Chimeric antigen receptor immune cell constructed based on cyclic peptide Peptide G and application of chimeric antigen receptor immune cell

Abstract

The invention provides a chimeric antigen receptor immune cell constructed on the basis of a small molecule cyclic peptide Peptide G (cyclic peptide G), and a preparation method and an application of the chimeric antigen receptor immune cell constructed on the basis of the small molecule cyclic peptide Peptide G. Specifically, the invention provides a chimeric antigen receptor (CAR) (PG-CAR) modified based on Peptide G. The CAR contains an extracellular binding domain, and the extracellular binding domain can specifically target MMP14 on the surface of a cell membrane. The CAR immune cell provided by the invention has relatively strong specificity and target affinity, relatively strong killing ability to target cells and high safety.

Description

Technical Field

[0001] The present invention belongs to the field of immune cell therapy, and specifically relates to the preparation and application of chimeric antigen receptor immune cells constructed based on cyclic peptide Peptide G. Background Art

[0002] Chimeric Antigen Receptor-T cell (CAR-T) therapy is a new cellular immunotherapy method that can enhance the immune response of T cells to cancer and improve the survival rate of cancer patients. After the patient is intravenously infused with CAR-T cells, the CAR-T cells can recognize the targeted antigens on tumor cells through chimeric antigen receptors, thereby selectively killing tumor cells. Unlike other treatment options, CAR-T cells are usually a one-time treatment, and the cells can maintain their activity for more than ten years after injection to exert their lasting effects. Among them, CD19 CAR-T cells have been reported to have significant efficacy in hematological malignancies. For solid tumors, the application of CAR-T cell therapy is greatly limited due to factors such as their inherent heterogeneity and immunosuppressive microenvironment.

[0003] Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases responsible for the degradation of extracellular matrix (ECM) components, and are involved in tissue repair, immune and pathological processes, including tumors, fibrosis and infection. MMP14, also known as membrane-type matrix metalloproteinase MT1-MMP, is located on chromosome 14q11 and was first cloned and identified by Sato et al. in 1994. MMP14 is one of the most typical members of the MMPS family. A large number of literatures have shown that MMP14 is widely expressed in tumor tissues, such as breast cancer, ovarian cancer, pancreatic cancer, liver cancer, sarcoma, etc. In addition to malignant tumor cells, tumor-associated fibroblasts, tumor-associated macrophages and endothelial cells also express MMP14. MMP14 has a wide range of recognition domains for ECM components, including collagen I and II, gelatin, fibronectin, fibrillin, laminin, fibrin, yolk nectin, entactin and aggrecan, which can induce tumors to acquire a mesenchymal phenotype during development and promote the invasion and migration of tumor cells. Based on the expression and key role of MMP14 in tumors, MMP14 is an important target for tumor treatment, suggesting that targeting MMP14 can be used as an effective strategy for CAR-T treatment of solid tumors.

[0004] Several ligands that specifically target MMP14 are disclosed in the art. Among them, the cyclic peptide peptide G is a ligand of MMP14 and can specifically bind to the receptor MMP14. MMP14 can cleave the Ln-332γ2 chain produced by various cancer cells at the tumor-stroma interface, resulting in increased invasiveness. Peptide G can effectively inhibit the hydrolysis of Ln-332γ2 chain by MMP14, with an effective rate of 64%. In addition, the natural TIMP allosteric NTIMP2 modified by Valeria Arkadash et al. D , targeting MMP14 to inhibit tumor metastasis. Other researchers have found that MMP14 depends on the MT-loop structure to promote cancer cell invasion, and based on this, a specific selective binding peptide MT1-AF7p targeting the MT-loop structure was developed.

[0005] Ligands / receptors are important sources for constructing CARs, but whether ligands / receptors can be constructed into well-functioning CARs requires a lot of research and attempts. There is an urgent need to develop CAR-T treatment technology targeting MMP14 in this field. Summary of the invention

[0006] The purpose of the present invention is to provide a chimeric antigen receptor immune cell specifically targeting MMP14 and a preparation and application method thereof.

[0007] In a first aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the CAR contains an extracellular binding domain, and the extracellular binding domain can specifically bind to MMP14 in a ligand receptor manner.

[0008] In another preferred embodiment, the extracellular binding domain has a cyclic peptide structure.

[0009] In another preferred embodiment, the cyclic peptide structure is based on the cyclic peptide structure of the small molecule cyclic peptide Peptide G.

[0010] In another preferred embodiment, the extracellular binding domain of the CAR contains, in addition to the first extracellular domain having a cyclic peptide structure targeting MMP14, a second extracellular domain targeting an additional target.

[0011] In another preferred embodiment, the additional target is a tumor-specific target.

[0012] In another preferred embodiment, the cyclic peptide structure is located at the end (ie, N-terminus) or in the middle of the extracellular binding domain.

[0013] In another preferred embodiment, the length of the cyclic peptide structure is 12 amino acids.

[0014] In another preferred embodiment, the cyclic peptide structure (or Peptide G peptide segment) has an amino acid sequence as shown in SEQ ID NO: 1, or its amino acid sequence is ≥85% identical to SEQ ID NO: 1, preferably ≥90% identical to SEQ ID NO: 1, or has 1, 2 or 3 amino acid differences compared with SEQ ID NO: 1.

[0015] In another preferred embodiment, the MMP14 is selected from the following group: cell membrane surface MMP14 (MT1-MMP).

[0016] In another preferred embodiment, the extracellular binding domain includes a Peptide G peptide segment, and the Peptide G peptide segment has an amino acid sequence as shown in SEQ ID NO:1.

[0017] In another preferred embodiment, the extracellular binding domain has an amino acid sequence as shown in SEQ ID NO:1.

