Fusion protein for enhancing adoptive immune efficacy and application thereof

Abstract

The invention provides a fusion protein for enhancing adoptive immune curative effect and application thereof. Specifically, the invention provides a fusion protein composed of a PD1 extracellular element or a TGF-beta extracellular element, a PD1 transmembrane segment or a TLR transmembrane segment, and a TLR intracellular segment. The fusion protein provided by the invention can convert an inhibitory signal of a tumor immune microenvironment into an activation signal, thereby promoting immune cell proliferation and enhancing the ability of immune cells to kill tumor cells. The invention also provides an engineered immune cell for simultaneously expressing the fusion protein and the chimeric antigen receptor, a preparation method of the engineered immune cell and the like.

Description

Technical Field

[0001] The invention belongs to DNA recombination technology and biomedicine, and in particular, relates to a fusion protein for enhancing the therapeutic effect of adoptive immunity and an application thereof. Background Art

[0002] Adoptive cellular immunotherapy has achieved great results in recent years. The U.S. Food and Drug Administration has approved cell therapy products targeting CD19 and BCMA for the clinical treatment of relapsed and refractory hematological tumors, including large B-cell lymphoma, mantle cell lymphoma, and multiple myeloma, providing a new and reliable treatment strategy that can prolong the patient's survival and quality of life. These adoptive immunotherapies approved by the FDA are all for the purpose of transforming autologous T cells to express chimeric antigen receptors, targeting specific tumor antigens to kill tumors and achieve anti-tumor effects.

[0003] The structure of chimeric antigen receptors is that the extracellular antigen binding domain is connected to the intracellular signal transduction domain through the hinge region and the transmembrane region. However, the efficacy of chimeric antigen receptor cell therapy for solid tumors is currently limited, and the inhibitory immune microenvironment of the tumor greatly limits the efficacy of chimeric antigen receptors.

[0004] Therefore, there is an urgent need in the art to develop a method that can better promote the proliferation of immune cells and the ability to kill tumor cells. Summary of the invention

[0005] The present invention provides a method for better promoting the proliferation of immune cells and the ability to kill tumor cells.

[0006] In the first aspect of the present invention, a fusion protein is provided, wherein the fusion protein has a structure of Formula I:

[0007] Z0-L1-Z1-L2-Z2-L3-Z3(I)

[0008] In the formula,

[0009] Z0 is signal peptide or none;

[0010] L1 is none or the first connecting peptide element;

[0011] Z1 is the PD1 extracellular element or the TGF-β extracellular element;

[0012] L2 is none, a second connecting peptide element, or a hinge region element;

[0013] Z2 is the PD1 transmembrane segment or the TLR transmembrane segment;

[0014] L3 is none or a third connecting peptide element; and

[0015] Z3 is the intracellular segment of TLR.

[0016] In another preferred embodiment, the intracellular segment of TLR is 90-100% of the complete intracellular segment of the corresponding TLR.

[0017] In another preferred embodiment, the TLR is selected from the following group: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

[0018] In another preferred embodiment, the Z2 and Z3 are from the same or different TLRs.

[0019] In another preferred embodiment, Z2 and Z3 are from the same TLR.

[0020] In another preferred embodiment, Z2 and Z3 are derived from TLR10, TCR1, PD1, CD28, or 4-1BB.

[0021] In another preferred embodiment, the L2 is a hinge region element.

[0022] In another preferred embodiment, the hinge region element and Z2 are derived from the same protein.

[0023] In another preferred embodiment, the hinge region element and Z3 are from the same TLR.

[0024] In another preferred embodiment, the TGR-β extracellular element is derived from TGR-βRII.

[0025] In another preferred embodiment, Z2 and Z3 in the fusion protein are a combination selected from the following groups:

[0026] (i) Z2 is the transmembrane segment of PD1, and Z3 is the intracellular segment of TLR10;

[0027] (ii) Z2 is the transmembrane segment of TLR10, and Z3 is the intracellular segment of TLR10.

[0028] In another preferred embodiment, the TLR10 intracellular segment is a full-length TLR10 intracellular segment, a mature form of the TLR10 intracellular segment, or an active fragment thereof.

[0029] In another preferred embodiment, when Z2 is derived from TLR10, the amino acid sequence of Z2 is as shown in SEQ ID NO:29.

[0030] In another preferred embodiment, when Z3 is derived from TLR10, the amino acid sequence of Z3 is shown in SEQ ID NO:31.

[0031] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes the fusion protein described in the first aspect of the present invention.

[0032] In another preferred example, when Z2 is derived from TLR10, the nucleotide sequence encoding Z2 is shown in SEQ ID NO:29.

[0033] In another preferred embodiment, when Z3 is derived from TLR10, the nucleotide sequence encoding Z3 is shown as SEQ ID NO:32.

[0034] In the third aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide described in the second aspect of the present invention.

[0035] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the third aspect of the present invention or the polynucleotide described in the second aspect of the present invention is integrated into its genome.

[0036] In another preferred embodiment, the host cells include prokaryotes and eukaryotes.

[0037] In another preferred embodiment, the host cell is a bacterium, preferably Escherichia coli.

[0038] In a fifth aspect of the present invention, a method for producing the fusion protein according to the first aspect of the present invention is provided, comprising the steps of:

[0039] (a) culturing the host cell according to the fourth aspect of the present invention under suitable expression conditions, thereby expressing the fusion protein according to the first aspect of the present invention;

[0040] (b) isolating and purifying the fusion protein expressed in step (a).

[0041] In a sixth aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell is a T cell or a NK cell, and the immune cell has the following characteristics:

[0042] (a) the immune cell expresses a chimeric antigen receptor (CAR), wherein the CAR targets a surface marker of a tumor cell; and

[0043] (b) The immune cells express the fusion protein described in the first aspect of the present invention.

[0044] In another preferred embodiment, the T cells include αβT cells, γδT cells, NKT cells, MAIT cells, or a combination thereof.

[0045] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0046] (i) Chimeric antigen receptor T cells (CAR-T cells);

[0047] (ii) Chimeric antigen receptor NK cells (CAR-NK cells).

[0048] In another preferred embodiment, a chimeric antigen receptor T cell (CAR-T cell) is provided, wherein the CAR-T cell has one or more of the following characteristics:

[0049] (a) the cell expresses a chimeric antigen receptor CAR, and the CAR targets a surface marker of a tumor cell; and

[0050] (b) When the CAR-T cells are contacted with an inducer, the CAR-T cells are induced to express the fusion protein described in the first aspect of the present invention.

[0051] In another preferred example, in the CAR cell, CAR and the fusion protein are expressed independently.

[0052] In another preferred embodiment, the "activation" refers to the binding of the CAR to the surface markers of tumor cells.

[0053] In another preferred embodiment, the "tumor surface marker" refers to a specific antigen on the surface of the tumor.

[0054] In another preferred embodiment, the chimeric antigen receptor CAR is located on the cell membrane of the engineered immune cell.

[0055] In another preferred embodiment, the chimeric antigen receptor CAR is located on the cell membrane of the CAR-T cell.

