

[0093] The seventh aspect of the present invention provides a method for treating tumors, comprising the steps of administering a safe and effective amount of the cells described in the second aspect of the present invention or the preparation described in the fourth aspect of the present invention to a desired subject, thereby treating the tumor of the subject.

[0094] In another preferred embodiment, when the dosage of the cells administered is 10 4 ~10 6 10 pieces / each person 6 ~10 10 pcs / 60kg.

[0095] 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 described in detail 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 listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 shows the structure of the CAR molecule.

[0097] Figure 2 shows the detection of the effects of different VL and VH sequences on the expression of CAR molecules.

[0098] Figure 3 shows the killing effect of TROP2 CAR-T cells with different scFvs on colorectal cancer cells.

[0099] Figure 4 shows the killing effect of TROP2 CAR-T cells with different hinge structures on colorectal cancer cells.

[0100] FIG5 shows the in vitro killing effect of BTRP003 on colorectal cancer cells.

[0101] FIG6 shows the in vitro killing effect of BTRP003 on pancreatic cancer cells.

[0102] FIG7 shows the in vitro killing effect of BTRP003 on gastric cancer cells.

[0103] FIG8 shows the in vitro killing effect of BTRP003 on esophageal cancer cells.

[0104] FIG9 shows the in vitro killing effect of BTRP003 on nasopharyngeal carcinoma cells.

[0105] FIG10 shows the in vitro killing effect of BTRP003 on breast cancer cells.

[0106] FIG11 shows the in vivo killing effect of BTRP003 on various solid tumors.

[0107] FIG12 shows the expression detection of BTRP003H under different oxygen concentrations.

[0108] FIG13 shows the IFN-γ release assay after BTRP003H was co-cultured with target cells under different oxygen concentrations.

[0109] Figure 14 shows the PD-1 expression detection after BTRP003H was co-cultured with target cells under different oxygen concentrations.

[0110] FIG15 shows the in vitro killing effect of BTRP003H on target cells under 100 μM CoCl2 simulated hypoxia conditions.

[0111] FIG16 shows the in vitro killing effect of BTRP003H on target cells under 1% oxygen conditions.

[0112] FIG17 shows the in vivo killing effect of BTRP003H on various solid tumors.

[0113] FIG18 shows the IFN-γ release assay after BTRP028H and BTRP029H were co-cultured with target cells under 100 μM CoCl 2 simulated hypoxia conditions. DETAILED DESCRIPTION

[0114] After extensive and intensive research and extensive screening, the present inventors have, for the first time, provided engineered immune cells targeting TROP2, as well as their preparation and application. Specifically, the present invention expresses a chimeric antigen receptor targeting TROP2 in these engineered immune cells. By targeting TROP2, these engineered immune cells possess excellent tumor cell-killing efficacy, while simultaneously eliminating tumor stem cells and reducing the risk of tumor immune escape and recurrence. This is the basis for the present invention.

[0115] 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 are equivalent to T cells (or T cells can be replaced by NK cells or TIL cells).

[0116] the term

[0117] 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.

[0118] 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.

[0119] 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.

[0120] Antibody

[0121] 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 complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from amino terminus to 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.

[0122] Antigen binding domain

[0123] As used herein, "antigen-binding domain" and "single-chain antibody fragment" refer to a Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment, or a VHH fragment of a single-domain antibody that has 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 single nucleotide chain.

[0124] In the present invention, the scFv comprises an antibody or an active fragment thereof that specifically recognizes the TROP2 antigen expressed by a tumor.

[0125] In addition, the immune cells of the present invention may further contain additional antibodies that specifically recognize antigens highly expressed in tumors, preferably single-chain antibodies or Fv antibodies.

[0126] Chimeric Antigen Receptor (CAR)

[0127] 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 costimulatory 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 by the intracellular domain, causing cell activation and 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 caused 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 costimulatory 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.

[0128] In one embodiment, the CAR of the present invention targets TROP2 and can specifically bind to TROP2 on the surface of tumor cells.

[0129] Chimeric antigen receptor T cells (CAR-T cells)

[0130] 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 that express TROP2, such as colorectal cancer, ovarian cancer, pancreatic cancer, breast cancer, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, and nasopharyngeal cancer.

[0131] 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 for 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; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

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

[0133] 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 that express TROP2, such as colorectal cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, breast cancer, liver cancer, bile duct cancer, and nasopharyngeal carcinoma.

[0134] Natural killer (NK) cells are a major type of immune effector cell that protects 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 possess enhanced anti-tumor cytotoxicity.

[0135] 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 storm; (3) they are extremely easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

[0136] Expression cassette

[0137] As used herein, "expression cassette" or "expression cassette of the present invention" refers to an expression cassette for expressing a CAR targeting TROP2. Typically, the expression cassette includes a coding sequence of a CAR operably linked to a promoter.

[0138] In the present invention, the expression cassette comprises a nucleic acid sequence encoding the TROP2-targeting CAR.

