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WO2013185552A1 - Récepteur antigénique chimérique indépendant à double signal et son utilisation - Google Patents

Récepteur antigénique chimérique indépendant à double signal et son utilisation Download PDF

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Publication number
WO2013185552A1
WO2013185552A1 PCT/CN2013/076774 CN2013076774W WO2013185552A1 WO 2013185552 A1 WO2013185552 A1 WO 2013185552A1 CN 2013076774 W CN2013076774 W CN 2013076774W WO 2013185552 A1 WO2013185552 A1 WO 2013185552A1
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chimeric antigen
cells
cell
cancer
antigen receptor
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PCT/CN2013/076774
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English (en)
Chinese (zh)
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钱其军
金华君
丁娜
俞德超
李林芳
吴孟超
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中国人民解放军第二军医大学东方肝胆外科医院
上海白泽生物科技有限公司
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Publication of WO2013185552A1 publication Critical patent/WO2013185552A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • the present invention relates to a chimeric antigen receptor, in particular a dual signal independent chimeric antigen receptor, the present invention also relates to an immunoreactive cell expressing said chimeric antigen receptor, and said immune response cell for use in the preparation of a therapeutic Use in drugs for malignant tumors and viral infectious diseases. Background technique
  • Adoptive cell therapy is a method of returning treated autologous or allogeneic immune cells (mainly autologous cells) to a tumor patient to enhance the patient's immune function and achieve therapeutic goals.
  • Current tumor ACT progresses rapidly, using tumor infiltrating lymphocytes
  • TIL tumor-infiltrating lymphocyte
  • the TCR-CD3 complex binds to the antigen peptide-MHC molecule and provides the first signal of T cell activation, which determines the kill specificity of T cells; the costimulatory molecule on the surface of T cells (such as CD28) binds to the corresponding ligand (such as B7).
  • the costimulatory molecule on the surface of T cells such as CD28
  • binds to the corresponding ligand such as B7.
  • the lack of or the decreased expression of the first signal stimulus source (such as MHC molecule) and the second signal ligand (such as B7) in tumor cells cannot effectively provide T cell activation-related signals, thereby activating T cell immune response. Therefore, it is necessary to genetically engineer T cells.
  • T cell receptor transgenic TCR
  • CAR chimeric antigen receptors
  • the TCR modification has a relatively large restriction.
  • the transgenic TCR chain may be mismatched with the patient's endogenous TCR chain, resulting in a decrease in the TCR density of tumor-reactive T cells on the surface of the T cell;
  • TCR recognizes the antigen presented by the MHC molecule, However, different patients have different MHC molecules, so it is necessary to isolate TCR specific for all MHC haplotypes, and the operability is low; 3 most TCRs cannot recognize carbohydrates or glycolipid antigens, and the antigen selection range is narrow; 4 The first signal related to T cell activation is provided, and the second signal is not provided, and the therapeutic effect is insufficient. Thus, chimeric antigen receptor CAR receptors are more favored.
  • Chimeric antigen receptor CAR consists of a scFv single-chain antibody (from the amino acid sequence of the VL region of the antibody and The amino acid sequence of the VH region is linked by Linker, and is constructed by a hinge structure linked to a transmembrane and intracellular signal structure derived from a TCR complex or an IgE high affinity receptor. T cells expressing CAR can react with antigens via a non-MHC restricted pathway.
  • CAR is not limited to protein antigens compared to protein antigens that can only be targeted by conventional TCRs, but also includes sugars and glycolipids, TAA, which are not as susceptible to mutations as protein antigens (Curr Opin Immunol 2009; 21 : 215-23; Blood 2010; 116: 1035-44; Cancer Res 2011; 71: 3175-81; J Cancer 2011; 2: 378-82). Since 1989, Eshhar and his colleagues first proposed the concept of CAR, which has undergone three different stages of development. The first generation of CAR receptors, including scFv fragments that specifically recognize tumor antigens, and intracellular activation signals by IT3 of CD3 or FcsRIy
  • the immunoreceptor tyrosine-based activation motifs are transmitted by signal chains.
  • the first-generation CAR receptor lacks the costimulatory signal of T cells, which leads to T cells only exerting transient effects, short time in the body and less secretion of cytokines.
