WO2019128998A1 - 自表达pd-1抗体并靶向间皮素的嵌合抗原受体修饰t细胞及其用途 - Google Patents

自表达pd-1抗体并靶向间皮素的嵌合抗原受体修饰t细胞及其用途 Download PDF

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WO2019128998A1
WO2019128998A1 PCT/CN2018/123560 CN2018123560W WO2019128998A1 WO 2019128998 A1 WO2019128998 A1 WO 2019128998A1 CN 2018123560 W CN2018123560 W CN 2018123560W WO 2019128998 A1 WO2019128998 A1 WO 2019128998A1
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seq
amino acid
antibody
cell
cancer
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French (fr)
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钱其军
金华君
江芏青
刘祥箴
何周
王超
崔连振
李林芳
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上海细胞治疗研究院
上海细胞治疗集团有限公司
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention belongs to genetic engineering and immunology, and relates to a chimeric antigen receptor-modified T cell targeting mesothelin and use thereof.
  • Cancer has become the number one killer of human health.
  • the fast pace of life, huge work pressure, unhealthy eating habits, and poor environment are all accomplices of cancer, making cancer's high incidence and rejuvenation trend more and more obvious.
  • the commonly used treatments are very limited, and it is still necessary to explore a more effective treatment to improve the survival rate and quality of life of cancer patients.
  • chimeric antigen receptor T cell therapy has achieved very good curative effect in malignant hematological tumors, and the complete remission rate of relapsed and refractory B cell leukemia is over 90%.
  • CAR-T cell Novartis's tissue lecleucel chimeric antigen receptor T cell
  • ALL acute lymphoblastic leukemia
  • CAR-T drug Novartis's tissue lecleucel chimeric antigen receptor T cell
  • Yescarta for the treatment of adult patients with certain types of large B-cell lymphoma.
  • the approval of CAR-T drugs has taken CAR-T treatment to a new level.
  • a chimeric antigen receptor is a synthetic receptor that typically comprises an extracellular antigen binding domain, a transmembrane hinge region, and an intracellular signal transduction region.
  • scFv single-chain fragment variable
  • TAA antibody-associated antigen
  • ITAM immunoreceptor tyrosine-based activation Motifs
  • the efficacy of CAR-T cells in the treatment of solid tumors is still insufficient.
  • the main reasons include: 1.
  • the solid tumor is highly heterogeneous, lacking cell surface targets suitable for CAR-T treatment; 2.
  • Solid tumors have a microenvironment that strongly inhibits immunity.
  • Mesothelin is a glycoprotein anchored to the plasma membrane by a phosphatidylinositol region (GPI), which is highly expressed in various tumor tissues and is expressed in a small amount in mesothelial cells of normal pleura, pericardium and peritoneum.
  • the mesothelin gene encodes a 69 kDa precursor protein that is processed to form a 40 kDa membrane-bound protein and a 31 kDa detached fragment called megakaryocyte promoting factor (MPF) and released out of the cell, which we usually call Peelin refers to a fragment anchored on a membrane, which can be divided into three regions, Region I, II, and III, according to its protein structure.
  • PD1 Programmed Death 1, Reprogrammed Cell Death Receptor 1
  • PD-1 and its ligand PD-L1/PD-L2 play important roles in the co-suppression and failure of T cells. Their interaction inhibits the proliferation of T cells regulated by costimulatory molecules and the secretion of cytokines, downregulated.
  • the expression of the anti-apoptotic molecule BCL-xl impairs the function of tumor-specific T cells, leading to the inability of some tumor patients to completely eliminate the tumor. Therefore, inhibition of the immune checkpoint PD-1/PD-L1 pathway is currently a new direction and target for the treatment of lymphoma.
  • PD-1 antibody blocking therapy has a certain degree of advanced or refractory melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, Hodgkin's lymphoma, ovarian cancer, etc. Therapeutic effect, but its high production cost and low response rate limit its clinical use.
  • T cell that self-expresses a PD1 antibody and targets mesothelin.
  • the expression cassette of the PD1 antibody and the expression cassette of the chimeric antigen receptor recognizing mesothelin are integrated into the genome of the T cell.
  • the amino acid sequence of the PD1 antibody is set forth in amino acid residues 21-495 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2.
  • the coding sequence of the PD1 antibody is set forth in the base sequence of positions 61-1488 of SEQ ID NO: 4, or as set forth in SEQ ID NO: 4.
  • the chimeric antigen receptor recognizing mesothelin comprises, optionally from the N-terminus to the C-terminus, an optional signal peptide, an anti-mesothelin III region single chain antibody, a hinge region, a transmembrane Region, intracellular costimulatory signal domain and intracellular signal domain.
  • the signal peptide is a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide; more preferably a CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide As shown in amino acid residues 1-22 of SEQ ID NO: 1.
  • the amino acid sequence of the scFv is as shown in amino acid residues 23-272 of SEQ ID NO: 1.
  • the hinge region is a CD8 hinge region or an IgG4 CH2CH3 hinge region; preferably, the amino acid sequence of the IgG4 CH2CH3 hinge region is amino acid residues 273-500 of SEQ ID NO: Show.
  • the transmembrane region is a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region.
  • a CD8 transmembrane region preferably having an amino acid sequence as shown in amino acid residues 501-528 of SEQ ID NO: 1.
  • the intracellular costimulatory signal domain comprises an intracellular domain of a costimulatory signaling molecule, including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase An intracellular domain of an inducible T cell costimulatory factor (ICOS) and a DNAX activator protein 10; preferably, the intracellular costimulatory signal domain is an intracellular domain of CD28; preferably, the amino acid sequence of the CD28 Shown as amino acid residues 529-569 of SEQ ID NO: 1.
  • the intracellular signal domain is a CD3 sputum intracellular signal domain or an Fc ⁇ RI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain is SEQ. ID NO: 1 shows the amino acid residues at positions 570-681.
  • the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 23-681 of SEQ ID NO: 1, or as shown in SEQ ID NO: 1; preferably, The coding sequence of the chimeric antigen receptor is shown as bases 67 to 2643 of SEQ ID NO: 3, or as shown in SEQ ID NO: 3.
  • the invention also provides a composition
  • a composition comprising: a vector comprising an expression cassette of a chimeric antigen receptor of the invention, the vector for integrating the expression cassette into the genome of a host cell; A vector comprising a coding sequence for a PD1 antibody or a complement thereof, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the amino acid sequence of the PD1 antibody is set forth in amino acid residues 21-495 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2.
  • the coding sequence of the PD1 antibody is set forth in the base sequence of positions 61-1488 of SEQ ID NO: 4, or as set forth in SEQ ID NO: 4.
  • the invention also provides a kit, the kit comprising:
  • a vector comprising an expression cassette of a chimeric antigen receptor of the present invention, which vector is used to integrate the expression cassette into the genome of a host cell;
  • a vector comprising a coding sequence of a PD1 antibody or a complement thereof, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the amino acid sequence of the PD1 antibody is set forth in amino acid residues 21-495 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 2.
  • the coding sequence of the PD1 antibody is set forth in the base sequence of positions 61-1488 of SEQ ID NO: 4, or as set forth in SEQ ID NO: 4.
  • the present invention also provides a pharmaceutical composition comprising the T cell of the present invention or the T cell and the PD1 antibody expressed thereby.
  • the invention also provides the use of a T cell or a T cell as described herein and a PD1 antibody thereof for the preparation of a medicament for the treatment or prevention of a malignancy.
  • the malignant tumor is a cancer whose surface of the cancer cell abnormally expresses mesothelin; preferably, the cancer is selected from the group consisting of: adenocarcinoma, mesothelioma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, Cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, rectal cancer, Hodgkin's lymphoma, pancreatic cancer or prostate cancer; more preferred
  • the cancer is a cancer in which mesothelin and CA125/MUC16 are simultaneously highly expressed.
  • Figure 1 Schematic diagram of the structure of PD-1 antibody and mesothelin chimeric antigen receptor gene.
  • Figure 2A Positive rates of three CAR-T cells: meso3CAR-2A-antiPD1, antiPD1-IRES-meso3CAR, and meso3CAR-antiPD1.
  • Figure 2B Antibody secretion of three CAR-T cells in meso3CAR-2A-antiPD1, antiPD1-IRES-meso3CAR, and meso3CAR-antiPD1.
  • Figure 3A Comparison of positive rates of meso3CAR-antiPD1 T cells constructed under different conditions of CAR and PD1 antibody plasmids.
  • Figure 3B Comparison of antibody secretion of meso3CAR-antiPD1 T cells constructed under different conditions of CAR and PD1 antibody plasmids.
  • Figure 4 Flow cytometry to detect PD1 expression on meso3CAR T and meso3CAR-antiPD1 T cells.
  • Figure 5 Killing of cervical cancer cell line Hela, ovarian cancer cell line SK-OV-3 and gastric cancer cell HGC-27 by meso3CAR-antiPD1 T cells.
  • Figure 6 Changes in the secretion of IL-2, IL-4, IL-6, IL-10, TNF- ⁇ and IFN- ⁇ cytokines by meso3CAR-antiPD1 stimulated by mesothelin antigen.
  • Figure 7 Therapeutic effect of meso3CAR T cells and meso3CAR-antiPD1 T cells on SK-OV-3 ovarian cancer xenograft models.
  • expression cassette refers to the entire element required for expression of a gene, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence.
  • coding sequence is defined herein as a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (eg, CAR, single chain antibody, hinge region, and transmembrane region).
  • the boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of the 5' end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • Fc fragment crystallizable (Fc) of an antibody
  • Fc fragment crystallizable
  • costimulatory molecule refers to a molecule that is present on the surface of an antigen presenting cell and that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal.
  • the proliferation of lymphocytes requires not only the binding of antigens, but also the signals of costimulatory molecules.
  • the costimulatory signal is transmitted to the T cells mainly by binding to the co-stimulatory molecule CD80 on the surface of the antigen presenting cells, and CD86 binds to the CD28 molecule on the surface of the T cell.