[0139] In the present invention, the CAR can be constitutively expressed or inducibly expressed.

[0140] 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.

[0141] In one embodiment, the expression cassette further comprises a promoter and / or a terminator, and the promoter may be a constitutive promoter or an inducible promoter.

[0142] carrier

[0143] The present invention also provides vectors containing the expression cassettes of the present invention. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for the long-term, stable integration of transgenes into the cellular genome and their replication as the genomes of daughter cells replicate. Lentiviral 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.

[0144] Typically, the expression cassette or nucleic acid sequence of the present invention can be connected to the promoter downstream and incorporated into an expression vector by conventional procedures. The vector can be integrated into the eukaryotic cell genome and replicated therewith. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0145] 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, and 5,589,466, which are incorporated herein by reference in their entirety.

[0146] 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.

[0147] 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 organism.

[0148] Many virus-based systems have been developed and used for gene transduction in 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 the 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 virus systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art.

[0149] 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, promoter function is maintained. In the thymidine kinase (tk) promoter, the intervals between promoter elements can be increased by 50bp before activity begins to decline. Depending on the promoter, it can be shown that individual elements can work together or independently to initiate transcription.

[0150] An example of a suitable promoter is the cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence high-level expression that can be operably connected thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to 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 linked to the inducible promoter when desired, or turn off expression when not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0151] The expression vector that is introduced into cell also can comprise any one or both in selectable marker gene or reporter gene, so that from being transfected or infected cell mass, identify and select expression cell by viral vector.In other respects, selectable marker can be carried on single section DNA and be used for cotransfection procedure.The flank of selectable marker gene and reporter gene both can have suitable regulatory sequence, so that can be expressed in host cell.Useful selectable marker gene comprises for example antibiotic resistance gene, such as neomycin etc.

[0152] Methods for introducing genes into cells and expressing genes in 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.

[0153] 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 lipofectamine transfection and cationic complex polyethyleneimine transfection.

[0154] 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 method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0155] 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).

[0156] In the case of using non-viral delivery system, exemplary delivery vehicle is liposome. Consider using lipid preparation, to introduce nucleic acid into host cell (in vitro, in vitro (ex vivo) or in vivo). On the other hand, this nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, be dispersed in the lipid bilayer of liposome, be attached to liposome through the connecting molecule that is associated with liposome and oligonucleotide, be trapped in liposome, be compounded with liposome, be dispersed in the solution that comprises lipid, mix with lipid, unite with lipid, be included in lipid as suspension, be included in micelle or be compounded with micelle, or be associated with lipid in other ways. The lipid, lipid / DNA or lipid / expression vector associated with composition are not limited to any concrete structure in solution. They also can be simply dispersed in solution, may form the aggregate of size or shape heterogeneity. Lipid is lipid substance, and it can be natural generation 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.

[0157] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0158] It should be understood that in the present invention, in addition to using lentivirus for transduction, CAR targeting TROP2 can also be expressed in immune cells such as T cells by directly transfecting mRNA or plasmids, or by expressing artificial transcription factors.

[0159] preparation

[0160] The present invention provides a formulation containing the engineered immune cells (such as CAR-T cells) described in the third aspect of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of the CAR-T cells in the formulation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0161] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or 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.

[0162] Therapeutic applications

[0163] The present invention provides therapeutic applications of cells (e.g., T cells) transduced with a vector (e.g., a lentiviral vector) containing the expression cassette of the present invention. The transduced T cells can target the surface marker TROP2 of tumor cells, synergistically and significantly improving their tumor cell killing efficiency.

[0164] 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 the mammal.

[0165] In one embodiment, the present invention comprises a type of cell therapy in which a patient's own T cells (or those of an allogeneic donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then infused back into the same patient. This approach minimizes the likelihood of a graft-versus-host reaction, as antigens are recognized by T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0166] 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 an adoptive immunotherapy step. Among them, the CAR-T cells can produce a specific immune response to tumor cells expressing the corresponding antigen. For example, the CAR-T cells of the present invention induce a specific immune response against tumor cells expressing TROP2.

[0167] Treatable cancers include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors. Cancer types 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, bile duct cancer, breast cancer, nasopharyngeal cancer, and the like.

[0168] 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 to a subject in need thereof a therapeutically effective amount of the CAR-T cells of the present invention.

[0169] The CAR-T cells of the present invention can 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. Briefly, the pharmaceutical compositions 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.

[0170] 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 may be determined by clinical trials.

[0171] When a "therapeutically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes 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 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 7 The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within the range). The T cell composition can also be administered multiple times at these doses. 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 dosage and treatment regimen for a specific patient can be easily determined by a person skilled in the art of medicine by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0172] Administration of the subject compositions 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, intravenously, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions can be injected directly into the tumor, lymph node, or site of infection.

[0173] In certain embodiments of the present 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 (e.g., before, simultaneously, or after) any number of related treatment modalities, including, but not limited to, treatment with 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 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 compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. 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 before or after surgery.

[0174] 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. Dosage ratios for human administration may be implemented according to practices accepted in the art. Typically, 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.