  • the second generation of CAR receptors adds an intracellular domain of a costimulatory signaling molecule to the first generation of CAR, providing two signals for T cell activation, including CD28, CD134/OX40, CD137/4-1BB, Lymphocyte-specific protein tyrosine kinase (LCK), inducible T-cell co-stimulator (ICOS) and
  • the DNAX-activation protein 10 (DAP10) and other domains enhance the proliferation of T cells and the secretion of cytokines, and increase IL-2, IFN- ⁇ and GM-CSF, thereby breaking the immunosuppression of the tumor microenvironment and prolonging AICD. (activate induced cell death, AICD).
  • the third-generation CAR receptor based on the second-generation CAR, adds an intracellular domain of another co-stimulatory signaling molecule, such as recombining a second-level co-stimulation between the co-stimulatory structure CD28 and the ITAM signal chain. Stimulating molecules such as 4-1BB produce a triple-signal CAR receptor, and third-generation CAR receptor-modified T cells have better effector function and survival time in vivo.
  • the first generation of CAR only provides the first signal of T cell activation
  • the second and third generation of CAR combines the two signals required for T cell activation
  • the second signal CD28 or / and The 4-1BB intracellular signal region is directly linked to the CD3z molecule, thereby bypassing the tumor cell.
  • the second signal such as B7, lacks the barrier that T cells cannot activate. The combination of the first signal and the second signal greatly improves the pair. T cell activation, proliferation and killing ability, so that its efficacy is greatly increased.
  • the present invention provides a dual signal independent chimeric antigen receptor, an immunoreactive cell expressing the chimeric antigen receptor, and the use of the immune response cell. specifically,
  • a first aspect of the invention relates to a dual-signal chimeric antigen receptor (dsCAR) consisting of two independent chimeric antigen receptors, each of which transmits two signals
  • Antigen receptor 1 is comprised of one or several (eg, two) chimeric antigens comprising a ligand capable of binding a tumor-specific antigen or a tumor-associated antigen, a transmembrane region, and an intracellular immunoreceptor tyrosine activation motif.
  • chimeric antigen receptor 2 consists of one or several (eg, two) ligands that bind to membrane receptors that are widely expressed by tumor cells, transmembrane regions, and intracellular domains of intracellular costimulatory signaling molecules. Combined with antigen receptor composition.
  • the two signals refer to a first signal and a second signal of T cell activation.
  • the two independent chimeric antigen receptors are those in which the chimeric antigen receptors that transmit the T cell activation first signal and the second signal are independent of each other, and transmit the first signal and the second signal of T cell activation, respectively.
  • the tumor-specific antigen or tumor-associated antigen is selected from the group consisting of CD19, CD20, CEA, GD 2 (also known as B4GALNT1, beta-l, 4-N-acetyl-galactosaminyl Transferase 1 ) , FR ( Flavin reductase ) , PSMA ( Prostate-specific membrane antigen ) , gpl00 ( PMEL premelanosome protein ) , CA9 ( carbonic anhydrase IX ) , CD171/L1-CAM , IL-13Ra2 , MART-1 (also known as melan -A ), ERBB2, NY-ESO-K, also known as CTAG1B, cancer/testis Antigen IB ) , MAGE ( Melanoma-associated antigen El ) family protein, BAGE ( B melanoma antigen family ) family protein, GAGE ( growth hormone releasing factor releasing factor
  • EGP-2, EGP-40, FBP, GD 3 also known as ST8SIA1, ST8 alpha-N-acetyl-neuraminide alpha- 2,8-sialyltransferase 1
  • PSCA promoting stem cell antigen
  • FSA also known as KIAA1109
  • PSA also known as KLK3 , kallikrein-related peptidase 3
  • HMGA2 fetal acetylcholine receptor
  • LeY also known as FUT3
  • EpCAM also known as FUT3
  • MSLN mesothelin
  • IGFR1 EGFR
  • EGFRvIII IGFR1
  • ERBB3, ERBB4, CA125 also known as MUC16, mucin 16, cell surface associated ), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR, vitamin D (1,25- dihydroxyvitamin D3) receptor), P2-MG (beta-2-microglobulin) and PROGRP ( GRP ga One or several of strin-releasing peptides.
  • the tumor-specific antigen or tumor-associated antigen is MUC1.