[0018] In another preferred embodiment, the Peptide G peptide segment has specific binding to MMP14.

[0019] In another preferred embodiment, the MMP14 is an MMP14 protein (MT1-MMP) located on the cell membrane surface (or membrane-bound).

[0020] In another preferred embodiment, the MMP14 protein is derived from human or non-human mammals.

[0021] In another preferred embodiment, the non-human mammals include: rodents (such as rats and mice), primates (such as monkeys); preferably primates.

[0022] In another preferred embodiment, the MMP14 protein is of human or monkey origin.

[0023] In another preferred embodiment, the MMP14 protein is of human origin.

[0024] In another preferred embodiment, the structure of the CAR is shown in Formula I below:

[0025] L-EB-H-TM-C-CD3ζ-RP(I)

[0026] In the formula,

[0027] Each "-" is independently a connecting peptide or a peptide bond;

[0028] L is none or a signal peptide sequence;

[0029] EB is the extracellular binding domain;

[0030] H is no or hinge region;

[0031] TM is the transmembrane domain;

[0032] C is no or co-stimulatory signaling molecule;

[0033] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

[0034] RP is none or reporter protein.

[0035] In another preferred embodiment, the reporter protein RP further comprises a self-cleavage recognition site at its N-terminus, preferably a T2A sequence.

[0036] In another preferred embodiment, the reporter protein RP is a fluorescent protein.

[0037] In another preferred embodiment, the reporter protein RP is mKate2 red fluorescent protein.

[0038] In another preferred embodiment, the amino acid sequence of the mKate2 red fluorescent protein is shown in SEQ ID NO:2.

[0039] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0040] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0041] In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID NO:3.

[0042] In another preferred embodiment, the H is the hinge region of a protein selected from the following group: CD8, CD28, CD137, or a combination thereof.

[0043] In another preferred embodiment, the H is a hinge region derived from CD8.

[0044] In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID NO:4.

[0045] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the following group: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0046] In another preferred embodiment, the TM is a transmembrane region derived from CD8.

[0047] In another preferred example, the amino acid sequence of the TM is shown in SEQ ID NO:5.

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

[0049] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from 4-1BB.

[0050] In another preferred example, the amino acid sequence of C is shown in SEQ ID NO:6.

[0051] In another preferred embodiment, the amino acid sequence of the cytoplasmic signal transduction sequence derived from CD3ζ is shown as SEQ ID NO:7.

[0052] In another preferred example, the amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO:8.

[0053] In the second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the chimeric antigen receptor as described in the first aspect of the present invention.

[0054] In the third aspect of the present invention, a vector is provided, wherein the vector contains the nucleic acid molecule as described in the second aspect of the present invention.

[0055] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

[0056] In another preferred embodiment, the vector is a lentiviral vector.

[0057] In another preferred embodiment, the vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0058] In another preferred embodiment, the vector is a pTomo lentiviral vector.

[0059] In another preferred embodiment, the vector further includes a member selected from the following group: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, etc.

[0060] In another preferred example, the vector comprises the nucleotide sequence shown in SEQ ID NO:9.

[0061] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector as described in the third aspect of the present invention or a chromosome in which an exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated or expresses the CAR as described in the first aspect of the present invention.

[0062] In the fifth aspect of the present invention, an engineered immune cell is provided, wherein the immune cell contains the vector or chromosome as described in the third aspect of the present invention, in which an exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated, or expresses the chimeric antigen receptor as described in the first aspect of the present invention.

[0063] In another preferred embodiment, the engineered immune cells are selected from the following group: T cells, NK cells, NKT cells, or macrophages.

[0064] In another preferred embodiment, the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).

[0065] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0066] In the sixth aspect of the present invention, a method for preparing the engineered immune cells as described in the fifth aspect of the present invention is provided, comprising the following steps: transducing the nucleic acid molecule as described in the second aspect of the present invention or the vector as described in the third aspect of the present invention into the immune cells, thereby obtaining the engineered immune cells.

[0067] In another preferred embodiment, the method further comprises the step of testing the function and effectiveness of the obtained engineered immune cells.

[0068] In the seventh aspect of the present invention, a pharmaceutical composition is provided, which contains the CAR as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, and / or the engineered immune cell as described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0069] In another preferred embodiment, the preparation is a liquid preparation.

[0070] In another preferred embodiment, the preparation is in the form of an injection.

[0071] In another preferred embodiment, the concentration of the engineered immune cells in the preparation is 1×10 3 -1×10 8cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0072] In the eighth aspect of the present invention, a CAR as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, or a host cell as described in the fourth aspect of the present invention, and / or an engineered immune cell as described in the fifth aspect of the present invention is provided for preparing a preparation for preventing and / or treating diseases associated with high expression of MMP14.

[0073] In another preferred embodiment, the MMP14 includes membrane-expressed MMP14.

[0074] In another preferred embodiment, the diseases associated with high expression of MMP14 include tumors, liver fibrosis, cardiovascular diseases, infectious diseases, etc.

[0075] In another preferred embodiment, the other diseases associated with abnormal expression of MMP14 include: diseases associated with abnormal expression of MMP14.

[0076] In another preferred embodiment, the high expression of MMP14 means that the expression level of MMP14 is ≥1.5 times, preferably ≥2 times, and more preferably ≥2.5 times the expression level under normal physiological conditions.

[0077] In another preferred embodiment, the diseases associated with abnormal expression of MMP14 include: tumors, fibrosis, cardiovascular diseases, infections, etc.

[0078] In another preferred embodiment, the disease is a malignant tumor with high expression of MMP14 (ie, MMP14 positive).

[0079] In another preferred embodiment, the tumor includes blood tumors and solid tumors.