[0056] In another preferred embodiment, the fusion protein is localized on the cell membrane of the CAR-T cell.

[0057] In another preferred embodiment, the structure of the CAR is as shown in Formula II:

[0058] L-ScFv-H-TM-C-CD3ζ(II)

[0059] In the formula,

[0060] L is none or a signal peptide sequence;

[0061] ScFv is the antigen binding domain;

[0062] H is none or hinge region;

[0063] TM is the transmembrane domain;

[0064] C is the co-stimulatory signaling domain;

[0065] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ (including wild type, or mutant / modified form thereof);

[0066] The "-" connects peptides or peptide bonds.

[0067] In another preferred embodiment, the scFv is a single-chain variable region sequence of an antibody targeting a tumor antigen.

[0068] In another preferred embodiment, the scFv is an antibody single-chain variable region sequence targeting an antigen selected from the following group: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD24, CD133, CD44, CD166, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, ErbB2, LMP1, EpCAM, VEGFR-1, Lewis-Y, ROR1, Claudin18.2, CEA or a combination thereof.

[0069] 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.

[0070] In another preferred embodiment, the H is selected from the hinge region of the following proteins: CD8, CD28, CD137, IgG, or a combination thereof.

[0071] In another preferred embodiment, the TM is selected from the transmembrane region of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or their mutants / modified forms, or their combinations.

[0072] In another preferred embodiment, the C is selected from the co-stimulatory domain of the following protein: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), GITR, OX40L, or its mutant / modified form, or a combination thereof.

[0073] In another preferred embodiment, C is a co-stimulatory domain derived from 4-1BB.

[0074] In another preferred embodiment, the nucleotide molecules of the chimeric antigen receptor and the fusion protein are one or more sequences selected from the following group from the 5' end to the 3' end:

[0075] (i) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:30 and SEQ ID NO:32;

[0076] (ii) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:32.

[0077] In another preferred embodiment, the amino acid molecules of the chimeric antigen receptor and the fusion protein are one or more sequences selected from the following group from the amino terminus to the carboxyl terminus:

[0078] (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:29 and SEQ ID NO:31;

[0079] (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:21 and SEQ ID NO:31.

[0080] In a seventh aspect of the present invention, a method for preparing the engineered immune cell according to the sixth aspect of the present invention is provided, comprising the following steps:

[0081] (A) providing an immune cell to be modified; and

[0082] (B) Transforming the immune cells so that the immune cells express the CAR molecules and the fusion protein described in the first aspect of the present invention, thereby obtaining the engineered immune cells described in the sixth aspect of the present invention.

[0083] In another preferred embodiment, in step (B), comprising:

[0084] (B1) introducing a first expression cassette expressing the CAR into the immune cell; and (B2) introducing a second expression cassette expressing the fusion protein into the immune cell;

[0085] The step (B1) can be performed before, after, simultaneously with, or alternately with step (B2).

[0086] In another preferred embodiment, a method for preparing the CAR-T cells of the present invention is provided, comprising the following steps:

[0087] (A) providing a T cell to be modified;

[0088] (B) Transforming the T cells so that the T cells express the CAR molecules and the fusion protein described in the first aspect of the present invention, thereby obtaining the engineered immune cells described in the sixth aspect of the present invention.

[0089] In another preferred embodiment, step (B) includes (B1) introducing a first expression cassette expressing the CAR into the T cell; and (B2) introducing a second expression cassette expressing the fusion protein described in the first aspect of the present invention into the T cell; wherein the step (B1) can be performed before, after, simultaneously, or alternately with step (B2).

[0090] In another preferred embodiment, when the T cells to be modified in step (A) already express CAR, then in step (B), (B2) a second expression cassette is introduced into the T cells.

[0091] In another preferred embodiment, the transcription directions of the first expression cassette and the second expression cassette are in the same direction (→→), in opposite directions (→←), or in opposite directions (←→).

[0092] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors.

[0093] In another preferred embodiment, the first expression cassette and the second expression cassette are located in the same vector.

[0094] In another preferred embodiment, when the first and second expression cassettes are located in the same vector, a third expression cassette for expressing a connecting peptide is further included between the first and second expression cassettes.

[0095] In another preferred embodiment, the connecting peptide is P2A.

[0096] In another preferred embodiment, the vector is a viral vector, and preferably the viral vector contains the first and second expression cassettes in tandem.

[0097] 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.

[0098] In another preferred embodiment, the vector is a pCDH series lentiviral vector.

[0099] In the eighth aspect of the present invention, a preparation is provided, which contains the engineered immune cells described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0100] In another preferred embodiment, the preparation contains the CAR-T cells described in the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0101] In another preferred embodiment, the preparation is a liquid preparation.

[0102] In another preferred embodiment, the dosage form of the preparation includes injection.

[0103] 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.

[0104] In the ninth aspect of the present invention, there is provided a use of the engineered immune cells as described in the sixth aspect of the present invention for preparing a drug or preparation for preventing and / or treating cancer or tumors.

[0105] In another preferred embodiment, the present invention provides a use of the CAR-T cells described in the sixth aspect of the present invention for preparing a drug or preparation for preventing and / or treating cancer or tumors.

[0106] In another preferred embodiment, the preparation contains CAR-T cells and a pharmaceutically acceptable carrier, diluent or excipient.

[0107] In another preferred embodiment, the tumor is selected from the following group: solid tumors (such as colon cancer, gastric cancer, pancreatic cancer, liver cancer), blood tumors (such as lymphoma, multiple myeloma), or a combination thereof.

[0108] In a tenth aspect of the present invention, a kit for preparing the engineered immune cells according to the sixth aspect of the present invention is provided, the kit comprising a container, and:

[0109] (1) a first nucleic acid sequence, wherein the first nucleic acid sequence contains a first expression cassette for expressing the CAR; and

[0110] (2) A second nucleic acid sequence, wherein the second nucleic acid sequence comprises a second expression cassette for co-expressing the fusion protein described in the first aspect of the present invention.

[0111] In another preferred embodiment, the present invention provides a kit for preparing the engineered immune cells according to the sixth aspect of the present invention, the kit comprising a container, and:

[0112] (1) a first nucleic acid sequence, wherein the first nucleic acid sequence contains a first expression cassette for expressing the CAR; and

[0113] (2) A second nucleic acid sequence, wherein the second nucleic acid sequence contains a second expression cassette for co-expressing the fusion protein.

[0114] In another preferred embodiment, the first and second nucleic acid sequences are independent or connected.

[0115] In another preferred embodiment, the first and second nucleic acid sequences are located in the same or different containers.

[0116] In another preferred embodiment, the first and second nucleic acid sequences are located on the same or different vectors.

[0117] In another preferred embodiment, the first and second nucleic acid sequences are located in the same vector.

[0118] In another preferred embodiment, when the first and second nucleic acid sequences are located in the same vector, a third nucleic acid sequence is included between the first and second nucleic acid sequences, and the third nucleic acid sequence contains a third expression cassette for expressing a connecting peptide.