  • the membrane receptor widely expressed by the tumor cell is selected from the group consisting of CD19, CD20, MUCl. EGFR, EGFRvIII. ERBB2, ERBB3, ERBB4, VEGFR1. VEGFR2, EpCAM, CD44 and One or several of IGFR.
  • the tumor-specific antigen or tumor-associated antigen is different from a membrane receptor widely expressed by tumor cells.
  • the ligand capable of binding to a membrane receptor widely expressed by a tumor cell is capable of simultaneously binding to two or more of the above membrane receptors, so that the present invention
  • the chimeric antigen receptor is capable of undergoing signal stimulation of a heterogeneous tumor cell population to prolong the effect time of the immune response cell.
  • the ligand capable of binding to a tumor cell to express a membrane receptor broadly refers to a ligand capable of binding to an EGFR family protein (including EGFR, ERBB2, and/or ERBB4) and the EGFR mutant EGFRvIII;
  • the ligand is HERIN.
  • a chimeric antigen receptor according to the first aspect of the invention wherein said intracellular immunoreceptor tyrosine activating motif comprises an immunoreceptor tyrosine activating motif signal chain selected from the group consisting of CD3 and FCSRIY;
  • the intracellular domain of the intracellular costimulatory signal molecule comprises an intracellular domain selected from the group consisting of CD28, CD134/OX40, CD137/4-lBB, LCK, ICOS, DAP10, preferably, the above.
  • the intracellular immunoreceptor tyrosine activation motif comprises
  • the intracellular domain of the intracellular costimulatory signaling molecule comprises the intracellular domain of CD28 and CD137/4-1BB.
  • a chimeric antigen receptor according to the first aspect of the invention wherein said transmembrane region refers to a portion of a membrane protein within a cell membrane, such as CD28, CD8, CD3, CD134, CD137, ICOS, and DAP10 transmembrane regions
  • the transmembrane regions of the two chimeric antigen receptors are different to prevent mismatching.
  • the transmembrane region of the chimeric antigen receptor 1 is a CD8 transmembrane region
  • the transmembrane region of the chimeric antigen receptor 2 is a CD28 transmembrane region.
  • the ligand is a molecule which specifically binds to the tumor-specific antigen or tumor-associated antigen, and a membrane receptor widely expressed by a tumor cell, and may be, for example, a protein.
  • a polypeptide or an antibody the antibody may be, for example, a monoclonal antibody, a single chain antibody, a Fab antibody, or the like, and in one embodiment of the invention, the antibody is a single chain antibody (ScFv); in another embodiment of the present invention
  • the ligand is a polypeptide, and in a specific embodiment, the polypeptide is a HERIN molecule.
  • the ligand is single copy or multiple copies, and the multiple copies are, for example, double copies.
  • a chimeric antigen receptor according to the first aspect of the invention which is co-expressed by a vector or separately expressed by two identical or different vectors. In one embodiment of the invention, it is co-expressed by a vector.
  • a protein precursor processing enzyme recognition sequence such as Furin-2A
  • Furin-2A is ligated between the two chimeric antigen receptors, and when the two chimeric antigen receptors are expressed, they are cleaved into two independent proteins. Transported to the cell membrane separately.
  • the vector of the present invention is a vector known in the art and can be used for protein cloning and expression, for example, a eukaryotic expression plasmid, a recombinant virus; and the eukaryotic expression plasmid can be, for example, pSV2, pRSV, pcDNA3.1, pCI and pVAXl.
  • the transposon plasmid; the recombinant virus may be, for example, a recombinant retrovirus, a recombinant lentivirus, or a recombinant adenovirus.
  • the vector is pcDNA3.1(+).
  • cells are genetically modified by introducing a vector expressing a chimeric antigen receptor into an immunoreactive cell to express a chimeric antigen receptor.
  • the method for introducing a vector into an immunoreactive cell can be a method commonly used in the art, for example, a gene gun method, a transfection method, an electrotransfer method, and a virus transduction method.
  • the chimeric antigen receptor 1 is a ScFv, a CD8 transmembrane region, a CD3 signal chain of MUC1.
  • the chimeric antigen receptor 2 is the amino acid sequence HERIN, the CD28 transmembrane region, the CD28 intracellular region, and the 4-1BB costimulatory peptide encoded by the eighth intron of the human Her2 gene.