[0080] In another preferred embodiment, the blood tumor is selected from the following group: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), etc., or a combination thereof.

[0081] In another preferred embodiment, the solid tumor is selected from the following group: breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian cancer, esophageal cancer, glioma, lung cancer, pancreatic cancer, prostate cancer, cutaneous neuroendocrine cancer, pleural mesothelioma, melanoma, oral cancer, osteosarcoma, or a combination thereof.

[0082] In the ninth aspect of the present invention, there is provided a use of the engineered immune cells as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention, for preventing and / or treating cancer or tumors.

[0083] In the tenth aspect of the present invention, a method for treating a disease is provided, comprising administering an effective amount of the engineered immune cells as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention to a subject in need of treatment.

[0084] In another preferred embodiment, the disease is a disease associated with abnormal expression of MMP14.

[0085] In another preferred embodiment, the disease is cancer or tumor.

[0086] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells (autologous cells) derived from the subject.

[0087] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).

[0088] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0089] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

[0091] 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

[0092] Figure 1 The schematic diagram of vector construction is shown. A is peptide G, MT1-AF7p and NTIMP2 D The amino acid sequence information of the control group plasmid CD19-CAR and the experimental group plasmid peptide G-CAR, MT1-AF7p-CAR and NTIMP2 D -Schematic diagram of the CAR structure, in which the signal peptide, hinge region, and transmembrane region are all derived from the human CD8 molecule, 4-1BB comes from human CD137, CD3ζ comes from human CD3, and mKate2 is a fluorescent marker used to detect CAR expression.

[0093] Figure 2The results of CAR infection efficiency test are shown. A represents CD19-CAR, peptide G-CAR, MT1-AF7p-CAR and NTIMP2 D -The results of cell fluorescence expression after CAR infection of T cells for 72 hours, NTD is untreated, BF is bright field, mKate2 is CAR fluorescence expression; B is the result of flow cytometry fluorescence expression.

[0094] Figure 3 A shows CD19-CAR T, peptide G-CAR T, MT1-AF7p-CAR T, and NTIMP2 D -CAR T cell killing effect on liver cancer cell lines SMMC7721 and HepG2. Figure 3 BF represents the killing effects of CD19-CAR T and peptide G-CAR T on pancreatic cancer, breast cancer, lung cancer, prostate cancer and glioma, respectively.

[0095] Figure 4 The release level of cytokine IFN-γ after 24 hours of co-incubation of CD19-CAR T, peptide G-CAR T with pancreatic cancer, breast cancer, lung cancer, prostate cancer and glioma cells is shown.

[0096] Figure 5 The results of MMP14 expression detection in liver cancer cell lines SMMC7721, HepG2 and MHCC97H are shown. A is flow cytometry detection of MMP14 expression. B is immunofluorescence detection of MMP14 expression level on the cell membrane.

[0097] Figure 6 The gradient killing results and cytokine release levels of peptide G-CAR T on different liver cancer cell lines are shown. Among them, AC is the gradient killing results of liver cancer cell lines SMMC7721, HepG2 and MHCC97H, respectively. DE is the release level of cytokines IFN-γ and TNF-α when E:T=5:1. E:T is the effector-target ratio of T cells to target cells.

[0098] Figure 7 The results of peptide G-CAR T killing MHCC97H cells overexpressing MMP14 and the release of IFN-γ and TNF-α are shown. A is the detection of MMP14 mRNA overexpression in MHCC97H. B is the immunofluorescence detection of MMP14 expression in MHCC97H cells overexpressing MMP14. C is the killing result of peptide G-CAR T on MHCC97H cells overexpressing MMP14. DE is the release level of cytokines IFN-γ and TNF-α.

[0099] Figure 8 The results of peptide G-CAR T cells killing the normal cell line HEK-293T are shown. HEK-293T does not express MMP14 and is not killed by peptide G-CAR T cells. Among them, A is the cell immunofluorescence detection of the expression level of MMP14 in HEK-293T cells. B is the killing detection of peptide G-CAR T on HEK-293T. DETAILED DESCRIPTION

[0100] After extensive and in-depth research and a large number of screenings, the inventors have developed for the first time a chimeric antigen receptor (CAR) with an extracellular binding domain having a specific cyclic peptide structure. Specifically, the present invention provides a preparation and application of chimeric antigen receptor immune cells constructed based on small molecule cyclic peptide peptide G. The experimental results show that the CAR-T cells of the present invention can efficiently and specifically target MMP14, thereby having a significant killing effect on target cells. The present invention was completed on this basis.

[0101] the term

[0102] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0104] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not occur.

[0105] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0106] "Transduction", "transfection", "transformation" or other terms used in this article refer to the process of transferring exogenous polynucleotides to host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to introduce exogenous polynucleotides into host cells (such as Escherichia coli).

[0107] "Gene expression" or "expression" refers to the process of gene transcription, translation and post-translational modification to produce the RNA or protein product of the gene.

[0108] "Polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and analogs. A polynucleotide may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to single-stranded and double-stranded molecules interchangeably. Unless otherwise indicated, the polynucleotides in any embodiment described herein include double-stranded forms and two complementary single strands that are known or predictable to constitute the double-stranded form.

[0109] Those skilled in the art will readily understand the meaning of all parameters, dimensions, materials and configurations described herein. Actual parameters, dimensions, materials and / or configurations depend on the specific application for which the invention is used. Those skilled in the art will appreciate that the embodiments or claims are given by way of example only, and within the scope of equivalents or claims, the scope of the embodiments of the invention is not limited to the scope specifically described and claimed.

[0110] Definitions and all definitions used herein should be understood to control over dictionary definitions or definitions in documents incorporated by reference.