  • B cells receive a costimulatory signal that can pass through a common pathogen component such as LPS, or through a complement component, or through activated antigen-specific Th cell surface CD40L.
  • "linker" or hinge is a polypeptide fragment that links between different proteins or polypeptides for the purpose of maintaining the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide.
  • exemplary linkers include linkers containing G and/or S, as well as, for example, Furin 2A peptide.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (KD) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • KD Affinity
  • pharmaceutically acceptable excipient refers to carriers and/or excipients that are compatible pharmacologically and/or physiologically to the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers
  • surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include, but are not limited to, sodium chloride.
  • the term "effective amount” refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition described herein.
  • subject or “patient” may refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog. , monkeys, cattle, horses, etc.
  • CAR chimeric antigen receptor
  • T cells immune cells
  • CAR typically comprises, in turn, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signaling region.
  • a polypeptide that binds to a tumor cell membrane antigen is capable of binding to a membrane antigen that is widely expressed by tumor cells with moderate affinity.
  • the polypeptide that binds to the tumor cell membrane antigen may be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or a Fab fragment.
  • single-chain antibody refers to an antibody fragment which is obtained by hinge-ligating an amino acid sequence of an antibody light chain variable region (VL region) and a heavy chain variable region (VH region), and having antigen-binding ability.
  • the single chain antibody of interest is from an antibody of interest.
  • Antibodies of interest may be human antibodies, including human murine chimeric antibodies and humanized antibodies. The antibody can be secreted or membrane anchored.
  • the present invention constructs a meso3CAR T cell which expresses a PD-1 antibody, and suppresses the PD-1/PD-L1 pathway at the immunological checkpoint while adopting adoptive immune cell therapy. It activates the function of residual tumor-specific T cells, increases the anti-tumor killing effect of endogenous cytotoxic T cells, and promotes the proliferation of CAR-T cells in vivo, thereby improving the efficacy of specific killing of tumors.
  • the Fc fragment of the PD-1 antibody designed by the present invention is a mutant IgG4 Fc, which avoids binding to the ⁇ -2 receptor on the surface of dendritic cells and is recognized and phagocytized by macrophages, so that self-expressing PD-1
  • the antibody's CAR-T cells function without causing an AICD response.
  • the present invention provides a PD-1 antibody comprising an anti-PD-1 single chain antibody and IgG4Fc.
  • the amino acid sequence of the IgG4 Fc is set forth in amino acid residues 267-495 of SEQ ID NO: 2; preferably, the coding sequence thereof is set at nucleotide sequence 797-1485 of SEQ ID NO: Shown.
  • the antibody light chain variable region (VL region) amino acid sequence of the anti-PD-1 single chain antibody is represented by amino acid residues 21 to 131 of SEQ ID NO: 2; The coding sequence thereof is shown in nucleotide sequences 61-393 of SEQ ID NO: 4.
  • the heavy chain variable region (VH region) amino acid sequence of the anti-PD-1 single chain antibody is set forth in amino acid sequence at positions 147-266 of SEQ ID NO: 2; preferably, the encoding The sequence is shown in nucleotide sequence 439-798 of SEQ ID NO:4.
  • the amino acid sequence of the anti-PD-1 single chain antibody is represented by amino acid residues 21 to 266 of SEQ ID NO: 2; preferably, the coding sequence thereof is SEQ ID NO: 4, item 61. -798 base sequence shown.
  • the PD-1 antibody further comprises a light chain signal peptide.
  • the PD-1 antibody comprises, in order from the N-terminus to the C-terminus, a light chain signal peptide, an anti-PD-1 single chain antibody, and an IgG4 Fc.
  • the amino acid sequence of the light chain signal peptide is represented by amino acid residues 1-20 of SEQ ID NO: 2; preferably, the coding sequence of the indicated light chain signal peptide is SEQ ID NO: 4 The base sequence of the 1-60th base is shown.
  • the amino acid sequence of the PD-1 antibody is set forth in amino acid sequence 21-495 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 4.
  • the invention also encompasses a coding sequence for the PD-1 antibody or a complement thereof, the coding sequence comprising at least the coding sequence for an IgG4 Fc described herein or a complement thereof.
  • the coding sequence of the PD-1 antibody comprises the sequence set forth in bases 61-1495 of SEQ ID NO: 4, preferably comprising the sequence set forth in SEQ ID NO:4.
  • the present invention also encompasses a nucleic acid construct comprising the coding sequence of the PD-1 antibody of the present invention or a complement thereof.
  • the nucleic acid construct is an expression vector or an integration vector for integrating the coding sequence or its complement into a host cell.
  • the invention also provides a host cell comprising a nucleic acid construct as described herein.
  • the invention also provides the use of the PD-1 antibody, its coding sequence or complementary sequence, a nucleic acid construct, and a host cell for the preparation or treatment of a malignancy, particularly a tumor associated with PD-1, including but Not limited to the various malignancies described herein.
  • the invention also provides a T cell modified by the meso3CAR gene and capable of expressing the PD-1 antibody, wherein the T cell can stably express the meso3CAR gene and the PD-1 antibody at a high level, and the exogenously expressed meso3CAR gene can accurately target the cell. Peel, enhances the proliferation of T cells and the secretion of cytokines, enhances the killing of tumor cells by CAR-T cells, and exerts anti-tumor effects by enhancing the immune response.
  • exogenously expressed PD-1 antibody can eliminate the immune escape of tumor cells, restore the phagocytosis of macrophages to tumor cells, promote the apoptosis of tumor cells, and exert an anti-tumor immune response.
  • the exogenous meso3CAR gene and the PD-1 antibody gene can be integrated into the genome of T cells via the PB transposase system, thereby stably and continuously expressing in T cells.
  • the T cells of the present invention which stably express the meso3CAR gene and the PD-1 antibody gene at a high level can be used for the treatment of various mesothelin-expressing malignant tumors.
  • the CAR of the invention typically contains an optional signal peptide sequence, an scFv that recognizes a mesothelin antigen, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.
  • a signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of newly synthesized proteins to the secretory pathway, often referred to as the N-terminal amino acid sequence in the newly synthesized polypeptide chain that directs transmembrane transfer (localization) of the protein. (Sometimes not necessarily at the N-terminus), it is responsible for directing proteins into subcellular organelles with different membrane structures.
  • the signal peptide can be a secreted signal peptide or a membrane-bound signal peptide.
  • the signal peptide is a CD8 signal peptide, a CD28 signal peptide or a CD4 signal peptide or a light chain signal peptide; more preferably a CD8 signal peptide.
  • the amino acid sequence of the CD8 signal peptide can be as shown in amino acid residues 1-22 of SEQ ID NO: 1; in certain embodiments, the coding sequence is set forth in bases 1-66 of SEQ ID NO: 3.
  • the scFv recognizing the mesothelin antigen described herein may be a single chain antibody directed against mesothelin antigen as is well known in the art.
  • the light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence of the single chain antibody are derived from an antibody directed against the mesothelin proximal membrane amino acid sequence.
  • the anti-mesothelin single chain antibody described herein is a single chain antibody directed against Region I or III of mesothelin.
  • the light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence of the single chain antibody are derived from an antibody against the amino acid sequence of mesothelin, Region I or III.
  • the amino acid sequence of mesothelin Region I is set forth in SEQ ID NO: 8; the amino acid sequence of mesothelin Region III is set forth in SEQ ID NO: 9.
  • An exemplary amino acid sequence of a single-chain antibody against mesothelin, Region I is set forth in SEQ ID NO: 10.
  • An exemplary amino acid sequence of a single-chain antibody against mesothelin-Region III is shown in amino acid residues 23 to 272 of SEQ ID NO: 1, and an exemplary coding sequence thereof is set to positions 67-816 of SEQ ID NO: 3.
  • the nucleotide sequence is shown.
  • mesothelin refers to a mesothelin fragment anchored on a membrane unless otherwise specified.
  • the hinge region refers to the region between the functional regions of the immunoglobulin heavy chain CH1 and CH2, which is rich in proline, does not form an alpha helix, is prone to stretching and is somewhat distorted, and is beneficial to the antigen binding site of the antibody. Complementary binding between epitopes.
  • a hinge region suitable for use herein may be selected from any of the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD4 or A variety.
  • the hinge region is preferably a hinge region that is longer than 50 amino acid residues, more preferably 80 amino acids or longer.
  • a CD8 alpha hinge region or an IgG4 Fc CH2CH3 hinge region is used herein.
  • the amino acid sequence of the exemplary IgG4 FcCH2CH3 hinge region is shown in amino acid residues 273-500 of SEQ ID NO: 1, and the coding sequence for the exemplary IgG4 FcCH2CH3 hinge region is shown in SEQ ID NO: 3, positions 817-1500.
  • the transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD8 transmembrane region
  • the amino acid sequence thereof is set forth in 501-528 of SEQ ID NO: 1; in certain embodiments, the coding sequence is set forth in bases 1501-1581 of SEQ ID NO: 3.
  • the intracellular co-stimulatory signal domain including the intracellular domain of the costimulatory signaling molecule may be selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), and inducible T cell costimulation. Intracellular domain of factor (ICOS) and DNAX activator protein 10 (DAP10).
  • the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28, preferably the amino acid sequence thereof is set forth in amino acid residues 529-569 of SEQ ID NO: 1, exemplary The coding sequence is shown as bases 1585-1707 of SEQ ID NO:3.
  • the intracellular signal domain is preferably an immunoreceptor tyrosine activation motif, which may be a CD3 sputum intracellular signal domain or an Fc ⁇ RI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain As described in SEQ ID NO: 1 at amino acid residues 570-681; in certain embodiments, the coding sequence is set forth in SEQ ID NO: 3, 1708-2043.
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, an optional CD8 signal peptide, an anti-mesothelin-Region III scFv, an IgG4 Fc CH2CH3 hinge region, a CD8 transmembrane region, The intracellular domain of CD28 and the CD3 intracellular signal domain; preferably, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 23-681 of SEQ ID NO: 1.