  • the amino acid sequence of the chimeric antigen receptor 1 comprises the amino acid sequence of SEQ ID NO: 31, and the amino acid sequence of the chimeric antigen receptor 2 comprises SEQ ID NO: The amino acid sequence shown by 33.
  • the amino acid sequence of the chimeric antigen receptor 1 is the amino acid sequence set forth in SEQ ID NO:31.
  • the amino acid sequence of the chimeric antigen receptor 2 is the amino acid sequence set forth in SEQ ID NO:33.
  • a second aspect of the invention relates to an engineered immunoreactive cell expressing the chimeric antigen receptor of any one of the first aspects of the invention.
  • the immunoreactive cell may be selected, for example, from a T cell, a monocyte, a natural killer cell (NK cell), a neutrophil cell; wherein the T cell can be, for example, For cytotoxic T lymphocytes, KT cells, helper T cells, or inhibitory/regulatory T cells.
  • a third aspect of the invention relates to a kit comprising the immunoreactive cell of any of the second aspects of the invention, and optionally instructions for use.
  • a fourth aspect of the invention relates to the chimeric antigen receptor of any one of the first aspect of the invention, or the immunoreactive cell of any one of the second aspect, for use in the preparation of a prophylactic and/or therapeutic malignancy and viral infectivity Use in medicines for diseases.
  • the malignant tumor can be any malignant tumor, such as lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct Cancer, biliary cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer or prostate cancer.
  • a malignant tumor such as lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct Cancer, biliary cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer or prostate cancer.
  • a fifth aspect of the invention relates to a method of modifying an immunoreactive cell, comprising the step of expressing the chimeric antigen receptor of any one of the first aspects of the invention on an immunoreactive cell.
  • the immune response cell is selected from the group consisting of a T cell, a monocyte, a natural killer cell, and a neutrophil; wherein the T cell can be, for example, a cytotoxic T lymphocyte or an NKT. Cells, helper T cells, or inhibitory/regulatory T cells.
  • a sixth aspect of the invention relates to a method of preventing and/or treating a malignant tumor and a viral infectious disease, the method comprising preventing or treating a subject in need thereof an effective amount of any one of the first aspects of the invention A step of a chimeric antigen receptor or an immune response cell according to any one of the preceding aspects.
  • the malignant tumor can be any malignant tumor, such as lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct Cancer, biliary cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer or prostate cancer.
  • a malignant tumor such as lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct Cancer, biliary cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer or prostate cancer.
  • the virus may be any virus that infects cells, such as HIV (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and Epstein-Barr virus ( Epstein-Barr virus), Papillomavirus, Herpesvirus or cytomegalovirus.
  • HIV HIV
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • Epstein-Barr virus Epstein-Barr virus
  • Papillomavirus Herpesvirus or cytomegalovirus.
  • chimeric antigen receptor in the present invention is an artificially engineered receptor capable of anchoring a specific molecule (such as an antibody) that recognizes a tumor antigen to an immune cell (such as a T cell), so that the immune cell recognizes a tumor antigen or virus. Antigen and cells that kill tumor cells or virus infection.
  • T cell activation-associated signal in the present invention means that two signals required for activation of T cells, that is, a TCR-CD3 complex on the surface of a T cell, bind to an antigen peptide-MHC molecule, and provide a first signal for T cell activation, which is determined. Killing specificity of T cells; co-stimulatory molecules on the surface of T cells (such as CD28) bind to the corresponding ligand (such as B7), providing a second signal of T cell activation, promoting T cell activation, proliferation and survival.
  • TAM immunoreceptor tyrosine activation motif
  • FcaR and FcRy etc.
  • co-stimulating molecule in the present invention means some adhesion molecules on the surface of an immune cell, such as CD28, CD134/OX40. CD137/4-1BB. CD40, etc., activated by binding to its ligand.
  • the second signal of immune cells enhances the proliferative capacity of immune cells and the secretory function of cytokines, prolonging the survival time of activated immune cells.
  • TSA tumor specific antigen
  • TAA tumor-associated antigen
  • single-chain antibody variable region fragment refers to an antibody fragment having the ability to bind antigen by linking the amino acid sequence of the VL region of the antibody and the amino acid sequence of the VH region via Linker.
  • EGFR refers to the human epidermal growth factor receptor, also referred to as ERBB1 or HER1, and its family members include EGFR, ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4).