[0175] The main advantages of the present invention include:

[0176] (1) Compared with existing TROP2 CAR-immune cells, the TROP2 CAR-immune cells of the present invention can improve tumor killing ability and prevent tumor immune escape and recurrence;

[0177] (2) Compared with existing TROP2 antibody drugs, the TROP2 CAR-immune cell effect of the present invention is sustained, does not require repeated administration, and has memory.

[0178] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in "Molecular Cloning: A Laboratory Manual" (Sambrook et al., New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

[0179] Unless otherwise stated, the reagents and materials used in the examples were commercially available.

[0180] Materials and Methods

[0181] Structural design of CAR molecules

[0182] The target gene structure of the lentiviral vector involved in this example is shown in FIG1 .

[0183] (1) TROP2 scFv 1 is composed of the following structures in series: VL3, linker, VH3;

[0184] (2) TROP2scFv 1′ consists of the following structures in series: VH3, linker, VL3;

[0185] (3) TROP2scFv 2 is composed of the following structures in series: VL1, linker, VH1;

[0186] (4) TROP2scFv 2′ consists of the following structures in series: VH1, linker, VL1;

[0187] (5) TROP2scFv 3 is composed of the following structures in series: VL2, linker, VH2;

[0188] (6) TROP2scFv 3′ consists of the following structures in series: VH2, linker, VL2;

[0189] (7) BTRP001 is composed of the following structures in series: human CD8 signal peptide [abbreviated as CD8(SP)], TROP2scFv1, human IgG hinge region [abbreviated as IgG(Fc)], human CD8 transmembrane domain [abbreviated as CD8(TM)], human 4-1BB intracellular costimulatory domain [abbreviated as 4-1BB(ID)], and human CD3ζ intracellular signal transduction domain [abbreviated as CD3ζ(ID)];

[0190] (8) BTRP001′ is composed of the following structures in series: CD8 (SP), TROP2scFv1′, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3ζ (ID);

[0191] (9) BTRP002 is composed of the following structures in series: CD8 (SP), TROP2scFv2, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3ζ (ID);

[0192] (10) BTRP002′ consists of the following structures in series: CD8 (SP), TROP2scFv 2′, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3ζ (ID);

[0193] (11) BTRP003 is composed of the following structures in series: CD8(SP), TROP2scFv2, human CD8 hinge region [abbreviated as CD8(hinge)], CD8(TM), 4-1BB(ID), and CD3ζ(ID);

[0194] (12) BTRP003H consists of the following structures in series: CD8 (SP), TROP2scFv 2, CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3ζ (ID), and oxygen-dependent degradation domain (ODD);

[0195] (13) BTRP028H is composed of the following structures in series: CD8(SP), TROP2scFv 2, CD8(hinge), human CD28 intracellular costimulatory domain [abbreviated as CD28(TM)], human CD28 intracellular costimulatory domain [abbreviated as CD28(ID)], 4-1BB(ID), CD3ζ(ID), ODD;

[0196] (14) BTRP029H consists of the following structures in series: CD8 (SP), TROP2scFv 2, CD8 (hinge), CD3ζ containing the extracellular region and transmembrane domain, 4-1BB (ID), and ODD;

[0197] (15) BTRP101 is composed of the following structures in series: CD8 (SP), TROP2scFv3, CD8 (hinge), CD8 (TM), 4-1BB (ID), and CD3ζ (ID);

[0198] (16) BTRP101′ is composed of the following structures in series: CD8 (SP), TROP2scFv 3′, CD8 (hinge), CD8 (TM), 4-1BB (ID), and CD3ζ (ID).

[0199] The amino acid or nucleotide sequences of the related structures are as follows:

[0200] TROP2 (SEQ ID NO: 1):

[0201] CD8(SP)(SEQ ID NO:2):MALPVTALLLPLALLLHAARPS

[0202] TROP2 scFv VL1 (SEQ ID NO: 3):

[0203] TROP2 scFv VL2 (SEQ ID NO: 4):

[0204] TROP2 scFv VH1 (SEQ ID NO: 5):

[0205] TROP2 scFv VH2 (SEQ ID NO: 6):

[0206] TROP2 scFv1 (SEQ ID NO:7): VL3-VH3

[0207] TROP2 scFv2 (SEQ ID NO:8): VL1-VH1

[0208] TROP2 scFv3 (SEQ ID NO:9): VL2-VH2

[0209] CD8(hinge)(SEQ ID NO:10):TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFA

[0210] IgG1(Fc) (SEQ ID NO: 11):

[0211] IgG4 (Fc) (SEQ ID NO: 12):

[0212] CD8(TM)(SEQ ID NO:13): CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR

[0213] 4-1BB(ID)(SEQ ID NO:14):KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0214] CD28(ID)(SEQ ID NO:15):RSKRSRLLHSDYMFMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0215] CD3ζ(ID) (SEQ ID NO: 16):

[0216] ODD (SEQ ID NO: 17):