[0119] In another preferred embodiment, the connecting peptide is P2A.

[0120] In another preferred embodiment, the vector is a viral vector, and preferably the viral vector contains the first and second nucleic acid sequences in tandem.

[0121] 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

[0122] Figure 1 The expression of CEA and PD-L1 on target cells in Example 1 of the present invention is shown.

[0123] Figure 2A schematic diagram of the CEA CAR molecular structure (P320, P370, P371 and P372) of the present invention is shown.

[0124] Figure 3 The CAR expression on the surface of CAR-T cells in Example 2 of the present invention is shown.

[0125] Figure 4 The expression of PD-1 on the surface of CAR-T cells in Example 2 of the present invention is shown.

[0126] Figure 5 The killing effect of CAR-T cells in Example 3 of the present invention is shown: (A) CAR-T cytotoxicity to BxPC-3.LUC cells; (B) CAR-T cytotoxicity to LS174T.LUC cells; (C) CAR-T cytotoxicity to COLO 320DM.LUC cells.

[0127] Figure 6 The cytokine detection in Example 3 of the present invention is shown: (A) BxPC-3.LUC; (B) LS 174T.LUC; (C) COLO 320DM.LUC.

[0128] Figure 7 The proliferation of CAR-T cells after multiple rounds of stimulation of the target cell LS174T in Example 4 of the present invention is shown: (A) proliferation multiple; (B) cell viability.

[0129] Figure 8 The in vivo efficacy of CAR-T cells in Example 5 of the present invention in Bxpc3 tumor xenograft mice is shown: (A) tumor size; (B) mouse survival curve; (C) mouse weight change.

[0130] Fig. 9 A schematic diagram of the CEA CAR molecular structure (P320, P372, P373, P374 and P375) of the present invention is shown.

[0131] Fig.10 The CAR expression on the surface of CAR-T cells in Example 6 of the present invention is shown.

[0132] Fig.11 The expression of PD-1 on the surface of CAR-T cells in Example 6 of the present invention is shown.

[0133] Fig.12 The killing effect of CAR-T cells in Example 7 of the present invention is shown: (A) CAR-T cytotoxicity to BxPC-3.LUC cells; (B) CAR-T cytotoxicity to LS174T.LUC cells; (C) CAR-T cytotoxicity to COLO 320DM.LUC cells.

[0134] Fig.13 The cytokine detection in Example 7 of the present invention is shown: (A) BxPC-3.LUC; (B) LS 174T.LUC; (C) COLO 320DM.LUC.

[0135] Fig.14 The in vivo efficacy of CAR-T cells in Example 8 of the present invention in ASPC-1 tumor xenograft mice is shown: (A) tumor size; (B) mouse survival curve; (C) mouse weight change.

[0136] Fig.15 A schematic diagram of the CEA CAR molecular structure (P320, P321 and P312) of the present invention is shown.

[0137] Fig.16 The CAR expression on the surface of CAR-T cells in Example 9 of the present invention is shown.

[0138] Fig.17 The killing effect of CAR-T cells in Example 10 of the present invention is shown: (A) CAR-T cell toxicity to MKN-45.LUC cells; (B) CAR-T cell toxicity to COLO 320DM.LUC cells.

[0139] Fig.18 The cytokine detection in Example 10 of the present invention is shown: (A) MKN-45.LUC; (B) COLO320DM.LUC.

[0140] Fig.19 The in vivo efficacy of CAR-T cells in Example 11 of the present invention in MKN-45 tumor xenograft mice is shown: (A) tumor size; (B) mouse survival curve; (C) mouse weight change. DETAILED DESCRIPTION

[0141] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly discovered a specific fusion protein and expressed it in CAR-T cells in combination with a specific CAR. The first domain of the fusion protein is a PD1 extracellular element or a TGF-β extracellular element, the second domain is a PD1 transmembrane segment or a TLR transmembrane segment, and the third domain is a TLR intracellular segment. The fusion protein can convert the inhibitory signal of the tumor immune microenvironment into an activation signal, thereby promoting the proliferation of immune cells and enhancing the ability to kill tumor cells.

[0142] Specifically, the experiments of the present invention show that chimeric antigen receptor (such as P372, P312) T cells containing specific fusion proteins have good tumor cell killing activity, especially tumor cell killing activity in vivo, can significantly inhibit tumor cell growth, and have good in vivo safety. On this basis, the present invention was completed.

[0143] 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 the immune cells express chimeric antigen receptor CAR, NK cells are equivalent to T cells (or T cells can be replaced by NK cells).

[0144] the term

[0145] 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.

[0146] 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.

[0147] The term "administering" refers to the physical introduction of 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.

[0148] Antibody

[0149] 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.

[0150] Antigen binding domain

[0151] As used herein, "antigen binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab') fragments, etc. 2Fragment, or single Fv fragment. Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment with all antigen binding sites. Generally, Fv antibody also contains a polypeptide linker between the VH and VL domains, and is able to form the structure required for antigen binding. The antigen binding domain is usually scFv (single-chain variable fragment). Single-chain antibodies are preferably an amino acid chain sequence encoded by a nucleotide chain.

[0152] In the present invention, the scFv comprises the extracellular domain of CEA or its active fragment which specifically recognizes the antigen highly expressed in tumor.

[0153] 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.

[0154] Chimeric Antigen Receptor (CAR)

[0155] 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.

[0156] Chimeric Antigen Receptor T Cells (CAR-T Cells)

[0157] As used herein, the terms "CAR-T cells", "CAR-T", and "CAR-T cells of the present invention" all refer to the engineered immune cells described in the seventh aspect of the present invention. The CAR-T cells of the present invention can be used to treat tumors, such as colon cancer, gastric cancer, liver cancer, pancreatic cancer, etc.

[0158] 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.

[0159] Chimeric Antigen Receptor NK Cells (CAR-NK Cells)

[0160] 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, such as colon cancer, gastric cancer, liver cancer, pancreatic cancer, etc.

[0161] 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.

[0162] 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.

[0163] TLR molecules

[0164] The Toll-like receptor family (TLRs) is a type of pattern recognition receptor that can recognize PAMPs (pathogen-associated molecular patterns) and trigger a complex cascade immune response in the human body. They are not only expressed on immune cells, but also in various tumor cells, participating in tumor immune surveillance, and also play a key role in inflammation, immune cell regulation, and proliferation.

[0165] To date, 10 TLRs (TLR1-10) have been identified in humans. Based on their cellular localization and the ligands sensed, the 10 human TLRs are functionally divided into two subgroups. One group consists of TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, which are located on the cell membrane and mainly recognize bacterial components such as lipids, lipoproteins, and proteins. The other group consists of TLR3, TLR7, TLR8, and TLR9, which are located in lysosomes and recognize microbial nucleic acids.