[0111] All references, patents, and patent applications herein are incorporated by reference with respect to the subject matter in which they are cited, in some cases including the entire document.

[0112] It should be understood that for any method described herein comprising more than one step, the order of the steps is not necessarily limited to the order described in the examples.

[0113] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0114] 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.

[0115] 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, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0116] MMP14

[0117] Molecular targeted therapy has become a hot topic in tumor treatment and has achieved effective results in clinical trials, such as targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF). However, the main cause of death in cancer patients is tumor metastasis to important organs, causing organ or system dysfunction in patients. Tumor metastasis depends on the interaction between tumor cells and their microenvironment, especially the surrounding extracellular matrix (ECM). This interaction contributes to the development of anti-tumor drug response and drug resistance, and is a major challenge in current cancer research. The balance between the synthesis and degradation of ECM components is a key regulator of tumor growth and metastasis. Studies have reported that MMP14 is a member of the matrix metalloproteinase family, which is mainly responsible for degrading extracellular matrix (ECM) components and is related to the regulation of tumor cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) and angiogenesis, making MMP14 a potential target for cancer treatment. MMP14 is widely expressed in tumor cells, cancer-associated fibroblasts, tumor-associated macrophages, and endothelial cells. In addition, MMP14 is mainly involved in the degradation of extracellular matrix (ECM), the processing of growth factors and receptors, the regulation of cell migration, invasion, proliferation and epithelial-mesenchymal transition (EMT), and the regulation of angiogenesis.

[0118] peptide G cyclic peptide

[0119] The cyclic peptide peptide G is a ligand of MMP14. It is a selective MMP14 peptide inhibitor obtained by Suojanen et al. using a phage display library. It consists of 12 amino acids and has the sequence of GACFSIAHECGA. Due to its specific binding to the receptor MMP14, peptide G does not affect the activity of many other MMPs. In the fluorescent peptide cleavage experiment, it showed an IC50 value of 150μM. Studies have shown that peptide G does not affect the adhesion, survival or proliferation of cancer cells, but can effectively inhibit their invasion and migration capabilities. In in vitro experiments, peptide G inhibits the activity of membrane-anchored MMP14 and soluble MMP14 to exert its effects. In vivo, the peptide G reduces the growth of tongue cancer and effectively prolongs the survival time of mice. In addition, studies have shown that peptide G specifically binds to MMP14 through a specific disulfide bond structure. When this special structure is changed, such as converting cysteine ​​in the sequence to serine, peptide G completely loses its activity and its affinity to MMP14 is significantly reduced. Therefore, the above studies show that the disulfide bond structure formed by a specific amino acid sequence is an important factor in the binding of MMP14.

[0120] The peptide G-based CAR of the present invention is a receptor / ligand-based CAR molecule, which has advantages such as moderate affinity and minimal immunogenicity compared to the CAR designed based on the single-chain antibody scFv sequence.

[0121] Chimeric Antigen Receptor (CAR) of the Present Invention

[0122] Chimeric antigen receptor (CAR) is composed of an extracellular antigen recognition region, a transmembrane region, and an intracellular co-stimulatory signal region.

[0123] The design of CAR has gone through the following process: The first generation of CAR has only one intracellular signal component CD3ζ or FcγRI molecule. Since there is only one activation domain in the cell, it can only cause short-term T cell proliferation and less cytokine secretion, but cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical efficacy. The second generation of CAR introduces a co-stimulatory molecule such as CD28, 4-1BB, OX40, and ICOS based on the original structure. Compared with the first generation of CAR, the function is greatly improved, further enhancing the persistence of CAR-T cells and the ability to kill tumor cells. On the basis of the second generation of CAR, some new immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and developed into the third and fourth generation of CAR.

[0124] The extracellular segment of CAR can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity and secretion of cytokines, thereby eliminating target cells. First, the patient's autologous cells (or allogeneic donors) are separated, activated and genetically modified to produce CAR immune cells, and then injected into the same patient. This method has a very low probability of graft-versus-host disease, and the antigen is recognized by immune cells in a non-MHC restricted manner.

[0125] CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. This high response rate cannot be achieved by any previous treatment method and has triggered a wave of clinical research around the world.

[0126] Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.

[0127] The extracellular domain includes a target-specific binding element. The extracellular domain can be an ScFv of an antibody based on antigen-antibody specific binding, or a natural sequence or a derivative thereof based on ligand-receptor specific binding. In the present invention, the extracellular binding domain peptide G of the chimeric antigen receptor can specifically bind to the MMP14 protein.

[0128] The intracellular domain includes a co-stimulatory signaling region and a zeta chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain that includes co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0129] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0130] The CAR of the present invention, when expressed in T cells, can be based on antigen binding specificity for antigen recognition. When it is combined with its associated antigen, it affects tumor cells, causing tumor cells not to grow, being prompted to die or otherwise affected, and causing the patient's tumor load to shrink or eliminate. Antigen binding domains are preferably fused with one or more intracellular domains from costimulatory molecules and ζ chains. Preferably, antigen binding domains are fused with CD8 hinge region, CD8 transmembrane region, 4-1BB costimulatory domains, and CD3ζ signaling domains combined intracellular domains.

[0131] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0132] The intracellular domain in the CAR of the present invention includes a 4-1BB co-stimulatory domain and a CD3ζ signaling domain.

[0133] In one embodiment of the present invention, the CAR is a CAR that can specifically target MMP14.