[0217] linker(SEQ ID NO:18):GGGGSGGGGSGGGGS

[0218] HRE1 (nucleotide sequence) (SEQ ID NO: 19): GGCCCTACGTGCTGTCTCACACAGCCTGTCTGAC

[0219] HRE2 (nucleotide sequence) (SEQ ID NO: 20): CCACAGTGCATACGTGGGCTCCAACAGGTCCTCTT

[0220] HRE3 (nucleotide sequence) (SEQ ID NO: 21): GTCGTGCAGGACGTGACA

[0221] TROP2scFv VL3 (SEQ ID NO:22):

[0222] TROP2scFv VH3 (SEQ ID NO:23):

[0223] CD3ζ (SEQ ID NO: 24):

[0224] CD28(TM)(SEQ ID NO:25): FWVLVVVGGVLACYSLLVTVAFIIFWV

[0225] Example 1 Preparation of Lentiviral Vectors

[0226] The above-mentioned nucleotide sequences were fully synthesized (Figure 1) and ligated into the lentiviral vector pCDH-EF1α-MCS plasmid via molecular cloning, enabling expression under the control of the human EF-1α promoter and Kozak sequence. Using the transfection reagent Lipofectamine 3000, the above-mentioned lentiviral expression plasmids were co-transfected into 293T cells with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE, and pMD2.G, respectively, according to the manufacturer's instructions. Viral supernatants were collected 48 and 72 hours after transfection to obtain viral concentrates, designated BTRP001, BTRP001', BTRP002, BTRP002', BTRP003, BTRP003H, BTRP028H, BTRP029H, BTRP101, and BTRP101', respectively, and stored at -80°C until further use. Finally, the activity titers of the above-mentioned lentiviral vectors were assayed using Jurkat cells.

[0227] Example 2 Preparation of CAR-T cells

[0228] Peripheral blood mononuclear cells (PBMCs) from healthy donors were revived in AIM V medium and stimulated with anti-human CD3 / CD28 antibodies to culture T cells. T cells were transduced with the aforementioned lentivirus at an MOI of 5 IU / ml. Seven days after transduction, expression of the corresponding CAR molecules was assayed. Ten days after transduction, the cells were harvested and stored in liquid nitrogen until further use. The resulting CAR-T cells retain the names of the lentiviruses: single-target CAR-T cells are BTRP001, BTRP001', BTRP002, BTRP002', BTRP003, BTRP003H, BTRP028H, BTRP029H, BTRP101, and BTRP101'. Cells not transduced with lentivirus are designated as Ctrl T cells.

[0229] Example 3 Detection of the Effect of Different VL and VH Sequences on TROP2 CAR Expression

[0230] To investigate the effects of different TROP2 scFvs and their VL and VH sequence on CAR expression, BTRP001, BTRP001', BTRP002, BTRP002', BTRP101, and BTRP101' cells were washed twice with FACS buffer. Anti-G4S antibody was added to the BTRP001 and BTRP001' groups and incubated at 4°C for 1 hour. After washing with FACS buffer, the expression rate of the TROP2 CAR molecule in each group was determined by flow cytometry.

[0231] FlowJo software was used for analysis, and the expression rates of various CAR-T cells are shown in Figure 2. BTRP001 and BTRP001' had low expression rates of only 29.9% and 11.2%. BTRP002 and BTRP101, scFvs composed of a VL-VH sequence, had higher expression levels of 63.0% and 77.5%, respectively. BTRP002' and BTRP101', scFvs composed of a VH-VL sequence, had lower expression levels of 4.46% and 0.79%, respectively.

[0232] The above results indicate that even with the same VL and VH, not all combinations can produce a CAR molecule that can be expressed normally. In subsequent examples, CAR structures containing TROP2scFv 2 and TROP2scFv 3 were used for research.

[0233] Example 4 Detection of target cells

[0234] In this example, the tumor cells used in the IncuCyte live cell dynamic imaging analysis experiment express a fluorescent protein reporter gene, while the tumor cells used in the real-time label-free dynamic cell analysis experiment (RTCA), LDH cytotoxicity experiment, or in vivo drug efficacy experiment do not express a fluorescent protein reporter gene. The tumor cells include colorectal cancer cells HT-29, HT-55, HCT-8, HCT-15, HCT-116, Caco-2, WiDr, DLD-1, and KM12; pancreatic cancer cells CFPAC-1, Capan-1, and Capan-2; gastric cancer cells Hs746T, HGC-27, and SNU-719; esophageal cancer cells TE-1, ECA-109, KYSE-140, KYSE-150, and KYSE-520; nasopharyngeal carcinoma cells C666-1 and SUNE-1; ovarian cancer cells Caov-3 and OVCAR-3; and breast cancer cells SK-BR-3 and MCF-7.

[0235] The target cells were washed twice with FACS buffer. PE-labeled anti-human TROP2 antibody was added to each target cell suspension and incubated at 4°C for 1 hour. Target cells incubated with IgG served as a negative control. TROP2 expression in target cells was determined by flow cytometry. Analyzed using FlowJo software, the results are shown in Table 1.