[0166] At present, the specific function of TLR10 is not very clear. TLR is a type I transmembrane protein composed of an N-terminal ligand recognition domain, a single transmembrane domain and a C-terminal signaling domain. The extracellular domain of TLR contains tandem copies of a motif, also known as a leucine-rich repeat structure (LRR), which is responsible for recognizing PAMPs. The intracellular signaling domain of TLR is homologous to the IL-1 receptor and is called the Toll / interukin-1 receptor (TIR) ​​domain. This signaling domain is required for initiating downstream signaling pathways. The two main signaling pathways associated with TLR activation include the MyD88-dependent pathway and the TRIF-dependent pathway.

[0167] PD-1, PD-L1, and TFG-β

[0168] The PD-1 (Programmed cell death-1) receptor is a type I transmembrane glycoprotein and a member of the CD28 receptor superfamily. However, unlike CD28, it is a monomer on the cell surface. Structurally, PD-1 consists of an extracellular immunoglobulin variable domain, a transmembrane domain, and a cytoplasmic tail responsible for the binding of signals to skeleton molecules. PD-L1 is the ligand of PD-1. PD-L1 is associated with the suppression of the immune system and can transmit inhibitory signals. Tumor cells can also express PD-L1. Once PD-1 and PD-L1 bind, they will transmit negative regulatory signals to T cells, causing T cells to be unable to recognize cancer cells, and tumor cells will thereby achieve "immune escape." This proves the key role of PD-1 in blocking anti-tumor immunity.

[0169] There are many studies on the application of PD-1 in cell therapy, such as knocking out PD-1 expression on CAR-T cells through genetic modification; or combining the extracellular domain of PD-1 with the intracellular domain of the activation signal of T cells to convert the inhibitory signal of PD-1 into the activation signal of T cells. Or allowing CAR-T cells to express soluble PD-1, etc., all of which have proven to make CAR-T more difficult to be exhausted, have stronger amplification ability, and have stronger tumor suppression ability in the body.

[0170] TGFβ, or transforming growth factor β, is a type of protein in cells that can both curb cancer formation and promote cancer cell growth. The TGFβ family transmits signals through receptors, and can be divided into type I receptors (TβRI), type II receptors (TβRII), and type III receptors (TβRIII) according to their structural and functional characteristics. The molecular weights of the three types of receptors are 53KDa, 70-80KDa, and 280-330KDa, respectively. TβRI and TβRII are single-pass transmembrane serine / threonine kinase receptors with intrinsic kinase activity, which are necessary for mediating TGFβ signal transduction. The main limitation of CAR-T's anti-tumor effect in solid tumors is the immunosuppressive environment of the tumor microenvironment (TME). By modifying TGFβ receptors or inhibiting the binding of TGFβ to receptors, excellent therapeutic effects are shown in cell therapy, and even good results are achieved in clinical practice. Therefore, the application of TGFβ in cell therapy has received widespread attention.

[0171] Expression cassette

[0172] As used herein, "expression cassette" or "expression cassette of the present invention" includes a first expression cassette and a second expression cassette. The expression cassette of the present invention is as described in the tenth aspect of the present invention, and the first expression cassette comprises a nucleic acid sequence encoding the CAR. The second expression cassette expresses the fusion protein described in the first aspect of the present invention.

[0173] In the present invention, the fusion protein of the present invention may be constitutively expressed.

[0174] In the case of induced expression, when the CAR-T cells are activated by the corresponding inducer, the second expression cassette expresses the fusion protein; thus, when the CAR-T cells of the present invention are not exposed to the corresponding inducer, the second expression cassette does not express the fusion protein.

[0175] In one embodiment, the first expression cassette and the second expression cassette further comprise a promoter and / or a terminator, respectively. The promoter of the second expression cassette may be a constitutive or inducible promoter.

[0176] Carrier

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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).

[0190] 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.

[0191] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0192] preparation

[0193] The present invention provides a composition containing the engineered immune cells (such as CAR-T cells) described in the seventh 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.

[0194] 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.

[0195] Therapeutic applications

[0196] The present invention includes therapeutic applications of cells (e.g., T cells) transduced by a vector (e.g., a lentiviral vector) containing the expression cassette of the present invention. The transduced T cells can target surface markers of tumor cells and express the fusion protein described in the first aspect of the present invention, synergistically and significantly improving their killing efficiency of tumor cells.

[0197] 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.

[0198] 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.

[0199] 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, wherein the CAR-T cells can induce a specific immune response to tumor cells that highly express the antigen recognized by the CAR antigen binding domain. For example, the CAR-T cells of the present invention cause a specific immune response to tumor cells that highly express CEA.

[0200] 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, and pancreatic cancer.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] When an "immunologically 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.

[0205] 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.

[0206] 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, simultaneously 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, simultaneously 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.

[0207] 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. 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.

[0208] The main advantages of the present invention include:

[0209] 1. The fusion protein of the present invention enables the immune cells of the present invention to kill tumor cells more efficiently, thereby significantly improving the therapeutic effect of tumors and reducing toxic side effects.

[0210] 2. The expression of the fusion protein of the present invention unexpectedly promotes the expression rate of CAR molecules in immune cells and promotes the proliferation of CAR-T cells.

[0211] The present invention will be further described below in conjunction with 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. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0212] Materials and Methods

[0213] CAR molecules and their structure

[0214] In the Examples, performance experiments of CEA CAR-T cells containing control fusion proteins were performed in addition to CEA CAR-T cells containing the preferred fusion protein of the present invention.

[0215] The CEA CAR molecules of the present invention respectively include the following partial structures: human CD8 signal peptide [abbreviated as CD8 (SP)], human anti-CEA single-chain antibody [abbreviated as CEAscFv], human CD28 hinge region [abbreviated as CD8 (hinge)], human CD28 transmembrane domain [abbreviated as CD8 (TM)] and human CD28 intracellular domain [abbreviated as CD8 (IC)], human 4-1BB intracellular domain [abbreviated as 4-1BB (ID)], human CD3ζ intracellular signal transduction domain [abbreviated as CD3ζ (ID)], self-cleavage peptide P2A, fusion protein of the present invention (TGF-β extracellular domain, PD1 extracellular domain, TGF-β extracellular domain, PD1 transmembrane domain [abbreviated as PD1 (TM)], TLR10 transmembrane domain [abbreviated as TLR10 (TM)], TGF-β transmembrane domain [abbreviated as TGF-β (TM)], TLR10 intracellular domain [abbreviated as TLR10 (IC)]).

[0216] The CEA CAR molecule as a control without fusion protein was named P320;

[0217] The CEA CAR molecules used as controls containing fusion proteins were named P370, P371, P373, P374, P375, P321, and P312.

[0218] The CEA CAR molecules containing the preferred fusion proteins of the present invention are named P372 and P312.

[0219] The specific structure of the CAR molecule is as follows Figure 2 , Fig. 9 and Fig.15 As shown, the details are as follows:

[0220] P320 is composed of CD8 (SP), CEAscFv, CD28 (hinge), CD28 (TM), CD28 (IC), and CD3ζ (ID) in series from its amino terminus to its carboxyl terminus.