  • the chimeric antigen receptor further comprises a CD8 signal peptide, preferably, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 1-22 of SEQ ID NO: 1.
  • the invention also encompasses chimeric antibody receptors and coding sequences thereof as described herein.
  • the intracellular signal domains and the like may be directly connected to each other or may be connected by a linker sequence.
  • the linker sequence can be a linker sequence suitable for use in antibodies well known in the art, such as linker sequences comprising G and S.
  • the linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, and 10 to 20 amino acid residues.
  • the linker sequence is a polyglycine linker sequence.
  • the amount of glycine in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, and 2 to 8.
  • the linker may also contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), styrene Amino acid (F), arginine (R), glutamine (Q), and the like.
  • a suitable cleavage site which necessarily introduces one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest.
  • promote expression of a recombinant protein obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or within the protein.
  • Other suitable regions include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like.
  • the amino or carboxy terminus of a CAR herein may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in this article.
  • the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags can be used to purify proteins.
  • polynucleotide sequences encoding the chimeric antigen receptor.
  • the polynucleotide sequence herein may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the polynucleotide sequences described herein can generally be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequences disclosed herein, and the relevant sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template.
  • the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the polynucleotide sequence encoding a fusion protein described herein is set forth in SEQ ID NO:3.
  • nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor described herein or a polynucleotide sequence encoding the PD-1 antibody, and one or operably linked to the sequences Multiple regulatory sequences.
  • the nucleic acid construct is an expression cassette.
  • the control sequence can be a suitable promoter sequence.
  • the promoter sequence is typically operably linked to the coding sequence of the protein to be expressed.
  • the promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated and hybrid promoters, and may be derived from an extracellular or heterologous source encoding the host cell. Or the gene of the intracellular polypeptide is obtained.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used herein.
  • the nucleic acid construct is a vector.
  • the coding sequence of the CAR or the coding sequence of the PD-1 antibody can be cloned into many types of vectors, for example, but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • the vector can be an expression vector.
  • the expression vector can be provided to the cells in the form of a viral vector.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism.
  • the invention employs a retroviral vector containing a replication initiation site, a 3'LTR, a 5' LTR, a coding sequence for a CAR described herein, or a PD-1 antibody A coding sequence, and optionally a selectable marker.
  • Suitable promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter sequences.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, heme Promoter and creatine kinase promoter. Further, an inducible promoter can also be considered.
  • SV40 prion 40
  • MMTV mouse breast cancer virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukemia virus promoter
  • EB virus immediate early promoter EB virus immediate early promoter
  • Russ sarcoma virus promoter avian leukemia virus promoter
  • an inducible promoter can also be considered.
  • an inducible promoter provides a molecular switch that is capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter upon expression of the term, and shutting down expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • various promoter sequences disclosed in CN201510021408.1 can be used, including but not limited to the CCEF promoter containing the mCMV enhancer, the hCMV enhancer, and the EF1 ⁇ promoter shown in SEQ ID NO: 5 of the application.
  • the selectable marker includes either or both of the selectable marker genes or reporter genes to facilitate identification and selection of the expressed cells from the population of cells infected by the viral vector.
  • Useful selectable marker genes include, for example, antibiotic resistance genes such as neo and the like.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes.
  • the coding sequence of the chimeric antigen receptor described herein and the coding sequence of the PD-1 antibody are separately cloned into a vector for integration of the nucleic acid sequence of interest into the genome of the host cell (also referred to as integration).
  • vectors especially transposon vectors.
  • the transposon vector is a eukaryotic expression vector comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty.
  • Such transposon vectors contain the 5' inverted terminal repeat (5' LTR) of the corresponding transposon and the 3' inverted terminal repeat (3' LTR) of the corresponding transposon.
  • the transposase can be a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system.
  • the sequences of the 5'LTR and 3'LTR in the vector are also correspondingly changed to sequences adapted to the transposition system, which can be readily determined by those skilled in the art.
  • LTR is the expression cassette for the CAR or antibody of the invention, including the corresponding promoter sequence, the coding sequence for the CAR or antibody, and the polyA tailing signal sequence.
  • the transposase is a transposase from a piggybac transposition system.
  • the 5' inverted terminal repeat and the 3' inverted terminal repeat of the transposon are the 5' inverted terminal repeat and the 3' inverted terminal repeat of the piggybac transposon, respectively.
  • the transposon 5' inverted terminal repeat is SEQ ID NO: 1 as described in CN 201510638974.7, the disclosure of which is incorporated herein by reference.
  • the transposon 3' inverted terminal repeat is as shown in CN 201510638974.7 SEQ ID NO: 4.
  • the piggybac transposase is a transposase comprising a c-myc nuclear localization signal coding sequence.
  • the coding sequence for the piggybac transposase is as shown in CN 201510638974.7 SEQ ID NO: 5.
  • the promoter of the transposase coding sequence can be a variety of promoters known in the art for controlling expression of the transposase coding sequence.
  • the expression of the transposase coding sequence is controlled using a CMV promoter.
  • the sequence of the CMV promoter can be as shown in CN 201510638974.7 SEQ ID NO: 6.
  • the vector of the present invention comprising the expression cassette of the chimeric antigen receptor is the pNB328 vector disclosed in CN 201510638974.7.
  • the coding sequence of the chimeric antigen receptor of the present invention can be prepared by a method conventional in the art and cloned into a suitable vector.
  • the vector for integrating a gene of interest into the genome of a host cell does not contain a transposase coding sequence.
  • such vectors can be obtained by removing the transposase coding sequence based on the pNB328 vector.
  • such vectors are used to integrate the coding sequence for the PD-1 antibody and the signal peptide coding sequence (e.g., the coding sequence for the light chain signal peptide) into the genome of the host cell.
  • the amino acid sequence of an exemplary light chain signal peptide is shown in amino acid residues 1-20 of SEQ ID NO: 2, and the coding sequence of an exemplary light chain signal peptide is SEQ ID NO: 4 bases 1-60. Shown.
  • a T cell modified with a meso3CAR gene and capable of expressing a PD-1 antibody described herein can be transduced into a transposase containing a chimeric antigen receptor expression cassette for integration into the T cell genome.
  • the T cell is transformed into a vector containing a chimeric antigen receptor coding sequence constructed using the pNB328 vector as a backbone vector and constructed using a pS328 vector (with no transposase coding sequence compared to pNB328) as a backbone vector.
  • the coding sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 3; the coding sequence of the PD-1 antibody is as set at bases 61-1488 of SEQ ID NO: 4.
  • the signal peptide of the PD-1 antibody is a light chain signal peptide.
  • amino acid sequence of an exemplary light chain signal peptide can be as shown in amino acid residues 1-20 of SEQ ID NO: 1; the coding sequence of an exemplary light chain signal peptide is nucleus 1 to 60 of SEQ ID NO: 4. The nucleotide sequence is shown.
  • the vector comprising a transposase coding sequence that incorporates a chimeric antigen receptor coding sequence in the T cell genome in turn comprises a 5' LTR, a promoter, a light chain peptide a coding sequence, a coding sequence for a scFv recognizing a mesothelin antigen (preferably a coding sequence for the scFv recognizing mesothelin Region III), a coding sequence for the IgG4 Fc CH2CH3 hinge region, a coding sequence for the CD8 transmembrane region, and an intracellular structure of CD28 a coding sequence of a domain, a coding sequence of a CD3 intracellular signal domain, a polyA tailing signal sequence, a coding sequence for a 3'LTR and a transposase, and a promoter thereof; the integration into the PD-genus described herein in the T cell genome
  • the vector containing the transposase coding sequence that incorporates a
  • the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the PD-1 antibody coding sequence is from 1 to 7:1 to 3, preferably from 1:1 to 3, more preferably 1: 1 to 2, more preferably 1:1.
  • Methods of transfection are routine methods in the art including, but not limited to, viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation.
  • the vector is transfected into a cell of interest using electroporation.
  • the cells of interest may be various T cells well known in the art including, but not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTLs), helper T cells, suppressor/regulatory T cells, ⁇ T cells, and cytokines.
  • T cells of mixed cell populations such as induced killer cells (CIK) and tumor infiltrating lymphocytes (TIL).
  • CIK induced killer cells
  • TIL tumor infiltrating lymphocytes
  • the T cell can be derived from a PBMC of a B cell malignancy patient.
  • the T cell is a primary cultured T cell.
  • the invention also provides a composition comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette for a PD-1 antibody described herein.
  • Suitable agents may also be included in the compositions including, but not limited to, transfection reagents.
  • the invention also provides a kit comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette of a PD-1 antibody described herein, or a composition described herein .
  • a reagent or instrument for transferring the vector into a cell can also be provided in the kit.
  • the expression cassettes described herein contain, in addition to the coding sequences for the CARs or antibodies described herein, at least the appropriate promoter and polyA tailing signal sequences.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a T cell or a T cell as described herein and a PD-1 antibody expressed thereby.
  • Suitable pharmaceutically acceptable carriers or excipients may be included in the pharmaceutical compositions.
  • the pharmaceutical composition contains a therapeutically or prophylactically effective amount of T cells.
  • the therapeutically or prophylactically effective amount of the T cell can be determined according to factors such as the condition of the patient.
  • the invention also provides the use of a T cell or a T cell as described herein and a PD-1 antibody thereof or a pharmaceutical composition thereof for the preparation of a medicament for the treatment or prevention of a malignancy.
  • the invention also provides a method of treating or preventing a malignant tumor, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a T cell of the invention.
  • a cancer suitable for treatment or prevention of a T cell described herein is preferably a cancer having abnormal expression of mesothelin on the surface of a cancer cell; preferably, the cancer is selected from the group consisting of: adenocarcinoma, mesothelioma, lung cancer, colon cancer, colon cancer, breast Cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, rectal cancer, Hodgkin's lymphoma, pancreatic cancer or Prostate cancer; more preferably, the cancer is a cancer in which mesothelin and CA125/MUC16 are simultaneously highly expressed.