[0236] Table 1 Detection of target protein expression rate of each tumor cell

[0237] Example 5 Detection of the Effects of Different scFvs on TROP2 CAR-T Cell Function

[0238] To study the effects of different scFv on the function of TROP2 CAR-T cells, 1×10 5 Colorectal cancer cells HT-29 and HCT116 were resuspended at a density of 100 μl / ml and seeded into 96-well plates at a volume of 100 μl per well. After being cultured overnight in the IncuCyte SX5 live cell imaging analysis system, BTRP003 and BTRP101 cells were added for co-culture at an effector-target ratio of 2:1, and the mCherry fluorescence signal of the target cells in each group was recorded in real time. After the co-culture, the changes in the mCherry fluorescence area of ​​the target cells in each group were calculated using 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 effect of each group.

[0239] The results are shown in Figure 3. Both BTRP003 and BTRP101 can effectively kill target cells. For HCT116 cells, the killing effect of BTRP003 is significantly better than that of BTRP101 (P<0.05).

[0240] Example 6 Detection of the Effects of Different Hinge Structures on TROP2 CAR-T Cell Function

[0241] To study the effects of different hinge structures on the function of TROP2 CAR-T cells, 1×10 5 Colorectal cancer cells Caco-2 were resuspended at a density of 100 μl / ml and inoculated into RTCA dedicated E-plate well plates at a volume of 100 μl per well. After being cultured overnight in the RTCA live cell real-time analysis system, BTRP002 and BTRP003 cells were added at an effector-target ratio of 1:1 for co-culture. The system records the impedance between the target cells in each group and the electrodes in the dedicated well plate in real time and converts it into a relative cell index. The lower the relative cell index, the fewer target cells in the group and the better the T cell killing effect. By comparing the relative cell index at the last time point, it is possible to statistically determine whether there is a significant difference in the killing effect of each group.

[0242] As shown in Figure 4, BTRP002 had a poor killing effect on target cells, with no significant difference compared to Ctrl T cells. In contrast, BTRP003 effectively killed target cells, significantly outperforming Ctrl T cells (P<0.05). This result suggests that while the IgG hinge and CD8 hinge structures each have a beneficial effect in some CAR molecules, the CD8 hinge has a superior function for TROP2 CAR-T cells.

[0243] Example 7: Cytotoxicity of TROP2 CAR-T cells against various solid tumor cells

[0244] 1×10 5 Colorectal cancer cells HT-29, HT-55, HCT-8, HCT-15, HCT116, Caco-2, WiDr, DLD-1, and KM12 were resuspended at a density of 100 μl / well and seeded into 96-well plates at a volume of 100 μl per well. After overnight culture using the IncuCyte SX5 Live Cell Imaging System, BTRP003 cells were added at a 2:1 effector-target ratio for co-culture. Real-time recording of the mCherry fluorescence signal of target cells in each group was performed. After co-culture, changes in the mCherry fluorescence area of ​​target cells in each group were calculated using IncuCyte software. Lower signal values ​​indicate smaller target cell area and greater T cell cytotoxicity. Comparison of the fluorescence signal values ​​at the final time point determined whether there was a significant difference in the cytotoxicity of BTRP003 cells compared to the negative control group (Ctrl T cells). The results are shown in Figure 5. BTRP003 can effectively kill various colorectal cancer cells, and the killing effect is significantly stronger than that of Ctrl T cells (P<0.05), indicating that the TROP2 CAR-T cells of the present invention are effective against colorectal cancer.

[0245] Using pancreatic cancer cells CFPAC-1 and Capan-2 as target cells, the cytotoxicity of BTRP003 cells was detected using a live cell imaging analysis system. As shown in Figure 6, BTRP003 was able to effectively kill various pancreatic cancer cells, with a significantly stronger cytotoxicity than Ctrl T cells (P < 0.05), indicating that the TROP2 CAR-T cells of the present invention are effective against pancreatic cancer.

[0246] Using gastric cancer cells Hs746T and HGC-27 as target cells, the killing effect of BTRP003 cells was detected using a live cell imaging analysis system. The results, as shown in Figure 7, show that BTRP003 can effectively kill various gastric cancer cells, and the killing effect is significantly stronger than that of Ctrl T cells (P < 0.05), indicating that the TROP2 CAR-T cells of the present invention are effective against gastric cancer.

[0247] 1×10 5Esophageal cancer cells TE-1, ECA-109, and KYSE-150 were resuspended at a density of 100 μl / ml and inoculated into E-Plate well plates at a volume of 100 μl per well. After being cultured overnight in the RTCA real-time label-free dynamic cell analysis system, BTRP003 cells were added at an effect-target ratio of 2:1 for co-culture. The resistance signal of the target cells in each group was recorded in real time and then converted into a cell index (cell index) by RTCA software analysis. The lower the cell index, the smaller the area occupied by the target cells in the group, and the better the T cell killing effect. By comparing the cell index of each group at the last time point, it can be statistically determined whether there is a significant difference in the killing effect of BTRP003 cells and the negative control group Ctrl T cells. As shown in Figure 8, BTRP003 can effectively kill various esophageal cancer cells, and the killing effect is significantly stronger than that of Ctrl T cells (P < 0.05), indicating that the TROP2 CAR-T cells of the present invention are effective against esophageal cancer.