[0134] It should be understood that the CAR of the present invention is mainly based on its extracellular domain (e.g., Peptide G peptide segment) with binding specificity to play a binding and killing effect. Elements connected to the extracellular domain (such as signal peptides, hinge regions, transmembrane regions, intracellular co-stimulatory signaling regions, and ζ chains) have been disclosed in the prior art and can be routinely selected by those skilled in the art. Therefore, in addition to the structure verified by the embodiment, the present invention also includes chimeric antigen receptors obtained by arbitrarily replacing these elements, which also have good binding force and killing effect.

[0135] Chimeric Antigen Receptor Immune Cells (CAR-Immune Cells)

[0136] In the present invention, a chimeric antigen receptor immune cell is provided, which comprises the chimeric antigen receptor of the present invention having a specific targeting peptide G receptor (preferably MMP14).

[0137] The chimeric antigen receptor immune cell of the present invention can be a CAR-T cell, or a CAR-NK cell, or a CAR-macrophage. Preferably, the chimeric antigen receptor immune cell of the present invention is a CAR-T cell.

[0138] 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 fifth aspect of the present invention.

[0139] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action process of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor markers, once the CAR gene construction targeting a certain tumor marker is completed, it can be widely used; (3) CAR can use both tumor protein markers and glycolipid non-protein markers, expanding the target range of tumor markers; (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.

[0140] 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 fifth aspect of the present invention. The CAR-NK cells of the present invention can be used for tumors with high expression of MMP14.

[0141] 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.

[0142] Compared with 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.

[0143] Carrier

[0144] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0145] The present invention also provides vectors comprising nucleic acid molecules of the present invention. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0146] In brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0147] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. 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. In another embodiment, the present invention provides a gene therapy vector.

[0148] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid 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, probe generation vectors and sequencing vectors.

[0149] 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, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0150] Many virus-based systems have been developed for transferring genes into 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 some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0151] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to start transcription.

[0152] An example of a suitable promoter is the immediate early 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 (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, 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 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 operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0153] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0154] Reporter genes are used to identify cells of potential transfection and to evaluate the functionality of regulatory sequences. Typically, reporter genes are genes that are not present in or expressed by receptor organisms or tissues, and that encode polypeptides whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into receptor cells, the expression of reporter genes is measured at the appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). In one embodiment of the invention, reporter genes are genes encoding mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared or commercially available using known techniques. Typically, constructs with a minimum of 5 flanking regions showing the highest level of reporter gene expression are identified as promoters. Such promoter regions may be connected to reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0155] 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, such as mammalian, bacterial, 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.

[0156] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, 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, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0157] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used 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.

[0158] 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).

[0159] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations 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. Nucleic acids associated with lipids 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 lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty 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.

[0160] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0161] preparation

[0162] The present invention provides a preparation containing the chimeric antigen receptor CAR described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention or the engineered immune cell described in the fifth 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.

[0163] 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.

[0164] Therapeutic applications

[0165] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target markers of tumor cells (preferably MMP14), synergistically activate T cells, and induce immune cell immune responses, thereby significantly improving their efficiency in killing tumor cells.

[0166] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-cell of the present invention to the mammal.

[0167] 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 has a very low probability of graft-versus-host disease, and antigens are recognized by T cells 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.

[0168] In one embodiment, the CAR-T cells of the present invention can undergo a robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, anti-MMP14 CAR-T cells cause a specific immune response to MMP14-positive cells.

[0169] Although the data disclosed herein specifically provide lentiviral vectors comprising a peptide G peptide segment, hinge and transmembrane regions, and 4-1BB and CD3ζ signaling domains, the present invention should be construed to include any number of variations to each of the components of the construct.

[0170] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors.Cancers may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors.The types of cancers treated with the CAR of the present invention include, but are not limited to, cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas.Also include adult tumors / cancers and childhood tumors / cancers.

[0171] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelo-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0172] Solid tumors are abnormal masses of tissue that do not usually contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, cancers, and lymphomas). Examples of solid tumors such as sarcomas and cancers include fibrosarcomas, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, liver cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian cancer and esophageal cancer, glioblastoma, lung cancer, melanoma, and other solid tumors with high expression of MMP14 (if there are other known cancers that highly express MMP14, they can be further supplemented as a basis for future revisions).

[0173] The examples of the present invention confirm that peptide G CAR-T has a killing effect on a variety of tumor cell lines expressing MMP14, and has excellent specificity. The cell killing experiment overexpressing MMP14 suggests that the killing effect of peptide G CAR-T is induced by binding to MMP14. Therefore, the CAR-T of the present invention can kill a wide range of target cells expressing MMP14, such as tumor cells that highly express MMP14.

[0174] The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0175] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0176] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0177] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0178] The present invention provides a method for treating a tumor, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof.

[0179] The CAR-modified 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 groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0180] 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-although the appropriate dosage can be determined by clinical trials.

[0181] When an "effective amount", "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, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 109 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6 The T cell composition can also be administered at these doses multiple times. The cells can be administered by using well-known injection techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0182] 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 (iv) 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 iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0183] 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 modalities (e.g., before, simultaneously or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for specific tumor patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the 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, 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 prior to or after surgery.

[0184] 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 6 to 1×10 10 The CAR-T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0185] The main advantages of the present invention include:

[0186] 1) Target specificity: MMP14 is lowly expressed on the cell membrane of normal cells, but significantly highly expressed on tumors and related fibroblasts. Hypoxia can stimulate the expression of MMP14 in tumor cells. Therefore, this CAR can specifically kill malignant cells with high expression of MMP14, while having little killing effect on normal cells.

[0187] 2) The present invention utilizes the binding mode of ligand and receptor, rather than the traditional scFv. The target MMP14 is highly conservative, and safety tests in animals, especially primates, can better reflect its safety in humans.