  • Herin in the present invention refers to a DNA sequence encoding the C-terminal 79 amino acids of Herstatin in the 8th intron of human Her2, which encodes the C-terminus of Herstatin in the 8th intron of human Her2.
  • the dual signal independent means that the chimeric antigen receptor that transmits the first signal of T cell activation and the chimeric antigen receptor of the second signal are independent of each other, respectively bind to the respective ligands, and respectively generate signals after binding. Transfer into the cells.
  • the Linker is a polypeptide fragment that links between different proteins or polypeptides, the purpose of which is to maintain the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide.
  • the polypeptide generally refers to a peptide chain molecule having a length of from 1 to 100 amino acids; a protein generally refers to a peptide chain molecule having a length of more than 100 amino acids.
  • the T cell is taken as an example, and the second generation and the third generation CAR which are the most popular in the world are reconstructed, and the first signal and the second signal are separated from the single CAR to construct a double Signal-independent chimeric antigen receptors (dsCAR).
  • the two CARs identify antigens from two different families of tumor cells, respectively, and transmit two signals related to T cell activation.
  • a CAR transmits a first signal related to T cell activation by binding a single-chain antibody or peptide of a tumor-specific antigen or a tumor-associated antigen to determine T cell killing specificity; another CAR is widely expressed by binding to tumor cells.
  • Single-chain antibodies or peptides of membrane receptors transmit a second signal associated with T cell activation, promoting T cell activation, proliferation, and survival. It is ensured that the T cells modified by dsCAR proliferate in a large amount in the tumor environment in which the first signal and the second signal source coexist, thereby specifically killing the tumor cells; and the normal cells do not simultaneously express the receptors of the two stimulation signals, even the normal cells.
  • Low expression of the first source only mild accidental injury, will not cause a "fall" over-expression of CAR+ T cells, leading to serious consequences; and in some normal tissue environments with only the second source, T The cells do not exert a killing effect, so the safety is greatly improved.
  • the present invention can avoid potential safety problems while maintaining the efficacy of second and third generation CAR.
  • the invention is equally applicable to other immune response cells, such as monocytes, NK cells, neutrophils.
  • the chimeric antigen receptor 2 of the present invention comprises a ligand capable of binding to a membrane receptor widely expressed by tumor cells, and thus is suitable for various malignant tumors, viral infectious diseases, and the same type of malignant cells or
  • the virus-infected cells express different membrane receptors due to heterogeneity, thereby achieving the effect of prolonging the effect time of the immune response cells.
  • Figure 1 Schematic diagram of CAR1 (CARIMUCI) binding to MUC1 and CAR2 (CAR2 EGFR ) binding to EGFR family proteins, and third generation CAR (G3-CAR MU ci) binding to MUC1.
  • CAR1 CARIMUCI
  • CAR2 CAR2 EGFR
  • G3-CAR MU ci third generation CAR
  • SP signal peptide
  • LI - L5 Linker 1 - Linker 5;
  • FIG. 2 Structure of the expression vector for CAR1 C1 CAR2 EGFR (abbreviation) pcDNA3.1-CARl: 2).
  • FIG. 3 Proliferation of Jurkat cells (Jurkat CAR1 , Jurkat CAR2 , Jurkat CAR1CAR2, and Jurkat 3G - CAR ) treated with different treatments after exposure to A431, MCF7, and U-20S.
  • FIG. 4 Secretion of INFry after co-culture with A431, MCF7, and U-20S cells by Jurkat cells (Jurkat CAR1 , Jurkat CAR2 , Jurkat CAR1CAR2, and Jurkat 3G - CAR ) treated with different treatments.