[0248] The killing effect of BTRP003 on nasopharyngeal carcinoma cells C666-1 and SUNE-1 was detected by LDH assay. 5 The target cells were resuspended at a density of 100 μl / ml and inoculated into a 96-well plate at a volume of 100 μl per well. After overnight culture, BTRP003 cells were added at different effector-target ratios for co-culture for 24 hours. Then, 50 μl / well of the cell supernatant was carefully aspirated and mixed with 50 μl of substrate solution. After half an hour of reaction, 50 μl of stop buffer was added and detected in a microplate reader. The results are shown in Figure 9. At different effector-target ratios, the killing efficiency of BTRP003 against both nasopharyngeal carcinoma cells was significantly higher than that of Ctrl T cells (P<0.05), indicating that the TROP2CAR-T cells of the present invention are effective against nasopharyngeal carcinoma.

[0249] Using breast cancer cells Hs746T and HGC-27 as target cells, the cytotoxicity of BTRP003 cells was detected using a live cell imaging analysis system. As shown in Figure 10, BTRP003 was able to effectively kill various breast cancer cells, with a significantly stronger killing effect than Ctrl T cells (P < 0.05), indicating that the TROP2 CAR-T cells of the present invention are effective against breast cancer.

[0250] Example 8 In vivo efficacy of TROP2 CAR-T cells against various solid tumors

[0251] The inhibitory effect of subcutaneous transplanted tumors in mice was tested using HT-55, WiDr, Capan-1, Capan-2, KYSE-140, KYSE-150, and SNU-719 as target cells and Ctrl T cells and BTRP003 cells as effector cells. For each target cell, 9 to 15 6-8 week old NOG mice were subcutaneously injected with 1×10 6 The corresponding cells. The average tumor volume is about 50mm 3 At the same time, the mice were randomly divided into vehicle control group, Ctrl T control group and BTRP003 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 designated as treatment day 0. Tumor volume and mouse body weight were measured 2-3 times per week.

[0252] As shown in Figure 11, in each subcutaneous model, the tumor volume of the vehicle control group and the Ctrl T control group gradually increased, while the tumor volume of the BTRP003 experimental group was significantly reduced, and the tumor completely disappeared within 7 to 20 days without recurrence, which was significantly different from the control group (P<0.05).

[0253] The above results indicate that the TROP2 CAR-T cells of the present invention can effectively inhibit the growth of various solid tumors in vivo, completely eliminate tumors, and inhibit tumor recurrence.

[0254] Example 9 Detection of expression of TROP2 CAR-T cells regulated by hypoxia

[0255] Hypoxia is a key feature of the tumor microenvironment, a condition of tumor hypoxia caused by the rapid growth of tumor cells and limited angiogenesis. Taking advantage of this characteristic, the CAR molecule was fused with the oxygen-dependent degradation domain (ODD) derived from the HIF1α protein. This domain has two oxygen-dependent ubiquitination sites, which can rapidly degrade the CAR molecule through the proteasome pathway under physiological normoxic conditions. The oxygen concentration in the tumor microenvironment is often less than 1%, and ODD cannot mediate ubiquitination modification. The stability of the CAR molecule is significantly improved and it can activate T cells, thereby activating CAR-T cells only in tumor lesions to ensure safety.

[0256] To investigate whether the TROP2 CAR molecule BTRP003H, which incorporates an ODD domain, can regulate its expression level depending on oxygen concentration, BTRP003H cells were cultured overnight in 1% and 5% oxygen environments. The cells were washed twice with FACS buffer, added with Protein L, and incubated at 4°C for 1 hour. After washing with FACS buffer, the expression rate of the TROP2 CAR molecule in each cell group was determined by flow cytometry.

[0257] FlowJo software was used to analyze the expression rates of various CAR-T cells, as shown in Figure 12. BTRP003 expressed at similarly high levels under both 1% and 5% oxygen conditions. In contrast, BTRP003H expressed only 5.44% under 5% oxygen; however, its expression increased significantly to 38.5% under 1% oxygen. This result demonstrates that the ODD domain indeed reduces TROP2 CAR expression under normoxic conditions and increases it under hypoxic conditions.

[0258] Example 10 Detection of activation and exhaustion of TROP2 CAR-T cells regulated by hypoxia

[0259] 1×10 5 293T and 293T-OE cells (overexpressing TROP2) were resuspended at a density of 100 μl / well and seeded into 96-well plates at a volume of 100 μl per well. After overnight culture, BTRP003H cells were added at an effector-target ratio of 5:1 and co-cultured overnight. The culture supernatant was collected and IFN-γ levels were measured by ELISA.