[0221] P370 is composed of CD8 (SP), CEAscFv, CD28 (hinge), CD28 (TM), CD28 (IC), CD3ζ (ID), P2A, PD1, PD1 (TM), and TLR10 (IC) in series from its amino terminus to its carboxyl terminus.

[0222] P371 is composed of CD8 (SP), CEAscFv, CD28 (hinge), CD28 (TM), CD28 (IC), CD3ζ (ID), P2A, PD1, CD28 (TM), and TLR10 (IC) in series from its amino terminus to its carboxyl terminus.

[0223] P372 is composed of CD8 (SP), CEAscFv, CD828 (hinge), CD28 (TM), CD28 (IC), CD3ζ (ID), P2A, PD1, TLR10 (TM), and TLR10 (IC) in series from its amino terminus to its carboxyl terminus.

[0224] P373 is composed of CD8 (SP), CEAscFv, CD8α (hinge), CD8α (TM), CD28 (IC), CD3ζ (ID), P2A, PD1, CD28 (TM), and CD28 (IC) in series from its amino terminus to its carboxyl terminus.

[0225] P374 is composed of CD8 (SP), CEAscFv, CD28 (hinge), CD28 (TM), CD28 (IC), CD3ζ (ID), P2A, PD1, TLR10 (TM), and 4-1BB (ID) in series from its amino terminus to its carboxyl terminus.

[0226] P375 is composed of CD8 (SP), CEAscFv, CD28 (hinge), CD28 (TM), CD28 (IC), CD3ζ (ID), P2A, PD1, PD1 (TM), and 4-1BB (ID) in series from its amino terminus to its carboxyl terminus.

[0227] P321 is composed of CD8 (SP), CEAscFv, CD28 (hinge), CD28 (TM), CD28 (IC), CD3ζ (ID), P2A, and TGF-βRII in series from its amino terminus to its carboxyl terminus.

[0228] P312 is composed of CD8 (SP), CEAscFv, CD28 (hinge), CD28 (TM), CD28 (IC), CD3ζ (ID), P2A, TGF-βRII, TGF-βRII (TM), and TLR10 in series from its amino terminus to the carboxyl terminus.

[0229] In another preferred embodiment, the preferred chimeric antigen receptor (CEACAR) nucleotide molecule containing the fusion protein of the present invention is one or more sequences selected from the following group from the 5' end to the 3' end:

[0230] (i) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:30 and SEQ ID NO:32;

[0231] (ii) SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:32.

[0232] In another preferred embodiment, the preferred chimeric antigen receptor (CEACAR) amino acid molecule containing the fusion protein of the present invention is one or more sequences selected from the following groups from the amino terminus to the carboxyl terminus:

[0233] (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:29 and SEQ ID NO:31;

[0234] (ii) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:21 and SEQ ID NO:31.

[0235] Example 1

[0236] Target cell construction and target cell CEA and PD-L1 expression

[0237] 1.1 Construction of target cells MKN-45.LUC, LS174T.LUC, BxPC-3.LUC and COLO320DM.LUC

[0238] HEK293T cells with good growth status were centrifuged and the medium was changed after digestion. 2×10 7 The cells were seeded in 15 cm culture dishes and cultured for 24 h.

[0239] The plasmid expressing luciferase (purchased from NovoPro, catalog number V006878) was mixed with the lentiviral packaging kit (Aikangde Biotechnology, catalog number LVP2MIX) and transfected into HEK293T cells. 2 The supernatant was collected after 48 h.

[0240] MKN-45 (purchased from Cobioer, catalog number: CBP60488), LS174T (purchased from ATCC, catalog number: CL-188), BxPC-3 (purchased from the cell bank of the Chinese Academy of Sciences, catalog number: TCHu 12) and COLO 320DM (purchased from Cobioer, catalog number: CBP60027) cells in good growth state were transduced with viruses and cultured for 3-5 days. Monoclonal cells expressing luciferase were sorted by flow cytometry and continued to be cultured for subsequent experiments.

[0241] 1.2 CEA and PD-L1 expression on target cells

[0242] MKN-45, LS174T, BxPC-3 and COLO 320DM were centrifuged and the medium was changed. 1E+05 cells were centrifuged respectively, and 100ul of PE anti-human CD274 (B7-H1, PD-L1) Antibody (Biolegend) was diluted 1:100 with DPBS; Recombinant Anti-CEACAM5 Antibody (APC) (Sino Biological) was diluted 1:50; and incubated at 4°C for 45min. After washing the cells 3 times, the cells were resuspended with DPBS, and the expression of CEA and PD-L1 was detected on a flow cytometer.

[0243] like Figure 1 As shown, MKN-45, LS174T and BxPC-3 all expressed CEA, while COLO 320DM did not. All cells expressed PD-L1.

[0244] Example 2 CAR virus preparation and CAR-T cell preparation

[0245] 2.1CAR retroviral packaging

[0246] HEK293T cells with good growth status were centrifuged and the medium was changed after digestion. 2×10 7 The cells were inoculated in a 15 cm culture dish and cultured for 24 h. The CAR plasmid (inserted into the vector after full gene synthesis, the vector sequence is shown in SEQ ID NO: XX) and the auxiliary plasmid gag-pol plasmid (purchased from NovoPro, catalog number V006593) and GALV plasmid (purchased from NovoPro, catalog number V000819) were pre-mixed with polyetherimide (PEI) at a ratio of 4:3:1, and then 0.5 ml of the transfection mixture was added dropwise to HEK293T cells (about 4×10 7 Place in a 37°C, 5% CO 2 The supernatant was collected after 24 h.

[0247] 2.2 CAR-T cell preparation

[0248] Fresh blood was collected from healthy individuals and peripheral blood mononuclear cells (PBMC) were separated using lymphocyte separation medium. The separated cells were resuspended in T cell culture medium (X-VIVO15 culture medium containing 300 IU / mL IL-2) and T Cell TransAct TM (Miltenyi) reagent and placed in a cell culture incubator for 48 h.

[0249] Novonectin protein (nearshore) was diluted to 20 ng / mL with DPBS and added to an untreated 12-well plate at 1 mL / well for overnight coating. The next day, the coating solution was removed and 2 mL / well of viral supernatant was added. The cells were centrifuged at 32°C and 2000 g for 120 min. The activated T cells for 2 days were centrifuged, resuspended in T cell culture medium, counted, and diluted to 2.5 × 10 5 cells / mL. Add 1mL / well to a 12-well plate and centrifuge at 32℃, 400g for 10min. Place in an incubator and culture overnight. Transfer to a new 12-well plate the next day and change the culture medium every 3 days.

[0250] On the 6th day, 1.2E+05 cells were centrifuged and 100ul of Biotinylated HumanCEACAM-5 / CD66e diluted 1:100 with DPBS was added. TMProtein, His Avitag, incubate at 4°C for 45 min. After washing the cells, add 100 ul of PE Streptavidin (Biolegend) diluted 1:100 with DPBS; PE anti-human CD279 (PD-1) Antibody (Biolegend) diluted 1:100, and incubate at 4°C for 45 min.

[0251] After washing the cells three times, resuspend the cells in DPBS and detect the expression of CAR or PD-1 on a flow cytometer.