  • Example 1 Construction of recombinant plasmids pNB328-meso3CAR, pS328-antiPD1, pNB328-meso3CAR-2A-antiPD1 and pNS328-antiPD1-IRES-meso3CAR
  • the coding sequence of the synthetic meso3CAR (SEQ ID NO: 3), the coding sequence of antiPD1 (SEQ ID NO: 4), and the coding sequence of meso3CAR-2A-antiPD1 were respectively commissioned by a commercial company (the coding sequence of 2A is SEQ ID NO: 5).
  • the amino acid sequence is shown in SEQ ID NO: 6 and the coding sequence of antiPD1-IRES-meso3CAR (the coding sequence of IRES is shown in SEQ ID NO: 7), and its structural pattern is shown in FIG.
  • Each sequence was inserted between pNB328, pS328 vector EcoRI and SalI cleavage sites.
  • pNB328 The structure and sequence of pNB328 are described in CN 201510638974.7, the entire contents of which are incorporated herein by reference.
  • the recombinant plasmid was identical to pNB328.
  • the recombinant plasmids were named pNB328-meso3CAR, pS328-antiPD1, pNB328-meso3CAR-2A-antiPD1, pNB328-antiPD1-IRES-meso3CAR.
  • the promoter sequence and the polyA tailing signal sequence are not shown in the respective structural pattern diagrams, which are located between the 5'LTR and the signal peptide sequence and before the 3' LTR, respectively.
  • Example 2 Construction of CAR T cells targeting mesothelin from PD1 antibody
  • PBMCs Peripheral blood mononuclear cells
  • the PBMCs were cultured for 2-4 h, and the unattached suspension cells were the initial T cells.
  • the suspension cells were collected into 15 ml centrifuge tubes, centrifuged at 1200 rmp for 3 min, the supernatant was discarded, physiological saline was added, and centrifuged at 1200 rmp for 3 min to abandon the physiology.
  • the a recombinant tube was added with the constructed recombinant plasmids pNB328-meso3CAR and pS328-antiPD1, 4ug, and the b tube was added with pNB328-meso3CAR-2A-antiPD1 plasmid 6ug, and the c tube was added to pNB328- antiPD1-IRES-meso3CAR plasmid 6ug; transfer the mixture into an electric rotor, put it into the electro-rotation instrument, select the required procedure, and perform electric shock; use the micro-pipette in the kit to transfer the cell suspension to the well In a six-well plate (containing 2% FBS in AIM-V), mix and incubate at 37 ° C in a 5% CO 2 incubator and add the stimulating factors IL-2 and meso/anti-CD28 after six hours, 37 Incubate for 3 to 4 days at °C, 5% CO2, observe the growth of T cells, and obtain self-expression PD1
  • Example 3 Comparison of CAR T cell positive rate and antibody secretion of targeted mesothelins from three self-expressing PD1 antibodies
  • the meso3CAR-antiPD1 T cells, meso3CAR-2A-antiPD1 T cells and antiPD1-IRES-meso3CAR T cells obtained in the examples were collected and divided into two parts, each 1 ⁇ 10 6 cells, washed twice with physiological saline, 100 ul physiological The cells were resuspended in saline, one portion was added with 1 ug of mesothelin-biotin, and the other was added without incubation at 30 °C for 30 minutes.
  • the saline was washed twice, and the cells were resuspended again with 100 ul of physiological saline, and 1 ul of streptomycin-PE antibody was added thereto, and incubated at 4 ° C for 30 minutes.
  • the saline was washed twice, and the upper machine was tested, and only the secondary antibody was added as a control. The result is shown in Fig. 2A.
  • ELISA detects the expression of antiPD1 antibody in the three T cells.
  • the OD value was measured at 450 nm on a microplate reader, and a standard curve was drawn to calculate the PD-1 antibody concentration.
  • the amount of pNBS328-meso3CAR and pS328-antiPD1 plasmids were set to 7 ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug, respectively, for CAR T
  • the cell was constructed and constructed in the same manner as in Example 2.
  • Example 5 The blocking effect of meso3CAR-antiPD1 T cells on their own PD1
  • the meso3CAR T cells and meso3CAR-antiPD1 T cells constructed in Example 2 were collected, and the expression of PD1 was detected using the flow antibody PE-mouse anti-human CD279 of BD, Flow Method Example 3.
  • meso3CAR-antiPD1 T which expresses the PD1 antibody can block its own PD1 expression.
  • Example 6 Comparison of killing function between meso3CAR T and meso3CAR-antiPD1 T cells
  • RTCA real-time unlabeled cell function analyzer
  • Target cell plating cervical cancer cell Hela, ovarian cancer cell SK-OV-3, gastric cancer HGC-27 (both purchased from American Type Culture Collection ATCC) were plated with detection electrodes at 10 4 cells/50 ⁇ l per well. Place the plate for a few minutes, wait for the cells to stabilize, then put them into the instrument, start step 2, and culture the cells;
  • step 3 After the target cells were cultured for 24 hours, the step 2 was suspended, and the effector cells were added, 50 ⁇ l per well, and the target ratio was set to 4:1, respectively, and the Mock T cells without the plasmid were used as a control, and step 3 was started. After continuing to co-culture for 24 hours, the cell proliferation curve was observed.