[0188] 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 for which specific conditions are not specified in the following examples 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 according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0189] Table 1 Summary of amino acid sequences involved in the present invention

[0190]

[0191]

[0192] Table 2 Cell lines

[0193]

[0194]

[0195] Example 1: Preparation of peptide G-CAR, MT1-AF7p-CAR, NTIMP2 D -CAR vector and CD19-CAR control vector

[0196] Peptide G, MT1-AF7p and NTIMP2 D The sequence was translated into a nucleotide sequence. Based on the nucleotide sequence of peptide G, human CD8 signal peptide, human CD8α hinge region, human CD8 transmembrane region, human 4-1BB intracellular region and human CD3ζ intracellular region gene sequence information, the sequence was designed and sent to Youkang Biotechnology Company to synthesize pTomo-peptide G-CAR, pTomo-MT1-AF7p-CAR and pTomo-NTIMP2 D -CAR recombinant expression plasmid. The recombinant plasmid was sequenced, the sequencing results were compared to confirm whether the plasmid was correct, and the enzyme digestion results were identified. The sequencing and enzyme digestion identification results both showed that the CAR coding sequence was correctly inserted into the predetermined position of the plasmid.

[0197] All plasmids were extracted using QIAGEN's endotoxin-free extraction kit. After purification, the plasmids were transfected into HEK293T cells using Beyotime lipo8000 transfection reagent for lentiviral packaging.

[0198] Example 2: Virus packaging

[0199] Culture HEK-293T cells in a 15 cm dish for virus packaging. When the confluence of HEK-293T cells is about 80%-90%, transfection is performed and 2 ml of Opti-MEM TM Plasmid mixture dissolved in culture medium (core plasmid 20ug, pCMVΔR8.9 10ug, PMD2.G 4ug); 2ml Opti-MEM TM Culture medium and 68ul of lipo8000 transfection reagent. After standing at room temperature for 5 minutes, the plasmid complex was added to the liposome complex and stood at room temperature for 20 minutes. The above mixture was added dropwise to the HEK-293T cell culture dish and incubated at 37°C for 6 hours before removing the culture medium. Re-add preheated complete culture medium. Collect the 48-hour and 72-hour viral supernatants and centrifuge at 3000rpm at 4°C for 15 minutes, then filter with a 0.45um filter membrane and centrifuge at 25000rpm at 4°C for 2.5 hours to concentrate the virus. The concentrated virus was dissolved with 30ul of virus lysis solution overnight, packaged and frozen in a -80°C refrigerator, and the virus titer was detected by qPCR.

[0200] Example 3: CAR-T cell preparation

[0201] Monocytes were separated from human peripheral blood using Ficoll separation solution, and CD3+T cells were separated and purified by RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies) kit, and then aliquoted and frozen in liquid nitrogen. Before preparing CAR-T cells, T cells were revived and activated with CD3 / CD28 magnetic beads (Life technology), and then 200U / ml IL-2 (PeproTech) was added. After stimulating culture for 48 hours, virus infection was performed. In the presence of lentiboost, lentivirus was used to infect T cells at MOI=20 to prepare CAR-T cells. The culture medium was replaced one day after infection.

[0202] Example 4: Detection of the positive rate of infected CAR-T cells by flow cytometry

[0203] CAR-T cells and NTD cells (control group) infected with the virus for 72 hours were collected by centrifugation, washed once with PBS and the supernatant was discarded. The cells were resuspended in PBS containing 2% FBS, and the positive rate was detected by flow cytometry.

[0204] The results of transfection efficiency are as follows Figure 2 shown.

[0205] The results showed that after the CAR-T2A-mKate2 fusion protein expressed by CAR-T cells was cut, the mKate2 protein formed showed red fluorescence in the cell. Flow cytometry for red fluorescence detection showed that the positive expression rate of CD19-CAR-T was about 64.5%, and the positive expression rate of peptide G-CAR-T was about 59.5%, and the positive rate levels of the two were comparable.

[0206] Example 5: Detection of the expression of MMP14 in each target cell

[0207] (1) Flow cytometry: Collect 1 million cells, fix the cells with 4% PFA at room temperature for 15 minutes, and wash them by centrifugation in 1X PBS; permeabilize the cells with 100% methanol on ice for 15 minutes, and wash them by centrifugation in 1X PBS; resuspend the cells with 100 μl of diluted primary antibody (1:300), incubate at room temperature for 1 hour; and wash them by centrifugation in 1X PBS. Discard the supernatant. Repeat the operation. Resuspend the cells with 100 μl of diluted fluorescent substance-conjugated secondary antibody (Cy3-anti-rabbit), incubate at room temperature in the dark for 30 minutes, and wash them by centrifugation in 1X PBS. Discard the supernatant. Repeat the operation. Resuspend the cells with 300 μl of 1X PBS and analyze them by flow cytometer.

[0208] (2) Cell immunofluorescence: Target cells were plated on a 24-well plate. After 24 hours, the cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes, washed three times with PBST for 5 minutes each time, blocked with 10% goat serum at room temperature for 1 hour, and incubated with an antibody that specifically recognizes MMP14 at four degrees overnight. The next day, the cells were washed three times with PBST for 5 minutes each time. The cells were incubated with a secondary antibody that specifically recognizes the primary antibody labeled with CY3 for 1 hour at room temperature. After washing three times with PBS, the nuclei were stained with DAPI. Confocal microscopy imaging was performed.

[0209] The results of MMP14 expression detection in each cell line are as follows Figure 5 As shown. By flow cytometry ( Figure 5 A) Immunofluorescence Figure 5 B) The expression of MMP14 in target cells was detected, and the results showed that SMMC7721 and HepG2 cells highly expressed MMP14, while MHCC97H cells lowly expressed MMP14.