[0252] The schematic diagram of the structure of the CEA CAR molecules (P320, P370, P371 and P372) selected in this example is as follows Figure 2 shown.

[0253] The expression of CAR and PD-1 on the surface of CAR-T cells is as follows Figure 3-4 As shown in the figure, 3 days after virus infection, the CAR positive rate was between 40% and 55%; P370, P371 and P372 CAR-T with PD-1 extracellular domain had much higher PD-1 expression than P320.

[0254] Example 3 In vitro killing experiment of CEA CAR-T cells on LS174T, BxPC-3 and COLO 320DM cells

[0255] 3.1 In vitro killing experiment of CEA CAR-T cells on LS174T, BxPC-3 and COLO 320DM cells

[0256] In this example, the function of CEA CAR effector cells in killing target cells was analyzed by in vitro luciferase assay.

[0257] LS174T, BxPC-3 and COLO 320DM cells expressing luciferase were used as target cells. The target cells were diluted to 2×10 5 cells / mL was inoculated into 96-well plates at 50 μL / well. CAR-T and blank control group cells were counted according to the effector-target ratio (E:T) of 8:1, 2:1, 1:2, 1:4, and 1:8, added to the corresponding 96-well plates, incubated for 16 hours, and then 50 μL of luciferase substrate (Promega) was added to quantify the remaining live target cells in each well by evaluating the remaining luciferase activity in each well.

[0258] The results are as follows Figure 5 As shown in AC, P370, P371, and P372 all had good in vitro killing effects on BxPC-3.LUC and LS174T.LUC cells, and their effects were significantly better than those of P320.

[0259] 3.2 Effect of CEA CAR-T on cytokine secretion of LS174T, BxPC-3 and COLO 320DM cells

[0260] The release of IL-2 and IFN-γ by CEA CAR under tumor cell stimulation was analyzed by ELISA test, indicating that the CAR cells expressing the present application have significant cytokine secretion ability.

[0261] CAR-T cells were co-incubated with LS174T, BxPC-3 and COLO 320DM cells, respectively. The cells were incubated for 16 hours at an effector-target ratio (E:T) of 8:1, 2:1, 1:2, 1:4, and 1:8, and the supernatant was collected by centrifugation. The cytokines in the supernatant were quantified using the Human IL-2 ELISA Kit II (BD Biosciences) and the Human IFN-γ ELISA Set (BD Biosciences). The absorbance of each well of the sample to be tested at 450 nm was read by a multi-mode microplate reader (Thermoscientific#Varioskan LUX), and the cytokine concentration corresponding to the absorbance of the sample to be tested was calculated according to the standard curve of the absorbance of the standard. For specific operations, refer to the instructions for the above kit.

[0262] The results are as follows Figure 6 As shown in AC, P370, P371 and P372 all released a large amount of cytokines to BxPC-3.LUC and LS174T.LUC and were higher than P320.

[0263] Example 4 Multiple rounds of stimulation of CEA CAR-T cells in vitro

[0264] In this example, LS174T target cells were co-incubated with CEA CAR multiple times to detect the proliferation and viability of CAR cells and analyze the proliferation ability of CEA CAR effector cells under tumor cell stimulation.

[0265] The CAR-T cells were centrifuged and counted, and 1.0E+06 cells were taken and added to LS 174T cells at an effector-target ratio (E:T) of 1:1, and incubated for 24 hours. The cells were centrifuged and cultured for 48 hours, and the number and activity of cells were measured. This was considered one round, and a total of 5 rounds were performed to count the expansion and activity of CAR-T cells.

[0266] The results are as follows Figure 7 As shown in AB, after 5 rounds of tumor cell stimulation, P372 and P370 expanded better than P371 and P320. There was little difference in cell viability during the 5 rounds of stimulation.

[0267] This example illustrates through in vitro experiments that cells expressing the CAR (P370, P372) described in this application have good targeted proliferation ability under tumor cell stimulation.

[0268] Example 5 In vivo efficacy evaluation of CEA CAR-T cells in BxPC-3 tumor xenograft mice

[0269] This example detects the killing / clearing ability of CEA CAR-T cells on pancreatic adenocarcinoma cells xenografted in NOG mice.

[0270] Select NOG mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) that meet the adaptive observation requirements, and inject 5×10 6 After 5 days of feeding, when the tumor volume was about 50 cubic millimeters, the cells were randomly divided into groups and injected with 5×10 6 CAR-T cells (prepared according to the method of Example 2, the solvent is X-VIVO15 culture medium) and non-virus-infected control T cells (the solvent is X-VIVO15 culture medium), the tumor diameter is measured twice a week with a vernier caliper, and the tumor volume is calculated as: V = 0.5 × a × b 2 , where a and b are the length and width of the tumor, respectively.

[0271] The results are as follows Figure 8 AC. In vivo efficacy experiments in Bxpc3 tumor xenograft mice:

[0272] P372 has the strongest tumor-suppressing effect and can continuously inhibit tumor growth, so it is the preferred one of the present invention;

[0273] P371 has strong cytotoxicity, and all mice in the group died on the 20th day after tumor infusion;

[0274] P370 had a certain anti-tumor effect, but the tumor continued to grow after 28 days.

[0275] Example 6 CAR virus preparation and CAR-T cell preparation

[0276] 6.1CAR Retroviral Packaging

[0277] HEK293T cells with good growth status were centrifuged and the medium was changed after digestion. 2×10 7The cells were inoculated in a 15 cm culture dish and cultured for 24 h. The CAR plasmid (inserted into the vector after full gene synthesis, the vector sequence is shown in SEQ ID NO: XX) and the auxiliary plasmid gag-pol plasmid (purchased from NovoPro, catalog number V006593) and GALV plasmid (purchased from NovoPro, catalog number V000819) were pre-mixed with polyetherimide (PEI) at a ratio of 4:3:1, and then 0.5 ml of the transfection mixture was added dropwise to HEK293T cells (about 4×10 7 Place in a 37°C, 5% CO 2 The supernatant was collected after 24 h.

[0278] 6.2 CAR-T cell preparation

[0279] Fresh blood from healthy individuals was collected and peripheral blood mononuclear cells (PBMC) were separated using lymphocyte separation medium. The separated cells were resuspended in T cell culture medium (X-VIVO15 culture medium containing 300 IU / mL IL-2) and TCell TransAct TM (Miltenyi) reagent and placed in a cell culture incubator for 48 h.

[0280] Novonectin protein (nearshore) was diluted to 20 ng / mL with DPBS and added to an untreated 12-well plate at 1 mL / well for overnight coating. The next day, the coating solution was removed and 2 mL / well of viral supernatant was added. The cells were centrifuged at 32°C and 2000 g for 120 min. The activated T cells for 2 days were centrifuged, resuspended in T cell culture medium, counted, and diluted to 2.5 × 10 5 cells / mL. Add 1mL / well to a 12-well plate and centrifuge at 32℃, 400g for 10min. Place in an incubator and culture overnight. Transfer to a new 12-well plate the next day and change the culture medium every 3 days.