  • Figure 5 Killing effect of differently treated Jurkat cells (Jurkat CAR1 , Jurkat CAR2 , Jurkat CAR1CAR2 and Jurkat 3G - CAR ) on A431, MCF7, U-20S. detailed description
  • amino acid sequence and coding sequence constituting each component of CAR the entire fused amino acid sequence and the coding DNA expression frame are spliced, wherein:
  • amino acid residue sequence of HERI is:
  • CD28 transmembrane and intracellular regions are:
  • CD28 transmembrane and intracellular regions (CD28) is:
  • the amino acid residue sequence of the 4-1BB costimulatory signal domain peptide (41BB) is:
  • the coding sequence of the 4-1BB costimulatory signal domain peptide (41BB) is:
  • the amino acid residue sequence of CD3 is:
  • CD3 The coding sequence of CD3 is:
  • amino acid residue sequence of MUC1 scFv-VH ( VHMUCI ) is:
  • the coding sequence of MUC1 scFv-VH ( VHMUCI ) is: GAGGTCCAGCTGCAGCAGTCAGGAGGAGGCTTGGTGCAACC
  • VLMUCI amino acid residue sequence
  • VLMUCI The coding sequence of MUC1 scFv-VL ( VLMUCI ) is:
  • CAAACTGACTGTCCTAGGATCCGAG SEQ ID NO: 12
  • CD8TM The amino acid residue sequence of the CD8 transmembrane region peptide (CD8TM) is:
  • CD8TM The coding sequence of the CD8 transmembrane region peptide (CD8TM) is:
  • amino acid residue sequence of signal peptide 1 is:
  • the signal peptide 1 coding sequence is:
  • the signal peptide 2 coding sequence is:
  • GGSGSGGSGSGGSGS (SEQ ID NO: 19)
  • Linker2 The amino acid residue sequence of Linker2 is:
  • EPKSCDKTHTCPPCPAPE (SEQ ID NO: 21)
  • Linker2 The coding sequence of Linker2 is:
  • Linker3 The amino acid residue sequence of Linker3 is:
  • Linker3 The coding sequence of Linker3 is:
  • the amino acid residue sequence of Linker 4 is:
  • the Linker 4 coding sequence is:
  • the amino acid residue sequence of Linker 5 is:
  • Linker 5 The coding sequence of Linker 5 is:
  • RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 29)
  • the coding sequence of Furin-2A is: CGTGCTAAACGAGCTCCTGTTAAACAGACTTTGAATTTTGAC CTTCTCAAGTTGGCGGGAGACGTCGAGTCCAACCCTGGGCCC
  • CARIMUCI is composed of signal-like l-VH M uci-Linkerl-VL M uci-Linker2-CD8TM-Linker3-CD3 fusion (see Figure 1), and its amino acid sequence is:
  • CAGGCCCTGCCCCCTCGC (SEQ ID NO: 32)
  • CAR2 EGFR is composed of the signal peptide 2-HERIN-Linker4-CD28-Linker5-41BB fusion (see Figure 1), and its amino acid sequence is:
  • the coding sequence of CAR2 EGFR is:
  • CAR1MUCICAR2 E GFR consists of CAR1MU C1 and CAR2 EGFR linked by Furin-2A.
  • the amino acid sequence is:
  • the coding sequence of CARl MUC1 CAR2 EeFR is:
  • the control G3-CAR MUC1 was composed of the signal peptide l-VH M uci-Linkerl-VL M uci-Linker2-CD28-Linker4-41BB-Linker3-CD3 fusion (see Figure 2), and its amino acid sequence was:
  • G3-CARMUCI The coding sequence of G3-CARMUCI is:
  • DNA coding sequence of ⁇ (SEQ ID NO: 32), DNA coding sequence of CAR2 EGFR (SEQ ID NO: 34), DNA coding sequence of CAR1 MUC1 CAR2 EGFR (SEQ ID NO: 36).
  • G3-CAR MUC1 The DNA coding sequence (SEQ ID NO: 38) was entrusted to Biotech Engineering (Shanghai) Co., Ltd. to synthesize its entire expression cassette and insert the pCDNA3.1 ( + ) vector ( Invitrogen ) EcoRl-Xbal site (see Figure 2). After transformation to E. coli (DH5a), after sequencing, the plasmid was extracted and purified using Qiagen's plasmid purification kit to obtain a high quality plasmid of each recombinant expression vector.
  • Example 2 Genetic modification of T cell lines
  • Example 3 High quality plasmids of each recombinant expression vector constructed and purified in Example 1 were transfected into Jurkat E6.1 (T lymphocyte strain, purchased from American Type Collection, ATCC) using Lipofectamine 2000 (Invitrogen), respectively. Two days later, the transfected Jurkat E6.1 cells were transferred to RPMI 1640 medium with neomycin and the cells were cloned by limiting dilution. After 21 days of screening, Jurkat E6.1 cell line Jurkat CAR1 , Jurkat CAR2 , Jurkat c dish with neomycin resistance and genetic modification by CAR1 MUC1 , CAR2 EGFR , CAR1 MUC1 CAR2 EGFR and G3-CAR M uci were established . AR2 and J U rkat G3 CAR .