  • the mes3R-antiPD1 T cells expressing the PD1 antibody were substantially identical to the meso3CAR T cells alone, and the antibody expression did not affect the CAR-T function.
  • Example 7 Comparison of cytokine release by meso3CAR and meso3CAR-antiPD1 T cells under the specific stimulation of mesothelin antigen
  • T cells into the culture cell supernatants were collected after 24h with BD TM CBA Human Th1 / Th2 cytokine Kit II detected by the three T-cell secretion of cytokines following stimulation mesothelin antigen, the following steps:
  • Th1/Th2 cytokine standard double dilution 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312.5pg/ml, 156pg/ml, 80pg/ml, 40pg/ml) , 20pg/ml, 0pg/ml) and 50ul of the sample to be tested (diluted by dilution 2 times);
  • Example 8 Therapeutic effect of meso3CAR and meso3CAR-antiPD1 T cells on a transplanted tumor model of ovarian cancer mice
  • mice There were 25 NSG completely immunodeficient mice at 4 to 6 weeks of age, with an average weight of 22 to 27 g, supplied by Biotech Biotech, and raised in SPF animal laboratory.
  • mice Inoculate SK-OV-3-luc cells subcutaneously in the right flank of the mouse, 0.1 ml/head. Seven days after the inoculation, the fluorescence intensity was observed by a living imager, and NSG immunodeficient mice were randomly divided into 5 groups. T cells of each group was injected with 1 ⁇ 10 7 th / 100ul, PBS group received 100ul PBS; the route of administration is intravenous injection.
  • mice The living state of the mice was observed daily and the tumor changes of the mice were observed by a living imager every 4 days.

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Abstract

本发明提供自表达PD-1抗体并靶向间皮素的嵌合抗原受体修饰T细胞及其用途。本发明的T细胞自表达PD1抗体并靶向间皮素，优选(1)含有表达识别间皮素的嵌合抗原受体的编码序列和PD-1抗体的编码序列；和/或(2)表达识别间皮素的嵌合抗原受体和PD-1抗体。本发明的T细胞在过继性免疫细胞治疗的同时，抑制免疫检查点PD-1/PD-L1通路，能原位激活残留的肿瘤特异性T细胞的功能，增加内源性细胞毒性T细胞的抗肿瘤杀伤效应，促进CAR-T细胞在体内的增殖，从而提高特异性杀伤肿瘤的疗效。

Description

自表达PD-1抗体并靶向间皮素的嵌合抗原受体修饰T细胞及其用途 技术领域
本发明属于基因工程学和免疫学，涉及一种靶向间皮素的嵌合抗原受体修饰T细胞及其用途。
背景技术
癌症现在已成为人类健康的头号杀手，快速的生活节奏、巨大的工作压力、不健康的饮食习惯、糟糕的环境都是癌症发生的帮凶，使得癌症的高发率和年轻化趋势也越来越明显。目前常用的治疗手段效果十分有限，仍需探索一个更加有效的治疗方法，来提高癌症患者的生存率和生存质量。
针对恶性肿瘤的免疫治疗近年来发展迅速，取得了令人瞩目的临床疗效。自2011年，Nature及临床肿瘤最顶级杂志JCO分别发表相同题目“肿瘤免疫治疗的时代已经来临”的评论文章(Nature.2011；480(7378):480；J Clin Oncol.2011；29(36):4828)，肿瘤免疫细胞治疗迎来新一轮的研究热潮。
嵌合抗原受体T细胞疗法作为肿瘤免疫治疗的重要分支之一，在恶性血液肿瘤中已经取得了非常好的疗效，对复发难治性B细胞白血病的完全缓解率超过90％。2017年8月，美国FDA批准了诺华的tisagenlecleucel嵌合抗原受体T细胞(CAR-T细胞)治疗用于治疗儿童和年轻成年患者急性淋巴细胞性白血病(ALL)，成为第一个批准上市的CAR-T药物。紧接着同年10月，美国FDA又宣布批准了Kite Pharma的CAR-T疗法Yescarta上市，治疗罹患特定类型的大B细胞淋巴瘤成人患者。CAR-T药物的陆续获批使得CAR-T治疗迈上了一个新的台阶。
嵌合抗原受体是一种人工合成受体，它通常包含胞外抗原结合域、跨膜铰链区和胞内信号转导区。通过将识别肿瘤相关抗原(tumor associated antigen,TAA)的抗体单链可变区(single-chain fragment variable，scFv)和胞内信号域“免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motifs，ITAM)”在体外进行基因重组。再通过病毒或其它载体系统将其导入T细胞中，这样经过基因改造的T细胞称之为CAR-T细胞。CAR-T细胞在体外经大规模扩增后，回输到患者体内，能够以非MHC 限制性的模式表现强效的抗癌作用。
但是，CAR-T细胞治疗实体瘤的疗效目前仍不足。主要原因包括：1、实体瘤异质性高，缺乏适合CAR-T治疗的细胞表面靶点；2、实体瘤具有强烈抑制免疫的微环境。
间皮素是一种通过磷脂酰肌醇区(GPI)锚定在细胞质膜上的糖蛋白，高表达于多种肿瘤组织中，少量表达于正常胸膜、心包和腹膜的间皮细胞中。间皮素基因编码一种69kDa的前体蛋白，经加工形成一个40kDa的膜结合蛋白和一个31kDa称之为巨核细胞促进因子(MPF)的脱落片断并释放出细胞外，我们通常所说的间皮素指的是锚定在膜上的片段，根据其蛋白结构可以分为Region I、II、III三个区域。它一方面可以通过其GPI结构域激活NFκB、MAPK和PI3K细胞内信号通路，促进细胞增殖，抵抗细胞凋亡；另一方面与其受体CA125/MUC16相互作用导致异常的细胞粘附，促进癌细胞转移。由于间皮素在正常组织中的分布有限而在多种恶性肿瘤(间皮瘤、卵巢癌、胰腺癌、胃癌、胆管癌等)中过表达，因此是一个很有潜力的肿瘤特异性治疗靶点。
目前，以间皮素为靶点的小分子药物、抗毒素已经取得了很好的效果，以此为靶点的CAR-T细胞研究也在如火如荼的展开，主要是针对胰腺癌(NCT01897415,NCT02465983)，间皮瘤(NCT01355965,NCT02414269)，肺癌和乳腺癌(NCT02414269)。
PD1(Programmed Death 1，重编程细胞死亡受体1)是调节性T细胞CD28家族的成员，属于免疫球蛋白超家族受体。PD-1和它的配体PD-L1/PD-L2在T细胞的共抑制和衰竭中起重要作用，他们相互作用抑制了共刺激分子调节的T细胞的繁殖和细胞因子的分泌，下调了抗凋亡分子BCL-xl的表达，削弱了肿瘤特异性T细胞的功能，导致一些肿瘤患者不能完全消除肿瘤。因此，抑制免疫检查点PD-1/PD-L1通路是目前治疗淋巴瘤的一个新的方向和靶标。PD-1抗体阻断治疗对晚期或难治性黑色素瘤、非小细胞肺癌、肾细胞癌、头颈鳞状上皮细胞癌、结直肠癌、霍奇金淋巴瘤、卵巢癌等都有一定程度的治疗效果，但是其较高的生产成本及应答率不高限制了它在临床中的运用。
发明内容
本文提供一种T细胞，所述T细胞自表达PD1抗体并靶向间皮素。
在一个或多个实施方案中，所述T细胞的基因组中整合了PD1抗体的表达框以 及识别间皮素的嵌合抗原受体的表达框。
在一个或多个实施方案中，所述PD1抗体的氨基酸序列如SEQ ID NO:2第21-495位氨基酸残基所示，或如SEQ ID NO:2所示。
在一个或多个实施方案中，所述PD1抗体的编码序列如SEQ ID NO:4第61-1488位碱基序列所示，或如SEQ ID NO:4所示。
在一个或多个实施方案中，所述识别间皮素的嵌合抗原受体从N端到C端依次含有任选的信号肽、抗间皮素III区单链抗体、铰链区、跨膜区、胞内共刺激信号域和胞内信号域。
在一个或多个实施方案中，所述信号肽为CD8信号肽、CD28信号肽、CD4信号肽或轻链信号肽；更优选地为CD8信号肽；优选地，所述CD8信号肽的氨基酸序列如SEQ ID NO:1第1-22位氨基酸残基所示。
在一个或多个实施方案中，所述scFv的氨基酸序列如SEQ ID NO:1第23-272位氨基酸残基所示。
在一个或多个实施方案中，所述铰链区为CD8铰链区或IgG4 CH2CH3铰链区；优选地，所述IgG4 CH2CH3铰链区的氨基酸序列如SEQ ID NO:1第273-500位氨基酸残基所示。
在一个或多个实施方案中，所述跨膜区为CD28跨膜区、CD8跨膜区、CD3ζ跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种；优选为CD8跨膜区，优选其氨基酸序列如SEQ ID NO:1第501-528位氨基酸残基所示。
在一个或多个实施方案中，所述胞内共刺激信号域包括共刺激信号分子的胞内结构域，包括CD28、CD134/OX40、CD137/4-1BB、淋巴细胞特异性蛋白酪氨酸激酶、诱导性T细胞共刺激因子(ICOS)和DNAX激活蛋白10的胞内结构域；优选地，所述胞内共刺激信号域为CD28的胞内结构域；优选地，所述CD28的氨基酸序列如SEQ ID NO:1第529-569位氨基酸残基所示。
在一个或多个实施方案中，所述胞内信号域为CD3ζ胞内信号域或FcεRIγ胞内信号域；优选为CD3ζ胞内信号域，优选地所述CD3ζ胞内信号域的氨基酸序列如SEQ ID NO:1第570-681位氨基酸残基所示。