[0260] The results are shown in Figure 13. For 293T cells, BTRP003H only released background levels of IFN-γ at different oxygen concentrations. For 293T-OE cells, BTRP003H IFN-γ release increased significantly with decreasing oxygen concentration (374.9±45.5 pg / ml at 21% oxygen, 1433.0±67.4 pg / ml at 5% oxygen, and 5355.1±537.3 pg / ml at 1% oxygen). This result demonstrates that BTRP003H activation is target-specific and oxygen concentration-dependent.

[0261] PD-1 can be used as a marker of T cell exhaustion, and its expression level correlates with the degree of exhaustion. CAR-T cells were collected from the co-cultured cells for flow cytometry. The cells were washed twice with FACS buffer, anti-PD-1 antibody was added, and the cells were incubated at 4°C for 1 hour. After washing with FACS buffer, the expression rate of PD-1 in each cell group was measured by flow cytometry.

[0262] The results are shown in Figure 14. For CD4+ T cells, PD-1 expression was similar between BTRP003H and Ctrl T cells (59.2% and 64.7%, respectively, at 1% oxygen, and 63.1% and 66.1%, respectively, at 21% oxygen), while PD-1 was significantly upregulated in BTRP003 cells (76.6% at 1% oxygen and 77.9% at 21% oxygen). For CD8+ T cells, PD-1 expression was also similar between BTRP003H and Ctrl T cells (5.67% and 6.29%, respectively, at 1% oxygen, and 5.34% and 6.27%, respectively, at 21% oxygen), while PD-1 was significantly upregulated in BTRP003 cells (22.4% at 1% oxygen and 17.2% at 21% oxygen). Since the structural difference between BTRP003H and BTRP003 is only the presence or absence of the ODD domain, the difference in depletion levels between BTRP003H and BTRP003 may originate from the ODD domain.

[0263] This unexpected discovery shows that ODD can not only regulate the expression of TROP2 CAR molecules according to oxygen concentration, but also significantly reduce the degree of exhaustion of CAR-T cells.

[0264] Example 11: Cytotoxicity of hypoxia-regulated TROP2 CAR-T cells against colorectal cancer cells

[0265] 1×10 5 Colorectal cancer cells (HCT116, Caco-2, and WiDr) were resuspended at a density of 100 μl / well and seeded into 96-well plates at a volume of 100 μl per well. After overnight culture using the IncuCyte SX5 Live Cell Imaging System, BTRP003 and BTRP003H cells were added at a 2:1 effector-target ratio for co-culture. 100 μM CoCl₂ was added to simulate hypoxia, and the mCherry fluorescence signal of target cells in each group was recorded in real time. After co-culture, changes in the mCherry fluorescence area of ​​target cells in each group were calculated using IncuCyte software. Lower signal values ​​indicate smaller target cell area and greater T cell cytotoxicity.

[0266] The results are shown in Figure 15. Both BTRP003H and BTRP003 can effectively kill colorectal cancer cells, and the killing effect is significantly stronger than that of Ctrl T cells (P<0.05).

[0267] Example 12: Cytotoxicity of Hypoxia-Regulated TROP2 CAR-T Cells on Various Tumor Cells

[0268] 1×10 5Pancreatic cancer cells Capan-2, esophageal cancer cells KYSE-150, and gastric cancer cells AGS and SNU-719 were resuspended at a density of 100 μl / well and seeded into RTCA-specific E-plates at a volume of 100 μl per well. After overnight incubation in an RTCA live cell real-time analysis system, BTRP003 and BTRP003H cells were added at a 2:1 effector-to-target ratio and co-cultured under 1% oxygen conditions. The system recorded the impedance between the target cells in each group and the electrodes in the dedicated well plate in real time and converted it into a relative cell index. A lower relative cell index indicates a lower number of target cells in that group and a higher T cell killing effect.

[0269] The results are shown in Figure 16. Both BTRP003 and BTRP003H can effectively kill target cells, which is significantly better than Ctrl T cells (P<0.05).

[0270] Example 13 In vivo tumor suppression effect of hypoxia-regulated TROP2 CAR-T cells

[0271] Capan-2 and CFPAC-1 were used as target cells, and Ctrl T, BTRP003, and BTRP003H cells were used as effector cells to test the inhibitory effect of subcutaneous transplanted tumors in mice. For each target cell, 2×10 6 The corresponding cells. The average tumor volume is about 50mm 3 At the same time, the mice were randomly divided into vehicle control group, Ctrl T control group, BTRP003 and BTRP003H experimental groups (4-5 mice in each group), and the vehicle (100 μl / mouse) or the corresponding T cells (5×10 6 The day of injection of the test substance was designated as treatment day 0. Tumor volume and mouse body weight were measured 2-3 times per week.