[0281] On the 6th day, 1.2E+05 cells were centrifuged and 100ul of Biotinylated HumanCEACAM-5 / CD66e diluted 1:100 with DPBS was added. TM Protein, His Avitag, incubate at 4°C for 45 min. After washing the cells, add 100 ul of PE Streptavidin (Biolegend) diluted 1:100 with DPBS; PE anti-human CD279 (PD-1) Antibody (Biolegend) diluted 1:100, and incubate at 4°C for 45 min.

[0282] After washing the cells three times, resuspend the cells in DPBS and detect the expression of CAR or PD-1 on a flow cytometer.

[0283] The schematic diagram of each CEA CAR molecular structure selected in this example is as follows Fig. 9 shown.

[0284] CAR expression and PD-1 expression on the surface of CAR-T cells Figure 10-11 As shown in the figure, 3 days after virus infection, the CAR positivity rates of P374 and P375 CAR-T were higher than those of P320, P372 and P373; P373, P374 and P375 CAR-T with PD-1 extracellular domain had much higher PD-1 expression than P320.

[0285] Example 7 In vitro killing experiment of CEA CAR-T cells against LS174T, BxPC-3 and COLO 320DM cells

[0286] 7.1 In vitro killing experiment of CEA CAR-T cells on LS174T, BxPC-3 and COLO 320DM cells

[0287] In this example, the function of CEA CAR effector cells in killing target cells was analyzed by in vitro luciferase assay.

[0288] LS174T, BxPC-3 and COLO 320DM cells expressing luciferase were used as target cells. The target cells were diluted to 2×10 5 cells / mL was inoculated into 96-well plates at 50 μL / well. CAR-T and blank control group cells were counted according to the effector-target ratio (E:T) of 8:1, 2:1, 1:2, 1:4, and 1:8, added to the corresponding 96-well plates, incubated for 16 hours, and then 50 μL of luciferase substrate (Promega) was added to quantify the remaining live target cells in each well by evaluating the remaining luciferase activity in each well.

[0289] The results are as follows Fig.12 As shown in AC, there is little difference in the killing effect of all CAR-T (P320, P372, P373, P374, P375) on BxPC-3.LUC and LS174T.LUC.

[0290] 7.2 Effect of CEA CAR-T on cytokine secretion of LS174T, BxPC-3 and COLO 320DM cells

[0291] The release of IL-2 and IFN-γ by CEA CAR under tumor cell stimulation was analyzed by ELISA test, indicating that the CAR cells expressing the present application have significant cytokine secretion ability.

[0292] CAR-T cells were co-incubated with LS174T, BxPC-3 and COLO 320DM cells, respectively. The cells were incubated for 16 hours at an effector-target ratio (E:T) of 8:1, 2:1, 1:2, 1:4, and 1:8, and the supernatant was collected by centrifugation. The cytokines in the supernatant were quantified using the Human IL-2 ELISA Kit II (BD Biosciences) and the Human IFN-γ ELISA Set (BD Biosciences). The absorbance of each well of the sample to be tested at 450 nm was read by a multi-mode microplate reader (Thermoscientific#Varioskan LUX), and the cytokine concentration corresponding to the absorbance of the sample to be tested was calculated according to the standard curve of the absorbance of the standard. For specific operations, refer to the instructions for the above kit.

[0293] The results are as follows Fig.13 As shown in AC, P320, P372, P373, P374 and P375 all released cytokines to LS174T.LUC, and P373 was slightly lower than that of CAR-T in other groups.

[0294] Example 8 In vivo efficacy evaluation of CEA CAR-T cells in ASPC-1 tumor xenograft mice

[0295] This example detects the killing / clearing ability of CEA CAR-T cells on mouse xenografted human metastatic pancreatic adenocarcinoma cells in NOG mice.

[0296] Select NOG mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) that meet the adaptive observation requirements, and inject 5×10 6 After 5 days of feeding, when the tumor volume was about 50 cubic millimeters, the cells were randomly divided into groups and injected with 5×10 6 CAR-T cells (prepared according to the method of Example 2, the solvent is X-VIVO15 culture medium) and non-virus-infected control T cells (the solvent is X-VIVO15 culture medium), the tumor diameter is measured twice a week with a vernier caliper, and the tumor volume is calculated as: V = 0.5 × a × b 2 , where a and b are the length and width of the tumor, respectively.

[0297] The results are as follows Fig.14 AC shows in vivo efficacy experiments in ASPC-1 tumor xenograft mice:

[0298] The tumor inhibition effect of P372 was significantly stronger than that of other groups, and it could continuously inhibit the growth of tumor cells. The tumor size of mice in the P372 group was about 16 mm on the 28th day. 3 , and thereafter no longer grow;

[0299] Tumors in other groups continued to grow after 21 days, and when the experiment was stopped (day 35), the tumor size exceeded 40 mm. 3 , and there is a trend of continued growth;

[0300] There was no mouse death in all CAR-T experimental groups, and the weight of mice in each group was relatively stable.

[0301] This example illustrates through in vivo experiments that cells expressing the CAR (eg, P372) of the present invention have significant in vivo anti-tumor activity

[0302] Example 9 CAR virus preparation and CAR-T cell preparation

[0303] 9.1CAR Retroviral Packaging

[0304] HEK293T cells with good growth status were centrifuged and the medium was changed after digestion. 2×10 7 The cells were inoculated in a 15 cm culture dish and cultured for 24 h. The CAR plasmid (inserted into the vector after full gene synthesis, the vector sequence is shown in SEQ ID NO: XX) and the auxiliary plasmid gag-pol plasmid (purchased from NovoPro, catalog number V006593) and GALV plasmid (purchased from NovoPro, catalog number V000819) were pre-mixed with polyetherimide (PEI) at a ratio of 4:3:1, and then 0.5 ml of the transfection mixture was added dropwise to HEK293T cells (about 4×10 7 Place in a 37°C, 5% CO 2 The supernatant was collected after 24 h.

[0305] 9.2 CAR-T cell preparation

[0306] Fresh blood from healthy individuals was collected and peripheral blood mononuclear cells (PBMC) were separated using lymphocyte separation medium. The separated cells were resuspended in T cell culture medium (X-VIVO15 culture medium containing 300 IU / mL IL-2) and TCell TransAct TM (Miltenyi) reagent and placed in a cell culture incubator for 48 h.

[0307] Novonectin protein (nearshore) was diluted to 20 ng / mL with DPBS and added to an untreated 12-well plate at 1 mL / well for overnight coating. The next day, the coating solution was removed and 2 mL / well of viral supernatant was added. The cells were centrifuged at 32°C and 2000 g for 120 min. The activated T cells for 2 days were centrifuged, resuspended in T cell culture medium, counted, and diluted to 2.5 × 10 5cells / mL. Add 1mL / well to a 12-well plate and centrifuge at 32℃, 400g for 10min. Place in an incubator and culture overnight. Transfer to a new 12-well plate the next day and change the culture medium every 3 days.