在一个或多个实施方案中，所述嵌合抗原受体的氨基酸序列如SEQ ID NO:1第23-681位氨基酸残基所示，或如SEQ ID NO:1所示；优选地，所述嵌合抗原受体的编码序列如SEQ ID NO:3第67-2043位碱基所示，或如SEQ ID NO:3所示。
本发明还提供一种组合物，所述组合物含有：含本发明所述嵌合抗原受体的表 达框的载体，所述载体用于将所述表达框整合到宿主细胞的基因组中；和含PD1抗体的编码序列或其互补序列的载体，所述载体用于将所述表达框整合到宿主细胞的基因组中。
在一个或多个实施方案中，所述PD1抗体的氨基酸序列如SEQ ID NO:2第21-495位氨基酸残基所示，或如SEQ ID NO:2所示。
在一个或多个实施方案中，所述PD1抗体的编码序列如SEQ ID NO:4第61-1488位碱基序列所示，或如SEQ ID NO:4所示。
本发明还提供一种试剂盒，所述试剂盒含有：
(1)含本发明所述嵌合抗原受体的表达框的载体，所述载体用于将所述表达框整合到宿主细胞的基因组中；和
(2)含PD1抗体的编码序列或其互补序列的载体，所述载体用于将所述表达框整合到宿主细胞的基因组中。
在一个或多个实施方案中，所述PD1抗体的氨基酸序列如SEQ ID NO:2第21-495位氨基酸残基所示，或如SEQ ID NO:2所示。
在一个或多个实施方案中，所述PD1抗体的编码序列如SEQ ID NO:4第61-1488位碱基序列所示，或如SEQ ID NO:4所示。
本发明还提供一种药物组合物，所述药物组合物含有本发明所述的T细胞或所述T细胞及其表达的PD1抗体。
本发明还提供本文所述的T细胞或所述T细胞及其表达的PD1抗体在制备治疗或预防恶性肿瘤的药物中的用途。优选地，所述恶性肿瘤为其癌细胞表面异常表达间皮素的癌症；优选地，所述癌症选自：腺癌、间皮瘤、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、黑色素瘤、非小细胞肺癌、肾细胞癌、头颈鳞状上皮细胞癌、直肠癌、霍奇金淋巴瘤、胰腺癌或前列腺癌；更优选地，所述癌症是间皮素和CA125/MUC16同时高表达的癌症。
附图说明
图1：PD-1抗体以及间皮素嵌合抗原受体基因结构模式图。
图2A：meso3CAR-2A-antiPD1、antiPD1-IRES-meso3CAR、meso3CAR-antiPD1三种CAR-T细胞阳性率。
图2B：meso3CAR-2A-antiPD1、antiPD1-IRES-meso3CAR、meso3CAR-antiPD1三种CAR-T细胞阳性率抗体分泌量。
图3A：CAR与PD1抗体质粒不同配比的条件下构建的meso3CAR-antiPD1 T细胞阳性率比较。
图3B：CAR与PD1抗体质粒不同配比的条件下构建的meso3CAR-antiPD1 T细胞抗体分泌量比较。
图4：流式检测meso3CAR T与meso3CAR-antiPD1 T细胞表面PD1表达。
图5：meso3CAR-antiPD1 T细胞对宫颈癌细胞Hela、卵巢癌细胞SK-OV-3和胃癌细胞HGC-27的杀伤。
图6：meso3CAR-antiPD1在间皮素抗原刺激下IL-2，IL-4，IL-6,IL-10，TNF-α和IFN-γ细胞因子分泌的变化。
图7：meso3CAR T细胞与meso3CAR-antiPD1 T细胞对SK-OV-3卵巢癌小鼠移植瘤模型的治疗效果。
具体实施方式
下面对本发明涉及的部分术语进行解释。
在本发明中，术语“表达框”是指表达一个基因所需的完整元件，包括启动子、基因编码序列、PolyA加尾信号序列。
术语“编码序列”在文中定义为核酸序列中直接确定其蛋白产物(例如CAR，单链抗体，铰链区和跨膜区)的氨基酸序列的部分。编码序列的边界通常是由紧邻mRNA 5’端开放读码框上游的核糖体结合位点(对于原核细胞)和紧邻mRNA 3’端开放读码框下游的转录终止序列确定。编码序列可以包括，但不限于DNA、cDNA和重组核酸序列。
术语“Fc”即抗体的可结晶段(fragment crystallizable,Fc)，是指位于抗体分子"Y"结构的柄部末端，包含抗体重链恒定区CH2和CH3结构域的肽段,是抗体与效应分子或者细胞相互作用的部位。
术语“共刺激分子”是指存在于抗原提呈细胞表面，能与Th细胞上的共刺激分子受体结合，产生协同刺激信号的分子。淋巴细胞的增殖不仅需要抗原的结合，还需要接受共刺激分子的信号。共刺激信号传递给T细胞主要是通过表达在抗原呈递细胞表面的共刺激分子CD80，CD86与T细胞表面的CD28分子结合。B细胞接受共刺激信号可以通过一般的病原体成分例如LPS，或者通过补体成分，或者通过激活了的抗原特异性的Th细胞表面的CD40L。
术语“接头”或铰链是连接不同蛋白或多肽之间的多肽片段，其目的是使所连接 的蛋白或多肽保持各自的空间构象，以维持蛋白或多肽的功能或活性。示例性的接头包括含有G和/或S的接头，以及例如Furin 2A肽。
术语“特异性结合”是指抗体或者抗原结合片段与其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合该抗原。“特异性识别”具有类似的含义。
术语“药学上可接受的辅料”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
术语“有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。
术语“疾病和/或病症”是指所述受试者的一种身体状态，该身体状态与本发明所述疾病和/或病症有关。
术语“受试者”或者“患者”可以指患者或者其它接受本发明药物组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。
术语“嵌合抗原受体”(CAR)是人工改造受体，能够将识别肿瘤细胞表面抗原的特异性分子(如抗体)锚定在免疫细胞(如T细胞)上，使免疫细胞识别肿瘤抗原或病毒抗原和杀死肿瘤细胞或病毒感染的细胞。CAR通常依次包含任选的信号肽、结合肿瘤细胞膜抗原的多肽如单链抗体、铰链区、跨膜区和胞内信号区。通常，结合肿瘤细胞膜抗原的多肽能够以中等亲和力结合肿瘤细胞广泛表达的膜抗原。结合肿瘤细胞膜抗原的多肽可以是天然多肽或人工合成多肽；优选地，人工合成多肽为单链抗体或Fab片段。
术语“单链抗体”(scFv)是指由抗体轻链可变区(VL区)氨基酸序列和重链可变区(VH区)氨基酸序列经铰链连接而成，具有结合抗原能力的抗体片段。在某些实施方案中，感兴趣单链抗体(scFv)来自感兴趣的抗体。感兴趣的抗体可以是人抗体，包括人鼠嵌合抗体和人源化抗体。抗体可以是分泌型或膜锚定型。
为了提高meso3CAR-T细胞治疗效果，本发明构建了一种自表达PD-1抗体的 meso3CAR T细胞，在过继性免疫细胞治疗的同时，抑制免疫检查点PD-1/PD-L1通路，能原位激活残留的肿瘤特异性T细胞的功能，增加内源性细胞毒性T细胞的抗肿瘤杀伤效应，促进CAR-T细胞在体内的增殖，从而提高特异性杀伤肿瘤的疗效。此外，本发明设计的PD-1抗体的Fc片段为突变型的IgG4 Fc，避免了与树突状细胞表面的γ-2受体结合而被巨噬细胞识别并吞噬，使自表达PD-1抗体的CAR-T细胞发挥功能的同时又不引起AICD反应。
因此，本发明提供一种PD-1抗体，所述PD-1抗体含有抗PD-1单链抗体和IgG4Fc。在某些实施方案中，所述IgG4Fc的氨基酸序列如SEQ ID NO:2第267-495位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:4第799-1485位碱基序列所示。
在某些实施方案中，所述抗PD-1单链抗体(scFv)中抗体轻链可变区(VL区)氨基酸序列如SEQ ID NO:2第21-131位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:4第61-393位碱基序列所示。在某些实施方案中，所述抗PD-1单链抗体中的重链可变区(VH区)氨基酸序列如SEQ ID NO:2第147-266位氨基酸序列所示；优选地，其编码序列如SEQ ID NO:4第439-798位碱基序列所示。在某些实施方案中，所述抗PD-1单链抗体的氨基酸序列如SEQ ID NO:2第21-266位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:4第61-798位碱基序列所示。
在某些实施方案中，所述PD-1抗体还含有轻链信号肽。在某些实施方案中，所述PD-1抗体从N端到C端，依次含有轻链信号肽、抗PD-1单链抗体和IgG4Fc。在某些实施方案中，所述轻链信号肽的氨基酸序列如SEQ ID NO:2第1-20位氨基酸残基所示；优选地，所示轻链信号肽的编码序列如SEQ ID NO:4第1-60位碱基序列所示。
在某些实施方案中，所述PD-1抗体的氨基酸序列如SEQ ID NO:2第21-495位氨基酸序列所示，或者如SEQ ID NO:4所示。
本发明还包括所述PD-1抗体的编码序列或其互补序列，所述编码序列至少包括本文所述的IgG4Fc的编码序列或其互补序列。在某些实施方案中，所述PD-1抗体的编码序列含有SEQ ID NO:4第61-1495位碱基序列所示的序列，优选含有SEQ ID NO:4所示的序列。
本发明还包括一种核酸构建物，所述核酸构建物含有本发明所述的PD-1抗体的编码序列或其互补序列。优选地，所述核酸构建物是表达载体或用于将所述编码序列或其互补序列整合入宿主细胞的整合载体。
本发明还提供一种宿主细胞，所述宿主细胞含有本文所述的核酸构建物。
本发明还提供所述PD-1抗体、其编码序列或互补序列、核酸构建物以及宿主细胞在制备治疗或预防恶性肿瘤中的用途，所述肿瘤尤其是与PD-1相关的肿瘤，包括但不限于本文所述的各种恶性肿瘤。
本发明还提供一种经meso3CAR基因修饰并能表达PD-1抗体的T细胞，该T细胞能高水平稳定的表达meso3CAR基因及PD-1抗体，外源表达的meso3CAR基因可以准确的靶向间皮素，增强T细胞的增殖能力及细胞因子的分泌，增强CAR-T细胞对肿瘤细胞的杀伤，并通过增强免疫反应，发挥抗肿瘤作用。同时，外源表达的PD-1抗体可以消除肿瘤细胞的免疫逃逸，恢复巨噬细胞对肿瘤细胞的吞噬作用，促进肿瘤细胞的凋亡，发挥抗肿瘤的免疫反应。此外，外源meso3CAR基因及PD-1抗体基因可经PB转座酶系统整合到T细胞的基因组中，从而在T细胞中稳定持续的表达。本发明高水平稳定表达meso3CAR基因及PD-1抗体基因的T细胞可用于多种间皮素高表达的恶性肿瘤的治疗。
本发明的CAR通常含有任选的信号肽序列、识别间皮素抗原的scFv、铰链区、跨膜区、胞内共刺激信号域和胞内信号域。
信号肽是引导新合成的蛋白质向分泌通路转移的短肽链(长度5-30个氨基酸)，常指新合成多肽链中用于指导蛋白质的跨膜转移(定位)的N-末端的氨基酸序列(有时不一定在N端)，它负责把蛋白质引导到细胞含不同膜结构的亚细胞器内。信号肽可以是分泌型信号肽或膜结合型信号肽。在本发明的某些实施方案中，信号肽为CD8信号肽、CD28信号肽或CD4信号肽或轻链信号肽；更优选地为CD8信号肽。CD8信号肽的氨基酸序列可如SEQ ID NO:1第1-22位氨基酸残基所示；在某些实施方案中，其编码序列如SEQ ID NO:3第1-66位碱基所示。