[0272] As shown in Figure 17, in each subcutaneous model, tumor volume gradually increased in both the vehicle and Ctrl T control groups, while tumor volume significantly decreased in both the BTRP003 and BTRP003H experimental groups. The tumors completely disappeared within 7 to 15 days without recurrence, significantly different from the control groups (P<0.05). These results demonstrate that hypoxia-regulated TROP2 CAR-T cells can effectively inhibit tumor growth in vivo.

[0273] Example 14 Structural Optimization of Hypoxia-Regulated TROP2 CAR Molecules

[0274] To further enhance the function of hypoxia-regulated TROP2 CAR-T cells, two modifications were performed based on BTRP003H: the CD28 costimulatory domain was added to the 4-1BB costimulatory domain to obtain BTRP028H; and the order of the 4-1BB costimulatory domain and the CD3ζ signaling domain was adjusted to obtain BTRP029H.

[0275] 1×10 5 293T-OE cells were resuspended at a density of 100 μl / well and seeded into 96-well plates at a volume of 100 μl per well. After overnight culture, BTRP003H, BTRP028H, and BTRP029H cells were added at an effector-target ratio of 5:1. 100 μM CoCl₂ was added to simulate hypoxia and the cells were cultured overnight. The culture supernatants were collected and assayed for IFN-γ levels using a bead-based immunoassay (CBA).

[0276] The results are shown in Figure 18. The IFN-γ release level of BTRP003H was 257.0±40.6 pg / ml. Although the IFN-γ release level of BTRP028H increased slightly (362.3±37.4 pg / ml), there was no significant difference (P>0.05). This indicates that for hypoxia-regulated TROP2 CAR-T cells, adding a co-stimulatory domain may not significantly enhance the activation function of the cells.

[0277] Surprisingly, BTRP029H significantly increased IFN-γ release (881.2±142.4 pg / ml), demonstrating significantly greater activation than BTRP003H and BTRP028H (P<0.05). Therefore, BTRP029H is a more optimal structure for hypoxia-regulated TROP2 CARs.

[0278] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and the equivalents of these changes and modifications also fall within the scope of the claims appended hereto.

Claims

1. A chimeric antigen receptor (CAR), characterized in that: The CAR targets TROP2, and its structure is shown in Formula Ia or Ib: L-TROP2scFv-H-TM-C1-C2-CD3ζ-D (Ia) L-TROP2scFv-H-TM-CD3ζ-C1-C2-D (Ib) In the formula, L is none or a signal peptide sequence; TROP2scFv is a single-chain antibody (scFv) targeting TROP2; H is no or hinge region; TM is the transmembrane domain; C1 is none or the first costimulatory domain; C2 is no or a second co-stimulatory domain; CD3ζ is absent or derived from the cytoplasmic signal transduction sequence of CD3ζ (including wild type, or mutant / modified form thereof); D is the absence or degradation domain (including wild type, or mutant / modified form thereof); The "-" is a connecting peptide or peptide bond.

2. The chimeric antigen receptor according to claim 1, wherein The TROP2 scFv has the following structure of Formula IIA or IIB: VH-VL (IIA); VL-VH (IIB); Wherein, VH is the heavy chain variable region of TROP2scFv, and VL is the light chain variable region of TROP2scFv; "-" indicates a connecting peptide or none.

3. The chimeric antigen receptor according to claim 1, wherein The amino acid sequence of VL is shown in SEQ ID NO: 3, 4 or 22.

4. The chimeric antigen receptor according to claim 1, wherein The amino acid sequence of VH is shown in SEQ ID NO: 5, 6 or 23.

5. The chimeric antigen receptor according to claim 1, wherein The amino acid sequence of the TROP2scFv is shown in SEQ ID NO: 7, 8 or 9.

6. The chimeric antigen receptor according to claim 1, wherein The amino acid sequence of ODD is shown in SEQ ID No:

17.

7. An engineered immune cell, characterized in that: The engineered immune cells are T cells, TIL cells, or NK cells, and the immune cells express the CAR of claim 1.

8. A method for preparing the engineered immune cell according to claim 7, 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 7, wherein the CAR targets the surface marker of tumor cells, wherein the CAR targets the surface marker TROP2 of tumor cells, and the TROP2scFv of the CAR that specifically binds to TROP2 is the antigen binding domain.

9. A preparation, characterized in that The preparation contains the engineered immune cells as described in claim 7, and a pharmaceutically acceptable carrier, diluent or excipient.

10. A use of the engineered immune cell according to claim 7, characterized in that: Used for preparing medicines or preparations for preventing and / or treating cancer.

11. The use according to claim 10, characterized in that The engineered immune cells are CAR-T cells.

12. The use according to claim 10, characterized in that The tumor includes a solid tumor.

13. The use according to claim 10, characterized in that: The tumor is a tumor with high, medium or low expression of TROP2.

14. A kit for preparing the engineered immune cell according to claim 7, characterized in that: The kit contains a container and a nucleic acid sequence located in the container; the nucleic acid sequence contains an expression cassette for expressing the CAR, wherein the antigen binding domain of the CAR specifically binds to TROP2.

15. The kit according to claim 14, characterized in that The engineered immune cells are engineered T cells.

Images