[0210] Example 6: Construction of target cells carrying luciferase

[0211] The luciferase fragment was amplified by PCR from the pGL3-luciferase plasmid, and then connected into the pTomo vector through XbaI and BamHI to construct the pTomo-lucferase plasmid. The IRES and puromycin fragments were amplified from the pTomo and PLkO.1 plasmids, respectively. The pTomo-luciferase-IRES-Puro plasmid was successfully constructed by connecting the three fragments. Lentivirus packaging and titer determination were as described above. Human embryonic kidney HEK-293T cells, human liver cancer cell lines HepG2, MHCC97H and SMMC7721, human pancreatic cancer cell lines BXPC3 and Mia-paca2, human breast cancer cell lines MDA-MB-231 and MDA-MB-468, human lung cancer cell lines H1299 and A549, human prostate cancer cell line PC3, and human glioma cell line T98G were infected at an MOI of 10. After 48 hours, HEK293T-lucif eras, HepG2-luciferase, MHCC97H-luciferase, SMMC7721-luciferase, BXPC3-luciferase, Mia-paca2-luciferase, MDA-M B-231-luciferase, MDA-MB-468-luciferase, H1299-luciferase, A549-luciferase, PC3-luciferase, T98G-luciferase cells.

[0212] Example 7: CAR-T kills tumor cells

[0213] After digestion and counting of HepG2-luciferase, SMMC7721-luciferase, BXPC3-luciferase, Mia-paca2-luciferase, MDA-MB-231-luciferase, MDA-MB-468-luciferase, H1299-luciferase, A549-luciferase, PC3-luciferase, and T98G-luciferase cells, the cell density was adjusted to 2×10 4 / ml. Take 100ul of cells and inoculate them in a 96-well plate. Adjust the cell density of CAR-T and NTD cells to 1×10 5 / ml, inoculated into a black 96-well plate at an E:T ratio of 5:1, with 100ul inoculated into each well. The target cells and T cells were mixed and incubated in an incubator for 24 hours.

[0214] HEK293T-luciferas, HepG2-luciferase, MHCC97H-luciferase and SMMC7721-luciferase cells were digested and counted and the cell density was adjusted to 2×10 4 / ml. Take 100ul of cells and inoculate them in a 96-well plate. Adjust the cell density of CAR-T and NTD cells to 1×10 5 / ml, inoculated into black 96-well plates according to different effector-target ratios, 100ul per well. Mix the target cells and T cells and incubate in an incubator for 24 hours.

[0215] Cell killing was detected using a Promega fluorescence detection kit. First, cells were treated with 20ul 1*PLB lysis buffer for 20 minutes, and 100ul substrate was added to each well and then detected immediately using a BioTek microplate reader.

[0216] Cytotoxic killing cell % = (1-fluorescence value of target cells with effector cells / fluorescence value of target cells without effector cells) × 100%

[0217] peptide G-CAR T, MT1-AF7p-CAR T and NTIMP2 D -CAR T cell killing results on liver cancer cell lines Figure 3 A. The results of peptide G-CAR T's killing effect on pancreatic cancer, breast cancer, lung cancer, prostate cancer and glioma cells are shown in Figure 3 As shown in BF, the results of cytokine IFN-γ release levels are as follows Figure 4 As shown in AF. The results showed that peptideG-CAR-T had a good killing effect on multiple tumor cell lines.

[0218] The gradient killing of target cells by peptide G-CAR-T is shown in Figure 6 As shown in AC, compared with the NTD group and CD19-CAR-T group, the killing effect of peptide G CAR-T cells on SMMC7721 and HepG2 liver cancer cells with high expression of MMP14 gradually increased with the increase of effector-target ratio (E:T).

[0219] Since peptide G-CAR T can effectively kill target cells with high expression of MMP14, subsequent experiments will be conducted with peptide G-CAR T, and CD19-CAR T will be used as a negative control.

[0220] Example 8: Cytokine Release

[0221] In this example, the release of cytokines when the CAR-T cells of the present invention were co-incubated with target cells was detected. The supernatant of the co-incubated cells in the cell killing experiment was used for detection.

[0222] The method is as follows: IFN gamma Human ELISA Kit (life technology) was used to detect IFN-γ in the supernatant of cells co-cultured with CAR-T cells and human liver cancer cell lines (HepG2-luciferase, MHCC97H-luciferase, SMMC7721-luciferase, SK-Hep1-luciferase), human pancreatic cancer cell lines BXPC3, Mia-paca2, breast cancer cell lines (MDA-MB-231-luciferase, MDA-MB-468-luciferase), lung cancer cell lines (H1299-luciferase, A549-luciferase), prostate cancer cell line PC3-luciferase and glioma cell line T98G-luciferase cells (E:T ratio of 5:1).

[0223] Dissolve the standard in Standard Dilution Buffer and perform gradient dilution to obtain 1000pg / ml, 500pg / ml, 250pg / ml, 125pg / ml, 62.5pg / ml, 31.2pg / ml, 15.6pg / ml, and 0pg / ml of standard.

[0224] Add 50ul Incubation buffer, 50ul test sample, and 50ul IFN-γbiotin conjugated solution to each well, mix well, and let stand at room temperature for 90 minutes.

[0225] Then follow these steps in order:

[0226] (1) Wash the wells 4 times with 1*Wash Buffer, each time for 1 minute.

[0227] (2) Add 100 μl of 1*Streptavidin-HRP solution to each well and let stand at room temperature for 45 minutes.

[0228] (3) Wash the wells 4 times with 1*Wash Buffer, each time for 1 minute.

[0229] (4) Add 100ul of stabilized chromogen and let stand at room temperature for 30 minutes.

[0230] (5) Add 100 μl of Stop solution to each well and mix well.