  • Example 3 Determination of proliferation of T cell strain after genetic modification
  • Jurkat CAR1 , Jurkat CAR2 , Jurkat CAR1CAR2 and J U rkat G3 CAR and unmodified Jurkat E6.1 cells (5xl0 5 /well, RPMI 1640 medium, containing 20U/ml IL-2) were added to pre-plated radiation Treatment (without disrupting the overall structure, the cells lose the activity of dividing proliferation, see Clin Cancer Res. 2011; 17(7): 1664-73) 6-well plates of A431, MCF7, U-20S (cells) Suspended Jurkat cells were counted on day 3 and day 7, respectively, from ATCC, 5xl0 5 /well.
  • dsCAR-modified T cells can proliferate in a large amount; when the first signal is present, the second signal is weak (such as MUC1 positive) , EGFR weakly positive, representing a small number of tumor cells), modified by dsCAR The cells can also proliferate effectively; when the first signal is weak, the second signal is present (such as MUC1 weak yang, EGFR weak yang, representing normal tissue cells), and dsCAR-modified T cells do not proliferate.
  • Example 4 Determination of IFNy secretion in T cell strain after genetic modification
  • the ⁇ secretion of Jurkat CAR1CAR2 is similar to that of Jurkat CAR1 , which is lower than that of Jurkat G3-CAR .
  • Jurkat CAR1CAR2 does not secrete IFNy, and Jurkat G3 - CAR can still Secreted more IFNy (see Figure 4).
  • Jurkat CAR1 , Jurkat CAR2 , Jurkat CAR1CAR2 and Jurkat G3-CAR and unmodified Jurkat E6.1 cells were plated with A431 at different target ratios (50:1, 25:1, 5:1, 1:1).
  • the method is as follows: The target cells are plated in 96-well plates (5 ⁇ 10 3 /well), medium background, volume correction, spontaneous LDH release from target cells, maximum LDH release from target cells, control cells spontaneous LDH release control wells, treatment group wells (groups) Prepare according to the kit instructions), repeat 3 wells per group, the final volume of each well is the same and not less than ⁇ 250g for 4 min, and incubated at 37 ° C, 5% C02 for at least 4 h. 45 min before centrifugation, lOx lysate was added to the largest release well of the target cells, and an equal amount of lysate was added to the volume-corrected well.
  • Cytotoxicity (%) [( D experimental well - D medium background well) - (D effector cell spontaneous LDH release well - D medium background well) - (D target cell spontaneous LDH release well - D medium background well) ⁇ / [ ( D target cell maximum LDH release hole - D volume correction hole) - (D target cell spontaneous LDH release hole - D medium background hole)] xl00 results show that Jurkat CAR1CAR2 can effectively kill MUC1 and EGFR double positive A431 Tumor cells; the killing effect of MCF7, which is highly positive for MUC1 and weakly positive for EGFR family protein, is similar to Jurkat CAR1 , lower than Jurkat G3-CAR ; U-20S cells with weak positive MUC1 and EGFR positive are not killed (see Figure 5).

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Abstract

L'invention concerne un récepteur antigénique chimérique indépendant à double signal, formé par deux types de CAR indépendants. Un type de CAR sert à transférer un premier signal associé à l'activation des lymphocytes T par combinaison d'un ligand d'un antigène spécifique à une tumeur ou d'un antigène associé à une tumeur, de manière à décider de la spécificité de destruction des lymphocytes T; et l'autre CAR sert à transférer un second signal associé à l'activation des lymphocytes T par combinaison d'un ligand de récepteurs membranaires abondamment exprimés par des cellules tumorales, de manière à favoriser l'activation, la prolifération et la survie des lymphocytes T. Des cellules à réaction immune exprimant le CAR et l'utilisation des cellules à réaction immune dans la préparation de médicaments, destinés au traitement de maladies tumorales malignes et infectieuses virales, sont également décrites.
PCT/CN2013/076774 2012-06-12 2013-06-05 Récepteur antigénique chimérique indépendant à double signal et son utilisation WO2013185552A1 (fr)

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