[0308] On the 6th day, 1.2E+05 cells were centrifuged and 100ul of Biotinylated HumanCEACAM-5 / CD66e diluted 1:100 with DPBS was added. TM Protein, His Avitag, incubate at 4°C for 45 min. After washing the cells, add 100 ul of PE Streptavidin (Biolegend) diluted 1:100 with DPBS.

[0309] After washing the cells three times, resuspend the cells in DPBS and detect CAR expression on a flow cytometer.

[0310] In this example, the schematic diagrams of the molecular structures of the selected CEA CARs are shown in Fig.15 shown.

[0311] The results are as follows Fig.16 As shown, 3 days after virus infection, the CAR positivity rates of P312 and P321 CAR-T were higher than that of P320.

[0312] Example 10 In vitro killing experiment of CEA CAR-T on MKN-45 and COLO 320DM cells

[0313] 10.1 In vitro killing experiment of CEA CAR-T on MKN-45 and COLO 320DM cells

[0314] In this example, the function of CEA CAR effector cells in killing target cells was analyzed by in vitro luciferase assay.

[0315] Luciferase-expressing MKN-45 and COLO 320DM cells were used as target cells. The target cells were diluted to 2×10 5 cells / mL was inoculated into 96-well plates at 50 μL / well. CAR-T and blank control group cells were counted according to the effector-target ratio (E:T) of 8:1, 2:1, 1:2, and 1:4, added to the corresponding 96-well plates, incubated for 16 hours, and then 50 μL of luciferase substrate (Promega) was added to quantify the remaining live target cells in each well by evaluating the remaining luciferase activity in each well.

[0316] The results are as follows Fig.17 As shown in AB, all CAR-Ts kill MKN-45.LUC, while all CAR-Ts do not kill COLO320DM.LUC.

[0317] 10.2 Effect of CEA CAR-T on cytokine secretion of MKN-45 and COLO 320DM cells

[0318] The release of IL-2 and IFN-γ by CEA CAR under tumor cell stimulation was analyzed by ELISA test, indicating that the CAR cells expressing the present application have significant cytokine secretion ability.

[0319] CAR-T cells were co-incubated with MKN-45 and COLO 320DM cells, respectively. The cells were incubated for 16 hours at effector-target ratios (E:T) of 8:1, 2:1, 1:2, and 1:4, and the supernatant was collected by centrifugation. The cytokines in the supernatant were quantified using Human IL-2 ELISA Kit II (BD Biosciences) and Human IFN-γ ELISA Set Kit (BD Biosciences).

[0320] The absorbance of each well of the sample to be tested at 450 nm was read by a multi-mode microplate reader (Thermoscientific#Varioskan LUX), and the cytokine concentration corresponding to the absorbance of the sample to be tested was calculated according to the standard curve of the absorbance of the standard. For specific operations, refer to the instructions of the above kit.

[0321] The results are as follows Fig.18 As shown in AB, P320, P312 and P321 all had strong cytokine release to MKN-45.LUC, while all CAR-Ts had almost no cytokine release to COLO 320DM.LUC.

[0322] Example 11 In vivo efficacy evaluation of CEA CAR-T cells in MKN-45 tumor xenograft mice

[0323] This example detects the killing / clearing ability of CEA CAR-T cells on xenografted human gastric cancer cells in NOG mice.

[0324] Select NOG mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) that meet the adaptive observation requirements, and inject 5×10 6 After 5 days of feeding, when the tumor volume was about 50 cubic millimeters, the cells were randomly divided into groups and injected with 5×10 6 CAR-T cells (prepared according to the method of Example 2, the solvent is X-VIVO15 culture medium) and non-virus-infected control T cells (the solvent is X-VIVO15 culture medium), the tumor diameter is measured twice a week with a vernier caliper, and the tumor volume is calculated as: V = 0.5 × a × b 2, where a and b are the length and width of the tumor, respectively.

[0325] The results are as follows Fig.19 AC shows that in the in vivo efficacy experiment in MKN-45 tumor xenograft mice, all CAR-T experimental groups had a strong tumor inhibition effect.

[0326] In the P320 group, the tumor volume of three out of five mice was zero on the 38th day, and one mouse died on the 45th day.

[0327] In the P321 group, on day 38, the tumor volume of three of the five mice was 0, and the tumor volume of the remaining two mice was stable at 14 mm 3 about.

[0328] In the P312 group, the tumor volumes of three out of five mice were zero on the 31st day, and the tumor volumes of all five mice were zero on the 34th day until the end of the experiment.

[0329] Therefore, this experiment confirmed that CAR-T with TLR intracellular region (such as P312) has better anti-tumor efficacy in vivo.

[0330] Sequence information of the present invention:

[0331]

[0332]

[0333]

[0334] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. 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. A fusion protein, It is characterized in that The fusion protein has a structure of Formula I: Z0-L1-Z1-L2-Z2-L3-Z3 (I) In the formula, Z0 is signal peptide or none; L1 is none or the first connecting peptide element; Z1 is the PD1 extracellular element or the TGF-β extracellular element; L2 is none, a second connecting peptide element, or a hinge region element; Z2 is the PD1 transmembrane segment or the TLR transmembrane segment; L3 is none or a third connecting peptide element; and Z3 is the intracellular segment of TLR.

2. The fusion protein according to claim 1, It is characterized in that Z2 and Z3 in the fusion protein are a combination selected from the following groups: (i) Z2 is the transmembrane segment of PD1, and Z3 is the intracellular segment of TLR10; (ii) Z2 is the transmembrane segment of TLR10, and Z3 is the intracellular segment of TLR10.

3. An isolated polynucleotide, It is characterized in that The polynucleotide encodes the fusion protein of claim 1.

4. A carrier, It is characterized in that The vector contains the polynucleotide according to claim 3.

5. A host cell, It is characterized in that The host cell contains the vector of claim 4 or the polynucleotide of claim 3 is integrated into its genome.

6. A method for producing the fusion protein according to claim 1, It is characterized in that Includes steps: (a) culturing the host cell according to claim 5 under suitable expression conditions, thereby expressing the fusion protein according to claim 1; (b) isolating and purifying the fusion protein expressed in step (a).

7. An engineered immune cell, It is characterized in that The engineered immune cells are T cells or NK cells, and the immune cells have the following characteristics: (a) the immune cell expresses a chimeric antigen receptor (CAR), wherein the CAR targets a surface marker of a tumor cell; and (b) The immune cell expresses the fusion protein according to claim 1.

8. A method for preparing the engineered immune cell according to claim 7, It is 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 the CAR molecule and the fusion protein of claim 1, thereby obtaining the engineered immune cells of claim 7.

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

10. A kit for preparing the engineered immune cell according to claim 7, It is characterized in that The kit comprises a container, and located within the container: (1) a first nucleic acid sequence, wherein the first nucleic acid sequence contains a first expression cassette for expressing the CAR; and (2) A second nucleic acid sequence, wherein the second nucleic acid sequence comprises a second expression cassette for co-expressing the fusion protein of claim 1.

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