[0231] (6) Detect the absorbance at 450 nm.

[0232] The detection of IFN-γ and TNF-α release is as follows Figure 4 AF and Figure 6 As shown in DE, compared with the NTD group and CD19-CAR-T group, the release of IFN-γ and TNF-α in the co-culture supernatant of the peptide G CAR-T group was significantly increased. To simplify the notation, ** (p < 0.01) in the figure represents that the peptide G CAR-T group has a statistically significant difference compared with the NTD group or CD19-CAR-T group.

[0233] Example 9: Effect of overexpression of MMP14 on the killing effect of peptide G CAR-T

[0234] According to the CDS sequence of MMP14, MMP14 overexpression plasmid (EX-Z7562-Lv105, ORFlentiviral expression clone) was purchased from Yijin Biotechnology and transiently transfected with MMP14 overexpression plasmid. One day before transfection, 500,000 target cells were inoculated into a six-well plate for culture. Before transfection, each well of the six-well plate was replaced with 2 ml of fresh culture medium (containing serum but no antibiotics); take two clean sterile centrifuge tubes and add 125 μl Then add 2.5 μg of plasmid DNA to one tube and mix it by blowing gently with a gun; add 5 μl of Lipo8000 to the other tube. TM After standing at room temperature for 5 minutes, add the culture solution containing DNA to the solution containing Lipo8000. TM In the culture medium of the transfection reagent, gently invert the centrifuge tube or gently blow with a gun to mix, let it stand at room temperature for 5 minutes, then add it to the 6-well plate and mix it. After 48 hours, detect the overexpression efficiency and perform peptide G CAR-T killing test.

[0235] The cell density of different liver cancer cell lines MHCC97H-Vector and MHCC97H-MMP14 was adjusted to 2×10 4 / ml. Take 100ul of cell suspension and inoculate it in a 96-well plate. Adjust the cell density of CAR-T / NTD cells to 1×10 5 , inoculated into a black 96-well plate at an E:T ratio of 5:1, with 100ul inoculated into each well. The target cells and T cells were mixed and incubated in an incubator for 24 hours.

[0236] The killing results of peptide G CAR-T on the liver cancer cell line MHCC97H after overexpression of MMP14 are shown in Figure 7 shown. Figure 7 A: qPCR detection of MMP14 mRNA level. Figure 7 B: Immunofluorescence detection of the expression level of MMP14 on the cell membrane. Figure 7 C shows the killing effect of peptide G CAR-T on the liver cancer cell line MHCC97H after overexpression of MMP14. Figure 7 DE is the release of IFN-γ and TNF-α after peptide GCAR-T kills the liver cancer cell line MHCC97H overexpressing MMP14. The results showed that compared with the control group MHCC97H-Vector, the killing effect of peptide G CAR-T cells on MHCC97H-MMP14 cells was significantly enhanced, and the release of IFN-γ and TNF-α was significantly increased.

[0237] Example 10: Peptide G CAR-T has no killing effect on non-tumor cells

[0238] HEK-293T cells are a cell line derived from normal human embryonic kidney. They were inoculated into black 96-well plates at effector-target ratios of 1:1, 2:1, 4:1, and 8:1. Peptide G CAR-T cells were co-incubated with HEK-293T-luciferase cells, and the killing of HEK-293T cells by peptide G CAR-T was detected by changes in fluorescence values.

[0239] The results are as follows Figure 8 shown. Figure 8 A: Cell immunofluorescence detection of MMP14 expression. Figure 8 B is the gradient killing effect of peptide G CAR on HEK-293T. The results suggest that peptide G CAR-T has no obvious killing effect on HEK-293T cells.

[0240] 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 chimeric antigen receptor (CAR), characterized in that The CAR contains an extracellular binding domain, and the extracellular binding domain can specifically bind to MMP14 in a ligand receptor manner.

2. The chimeric antigen receptor according to claim 1, wherein The extracellular binding domain includes a Peptide G peptide segment, and the Peptide G peptide segment has an amino acid sequence as shown in SEQ ID NO:

1.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that The structure of the CAR is shown in Formula I below: L-EB-H-TM-C-CD3ζ-RP(I) In the formula, Each "-" is independently a connecting peptide or a peptide bond; L is none or a signal peptide sequence; EB is the extracellular binding domain; H is no or hinge region; TM is the transmembrane domain; C is no or co-stimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; RP is none or reporter protein.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the chimeric antigen receptor according to claim 1.

5. A carrier, characterized in that The vector contains the nucleic acid molecule as claimed in claim 4.

6. A host cell, characterized in that The host cell contains the vector as described in claim 5 or the chromosome in which the exogenous nucleic acid molecule as described in claim 4 is integrated or expresses the CAR as described in claim 1.

7. An engineered immune cell, characterized in that: The immune cell contains the vector as described in claim 5 or the chromosome in which the exogenous nucleic acid molecule as described in claim 4 is integrated or expresses the CAR as described in claim 1.

8. A method for preparing the engineered immune cell according to claim 7, characterized in that: The following steps are involved: The nucleic acid molecule according to claim 4 or the vector according to claim 5 is transduced into immune cells to obtain the engineered immune cells.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the CAR as described in claim 1, the nucleic acid molecule as described in claim 4, the vector as described in claim 5, the host cell as described in claim 6, and / or the engineered immune cell as described in claim 7, and a pharmaceutically acceptable carrier, diluent or excipient.

10. A use of the CAR according to claim 1, the nucleic acid molecule according to claim 4, the vector according to claim 5, or the host cell according to claim 6, and / or the engineered immune cell according to claim 7, characterized in that: Used for preparing preparations for preventing and / or treating diseases associated with high expression of MMP14.

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