本文所述的识别间皮素抗原的scFv可以是本领域周知的针对间皮素抗原的单链抗体。优选的是，该单链抗体的轻链可变区氨基酸序列和重链可变区氨基酸序列来自针对间皮素近膜端氨基酸序列的抗体。优选地，本文所述的抗间皮素单链抗体是针对间皮素的Region I或III的单链抗体。优选的是，该单链抗体的轻链可变区氨基酸序列和重链可变区氨基酸序列来自针对间皮素Region I或III的氨基酸序列的抗体。在某些实施方案中，间皮素Region I的氨基酸序列如SEQ ID NO:8所示；间皮素Region III的氨基酸序列如SEQ ID NO:9所示。示例性的抗间皮素Region I的单链抗体的氨基酸序列如SEQ ID NO:10。示例性的抗间皮素Region III的单链抗体的氨基酸序列如SEQ ID NO:1第23-272位氨基酸残基所示，其示例性的编码序列如SEQ ID NO:3第 67-816位核苷酸序列所示。本文中，如果没有特别说明，间皮素指锚定在膜上的间皮素片段。
本文中，铰链区指免疫球蛋白重链CH1和CH2功能区之间的区域，该区富含脯氨酸，不形成α螺旋，易发生伸展及一定程度扭曲，有利于抗体的抗原结合部位与抗原表位间的互补性结合。适用于本文的铰链区可选自CD8的胞外铰链区、IgG1 Fc CH2CH3铰链区、IgD铰链区、CD28的胞外铰链区、IgG4 Fc CH2CH3铰链区和CD4的胞外铰链区的任意一种或多种。铰链区优选是长50个氨基酸残基以上、更优选长80个氨基酸以上的铰链区。在某些实施方案中，本文使用CD8α铰链区或IgG4 Fc CH2CH3铰链区。示例性的IgG4 FcCH2CH3铰链区的氨基酸序列SEQ ID NO:1第273-500位氨基酸残基所示，示例性的IgG4 FcCH2CH3铰链区的编码序列如SEQ ID NO:3第817-1500位所示。
跨膜区可以是CD28跨膜区、CD8跨膜区、CD3ζ跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种；优选为CD8跨膜区，优选其氨基酸序列如SEQ ID NO:1第501-528所示；在某些实施方案中，其编码序列如SEQ ID NO:3第1501-1584位碱基所示。
胞内共刺激信号域包括共刺激信号分子的胞内结构域可选自CD28、CD134/OX40、CD137/4-1BB、淋巴细胞特异性蛋白酪氨酸激酶(LCK)、诱导性T细胞共刺激因子(ICOS)和DNAX激活蛋白10(DAP10)的胞内结构域。在某些实施方案中，所述共刺激信号分子的胞内结构域为CD28的胞内结构域，优选其氨基酸序列如SEQ ID NO:1第529-569位氨基酸残基所示，示例性的编码序列如SEQ ID NO:3第1585-1707位碱基所示。
胞内信号域优选为免疫受体酪氨酸活化基序，可以是CD3ζ胞内信号域或FcεRIγ胞内信号域；优选为CD3ζ胞内信号域，优选地所述CD3ζ胞内信号域的氨基酸序列如SEQ ID NO:1第570-681位氨基酸残基所述；在某些实施方案中，其编码序列如SEQ ID NO:3第1708-2043所示。
在某些实施方案中，所述嵌合抗原受体从N端到C端依次含有：任选的CD8信号肽、抗间皮素Region III的scFv、IgG4 Fc CH2CH3铰链区、CD8跨膜区、CD28的胞内结构域和CD3ζ胞内信号域；优选地，所述嵌合抗原受体的氨基酸序列如SEQ ID NO:1第23-681位氨基酸残基所示。在某些实施方案中，所述嵌合抗原受体还含有CD8信号肽，优选地，该嵌合抗原受体的氨基酸序列如SEQ ID NO:1第1-22位氨基酸残基所示。
应理解，本发明也包括本文所述的嵌合抗体受体及其编码序列。
形成本文嵌合抗原受体的上述各部分，如信号肽、抗间皮素单链抗体的轻链可变区和重链可变区、铰链区、跨膜区、胞内共刺激信号域和胞内信号域等，相互之间可直接连接，或者可通过接头序列连接。接头序列可以是本领域周知的适用于抗体的接头序列，例如含G和S的接头序列。接头的长度可以是3～25个氨基酸残基，例如3～15、5～15、10～20个氨基酸残基。在某些实施方案中，接头序列是多甘氨酸接头序列。接头序列中甘氨酸的数量无特别限制，通常为2～20个，例如2～15、2～10、2～8个。除甘氨酸和丝氨酸来，接头中还可含有其它已知的氨基酸残基，例如丙氨酸(A)、亮氨酸(L)、苏氨酸(T)、谷氨酸(E)、苯丙氨酸(F)、精氨酸(R)、谷氨酰胺(Q)等。
应理解，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的氨基酸序列末端引入了一个或多个不相干的残基，而这并不影响目的序列的活性。为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸等。因此，本文的CAR的氨基端或羧基端还可含有一个或多个多肽片段，作为蛋白标签。任何合适的标签都可以用于本文。例如，所述的标签可以是FLAG，HA，HA1，c-Myc，Poly-His，Poly-Arg，Strep-TagII，AU1，EE，T7，4A6，ε，B，gE以及Ty1。这些标签可用于对蛋白进行纯化。
本文还包括编码所述嵌合抗原受体的多核苷酸序列。本文的多核苷酸序列可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。
本文所述的多核苷酸序列通常可以用PCR扩增法获得。具体而言，可根据本文所公开的核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增得到有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。例如，在某些实施方案中，编码本文所述融合蛋白的多核苷酸序列如SEQ ID NO:3所示。
本文还包括核酸构建物，其含有本文所述的编码所述嵌合抗原受体的多核苷酸序列或编码所述PD-1抗体的多核苷酸序列，以及与这些序列操作性连接的一个或多个调控序列。在某些实施方案中，所述核酸构建物是表达框。
调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列 操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。
调控序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本文。
在某些实施方案中，所述核酸构建物是载体。具体而言，可将本文CAR的编码序列或PD-1抗体的编码序列克隆入许多类型的载体，例如这些类型的载体包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。载体可以是表达载体。表达载体可以以病毒载体形式提供给细胞。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。
通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。例如，在某些实施方案中，本发明使用逆转录病毒载体，该逆转录病毒载体含有复制起始位点，3’LTR，5’LTR，本文所述CAR的编码序列或PD-1抗体的编码序列，以及任选的可选择的标记。
合适的启动子包括但不限于即时早期巨细胞病毒(CMV)启动子序列。该启动子序列是能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、EB病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，也可考虑使用诱导型启动子。诱导型启动子的使用提供了分子开关，其能够在期限表达时打开可操作地连接诱导型启动子的多核苷酸序列的表达，而在当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
在某些实施方案中，可使用CN201510021408.1所公布的各种启动子序列，包括但不限于该申请SEQ ID NO:5所示的含mCMV增强子、hCMV增强子和EF1α启动子的CCEF启动子；SEQ ID NO:7所示的含CD3e增强子、mCMV增强子、hCMV增强子和EF1α启动子的TCEF启动子；SEQ ID NO:8所示的含mCMV增强子、hCMV增强子和含内含子的EF1α启动子的CCEFI启动子；SEQ ID NO:3所示的含CD3e增 强子和含内含子的EF1α启动子的TEFI启动子；以及SEQ ID NO:3所示的含CD3e增强子、mCMV增强子、hCMV增强子和含内含子的EF1α启动子的TCEFI启动子。本文将该申请的全部内容以引用的方式纳入本文。
可选择的标记包括可选择的标记基因或报道基因中的任一个或两者，以便于从被病毒载体感染的细胞群中鉴定和选择表达细胞。有用的可选择标记基因包括例如抗生素抗性基因，诸如neo等。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白基因的基因。
在某些实施方案中，将本文所述嵌合抗原受体的编码序列和PD-1抗体的编码序列分别克隆到用于将目的核酸序列整合到宿主细胞的基因组中的载体(也称为整合载体)中，尤其是转座子载体。在某些实施方案中，该转座子载体是含有选自piggybac、sleeping beauty、frog prince、Tn5或Ty的转座元件的真核表达载体。这类转座子载体含有相应转座子的5’反向末端重复序列(5’LTR)和相应转座子的3’反向末端重复序列(3’LTR)。转座酶可以是来自piggybac、sleeping beauty、frog prince、Tn5或Ty转座系统的转座酶。当使用来自不同转座系统的转座酶时，所述载体中的5’LTR和3’LTR的序列也相应改变为与该转座系统适配的序列，这可由本领域技术人员容易地确定。在5’LTR和3’LTR之间是本发明的CAR或抗体的表达框，包括相应的启动子序列、CAR或抗体的编码序列以及polyA加尾信号序列。
在某些实施方案中，转座酶是来自piggybac转座系统的转座酶。因此，在这些实施方案中，转座子5’反向末端重复序列和3’反向末端重复序列分别为piggybac转座子的5’反向末端重复序列和3’反向末端重复序列。在某些实施方案中，转座子5’反向末端重复序列如CN 201510638974.7(本文将其内容以引用的方式纳入本文)SEQ ID NO:1所示。在某些实施方案中，转座子3’反向末端重复序列如CN 201510638974.7SEQ ID NO:4所示。在某些实施方案中，piggybac转座酶为含c-myc核定位信号编码序列的转座酶。在某些实施方案中，piggybac转座酶的编码序列如CN 201510638974.7SEQ ID NO:5所示。
转座酶编码序列的启动子可以是本领域已知的用于控制转座酶编码序列表达的各种启动子。在某些实施方案中，使用CMV启动子控制转座酶编码序列的表达。CMV启动子的序列可如CN 201510638974.7 SEQ ID NO:6所示。
在某些实施方案中，本发明含嵌合抗原受体的表达框的载体为CN 201510638974.7所公开的pNB328载体。可采用本领域常规的方法制备本发明的嵌合抗原受体的编码序列，并将其克隆入合适的载体中。
在某些实施方案中，所述用于将目的基因整合到宿主细胞的基因组中的载体不含有转座酶编码序列。例如，可在pNB328载体的基础上除去转座酶编码序列即可获得这类载体。通常，用这类载体将PD-1抗体的编码序列及信号肽编码序列(如轻链信号肽的编码序列)整合到宿主细胞的基因组中。示例性的轻链信号肽的氨基酸序列如SEQ ID NO：2第1-20位氨基酸残基所示，示例性的轻链信号肽的编码序列如SEQ ID NO：4第1-60位碱基所示。
在某些实施方案中，本文所述的经meso3CAR基因修饰并能表达PD-1抗体的T细胞可转入：用于在T细胞基因组中整合入嵌合抗原受体表达框的含转座酶编码序列的载体，和用于在T细胞基因组中整合入本文所述的PD-1抗体的表达框的不含转座酶编码序列的载体。
优选地，所述T细胞转入了以pNB328载体为骨架载体构建的含嵌合抗原受体编码序列的载体以及以pS328载体(与pNB328相比不含转座酶编码序列)为骨架载体构建的含PD-1抗体编码序列的载体。在某些实施方案中，所述嵌合抗原受体的编码序列如SEQ ID NO:3所示；所述PD-1抗体的编码序列如SEQ ID NO:4第61-1488位碱基序列。在某些实施方案中，所述含PD-1抗体的编码序列的载体中，PD-1抗体的信号肽为轻链信号肽。示例性的轻链信号肽的氨基酸序列可如SEQ ID NO:1第1-20位氨基酸残基所示；示例性的轻链信号肽的编码序列如SEQ ID NO:4第1-60位核苷酸序列所示。更具体而言，在某些实施方案中，所述在T细胞基因组中整合入嵌合抗原受体编码序列的含转座酶编码序列的载体依次含有5’LTR、启动子、轻链号肽编码序列、识别间皮素抗原的scFv的编码序列(优选为识别间皮素Region III的scFv的编码序列)、IgG4 Fc CH2CH3铰链区的编码序列、CD8跨膜区的编码序列、CD28胞内结构域的编码序列、CD3ζ胞内信号域的编码序列、polyA加尾信号序列、3’LTR和转座酶的编码序列及其启动子；所述在T细胞基因组中整合入本文所述的PD-1抗体的编码序列的不含转座酶编码序列的载体在5’LTR和3’LTR之间依次含有启动子、轻链信号肽的编码序列、PD-1抗体的编码序列和polyA加尾信号序列。
优选地，转染时，含嵌合抗原受体编码序列的载体与含PD-1抗体编码序列的载体的质量比为1～7：1～3，优选1：1～3，更优选1：1～2，更优选1：1。
转染的方法为本领域常规的方法，包括但不限于：病毒转导、显微注射、粒子轰击、基因枪转化和电转等。在某些实施方案中，采用电转将所述载体转染感兴趣的细胞中。
感兴趣的细胞可以是本领域周知的各种T细胞，包括但不限于外周血T淋巴细 胞、细胞毒杀伤T细胞(CTL)、辅助T细胞、抑制/调节性T细胞、γδT细胞以及细胞因子诱导的杀伤细胞(CIK)、肿瘤浸润淋巴细胞(TIL)等混合细胞群体的T细胞。在某些实施方案中，T细胞可来源于B细胞恶性肿瘤患者的PBMC。在某些实施方案中，T细胞为原代培养T细胞。
本发明还提供一种组合物，所述组合物含有含本文所述嵌合抗原受体表达框的载体和含本文所述PD-1抗体的表达框的载体。该组合物中还可含有合适的试剂，包括但不限于转染用的试剂。
本发明还提供一种试剂盒，所述试剂盒含有含本文所述嵌合抗原受体表达框的载体和含本文所述PD-1抗体的表达框的载体，或者含有本文所述的组合物。试剂盒中还可配有将所述载体转入细胞中的试剂或仪器。
应理解，本文所述的表达框除含有本文所述的CAR或抗体的编码序列外，还至少含有合适的启动子和polyA加尾信号序列。
本发明还提供一种药物组合物，所述药物组合物含有本文所述的T细胞或所述T细胞及其表达的PD-1抗体。药物组合物中可含有合适的药学上可接受的载体或辅料。药物组合物中含有治疗或预防有效量的T细胞。可根据患者的病情等因素确定T细胞的治疗或预防有效量。
本发明还提供本文所述的T细胞或所述T细胞及其表达的PD-1抗体或其药物组合物在制备治疗治疗或预防恶性肿瘤的药物中的用途。本发明还提供恶性肿瘤的治疗或预防方法，所述方法包括给予需要的对象治疗或预防有效量的本发明所述的T细胞。适用于本文所述T细胞进行治疗或预防的癌症优选癌细胞表面异常表达间皮素的癌症；优选地，所述癌症选自：腺癌、间皮瘤、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、黑色素瘤、非小细胞肺癌、肾细胞癌、头颈鳞状上皮细胞癌、直肠癌、霍奇金淋巴瘤、胰腺癌或前列腺癌；更优选地，所述癌症是间皮素和CA125/MUC16同时高表达的癌症。
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买获得的常规产品。全文及附图中的“Meso3CAR”和“meso3CAR”表示相同含义。
实施例1：重组质粒pNB328-meso3CAR、pS328-antiPD1、pNB328-meso3CAR-2A-antiPD1和pNS328-antiPD1-IRES-meso3CAR的构建
分别委托商业公司人工合成meso3CAR的编码序列(SEQ ID NO:3)、antiPD1的编码序列(SEQ ID NO:4)、meso3CAR-2A-antiPD1的编码序列(2A的编码序列如SEQ ID NO:5所示，氨基酸序列如SEQ ID NO:6所示)和antiPD1-IRES-meso3CAR的编码序列(IRES的编码序列如SEQ ID NO:7所示)，其结构模式如图1所示。将各序列分别装入pNB328、pS328载体EcoRI和SalI酶切位点之间(pNB328的结构及序列参见CN 201510638974.7，本文将其全部内容以引用的方式纳入本文；与pNB328相比，pS328缺少PB转座子，其它元件与pNB328相同)，构建出的重组质粒分别命名为pNB328-meso3CAR、pS328-antiPD1、pNB328-meso3CAR-2A-antiPD1、pNB328-antiPD1-IRES-meso3CAR。各结构模式图中未示出启动子序列和polyA加尾信号序列，其分别位于5’LTR和信号肽序列之间以及3’LTR之前。
实施例2：自表达PD1抗体的靶向间皮素的CAR T细胞构建
外周血单核细胞(PBMCs)由Ficoll分离法分离获得。将PBMC贴壁培养2-4h，其中未贴壁的悬浮细胞即为初始T细胞，将悬浮细胞收集到15ml离心管中，1200rmp离心3min，弃上清，加入生理盐水，1200rmp离心3min,弃生理盐水，并重复此步骤；取四个1.5ml离心管，每管加入5×10 6个细胞，编号a、b、c，1200rmp离心3min，弃上清，取电转试剂盒(来自Lonza公司)，a、b、c管按比例加入电转试剂共100ul，a管加入构建好的重组质粒pNB328-meso3CAR和pS328-antiPD1各4ug，b管加入pNB328-meso3CAR-2A-antiPD1质粒6ug，c管加入pNB328-antiPD1-IRES-meso3CAR质粒6ug；将混合液转移至电转杯中，放入电转仪，选取所需程序，进行电击；使用试剂盒中的微量吸管将电转好的细胞悬液转移到加好培液的六孔板中(含2％FBS的AIM-Ⅴ培液)，混匀，置于37℃，5％CO2培养箱培养，六小时后加入刺激因子IL-2和meso/anti-CD28，37℃，5％CO2培养3～4天，观察T细胞的生长情况，获得自表达PD1抗体并靶向间皮素的meso3CAR-antiPD1 T细胞、meso3CAR-2A-antiPD1 T细胞、antiPD1-IRES-meso3CAR T细胞。
此外，在另一离心管中加入5×10 6个细胞，编号d，1200rmp离心3min，弃上清，取电转试剂盒(来自Lonza公司)，按比例加入电转试剂共100ul，并加入6ug对照质粒(pNB328-meso3CAR)，按前文所述方法构建得到对照T细胞，即meso3CAR T细胞。
实施例3：比较三种自表达PD1抗体的靶向间皮素的CAR T细胞阳性率以及抗体分泌
1、流式检测CAR T细胞阳性率
收集实施例获得的meso3CAR-antiPD1 T细胞、meso3CAR-2A-antiPD1 T细胞和antiPD1-IRES-meso3CAR T细胞，各分为两份，每份1×10 6个细胞，生理盐水洗涤两遍，100ul生理盐水重悬细胞，一份加入1ug的间皮素-生物素，另一份不加，4℃孵育30分钟。生理盐水洗涤两遍，再次用100ul生理盐水重悬细胞，加入1ul的链霉素-PE抗体，4℃孵育30分钟。生理盐水洗涤两遍，上机检测，以只加二抗的为对照，结果如图2A。
2、ELISA检测所述三种T细胞的antiPD1抗体表达量
①用包被液将PD1抗原稀释至0.5ug/ml(5ul+1ml包被液)，100ul/孔包被酶标反应板，4℃过夜。
②用PBST清洗5遍，每次3分钟，用吸水纸拍干，200ul/孔。
③每孔加封闭液100ul，37℃孵育1小时。
④用PBST清洗5遍，每次3分钟，用吸水纸拍干，200ul/孔。
⑤加入样品及标准品，100ul/孔，设复孔和对照孔，37℃孵育1小时。
⑥用PBST清洗5遍，每次3分钟，用吸水纸拍干，200ul/孔。
⑦封闭液将IgG F4 HRP1：30000稀释，100ul/孔，37℃孵育45分钟。
⑧用PBST清洗5遍，每次3分钟，用吸水纸拍干，200ul/孔。
⑨加入显色液TMB，100ul/孔，37℃避光显色10-15min。
⑩加入终止液终止反应，50ul/孔。
酶标仪上450nm处测OD值，绘制标准曲线，计算PD-1抗体浓度。
结果如图2B所示。
实施例4：pNBS328-meso3CAR与pS328-antiPD1质粒配比
分别将pNBS328-meso3CAR与pS328-antiPD1质粒的量设置为1ug+7ug、2ug+6ug、3ug+5ug、4ug+4ug、5ug+3ug、6ug+2ug、7ug+1ug这7种配比，进行CAR T细胞构建，构建方法同实施例2。
采用实施例3所述方法检测7种配比下构建得到的CAR T细胞的阳性率及抗体 分泌量，结果分别如图3A、3B。
实施例5：meso3CAR-antiPD1 T细胞对自身PD1的封闭作用
收集实施例2构建得到的meso3CAR T细胞和meso3CAR-antiPD1 T细胞，使用BD的流式抗体PE-鼠抗人CD279检测PD1的表达，流式方法实施例3。
结果如图4所示，自表达PD1抗体的meso3CAR-antiPD1 T可以封闭自身的PD1表达。
实施例6：meso3CAR T与meso3CAR-antiPD1 T细胞的杀伤功能对比
采用实时无标记细胞功能分析仪检测实施例2构建得到的meso3CAR T与meso3CAR-antiPD1 T细胞在体外对肿瘤细胞的杀伤作用。
具体而言，选取MHC class I分型匹配的效应细胞与靶细胞，应用艾森公司的实时无标记细胞功能分析仪(RTCA)检测上述两种CAR-T细胞的体外杀伤活性，具体步骤如下：
(1)调零：每孔加入50μl DMEM或1640培养液，放入仪器中，选择step 1，调零；
(2)靶细胞铺板：宫颈癌细胞Hela，卵巢癌细胞SK-OV-3、胃癌HGC-27(均购买于美国菌种保藏中心ATCC)按每孔10 4个细胞/50μl铺在含有检测电极的板中，放置数分钟，待细胞稳定一下，再放入仪器中，开始step 2，培养细胞；
(3)加入效应细胞：靶细胞培养24h后，暂停step 2，加入效应细胞，每孔50μl，效靶比分别设置为4:1，以未转质粒的Mock T细胞作为对照，开始step 3，继续共培养24h后，观察细胞增殖曲线。
结果如图5所示，自表达PD1抗体的meso3CAR-antiPD1 T细胞与单独的meso3CAR T细胞杀伤功能基本一致，抗体表达并没有影响CAR-T功能。
实施例7：meso3CAR与meso3CAR-antiPD1 T细胞在间皮素抗原的特异性刺激下细胞因子释放对比
用2ug/ml的间皮素抗原包被96孔板，4℃包被过夜，PBS清洗3遍，分别加入1×10 5个实施例2构建得到的meso3CAR、meso3CAR-antiPD1 T细胞以及对照的Mock T细胞(转入，培养24h后收集细胞上清。用BD TMCBA Human Th1/Th2 Cytokine Kit II检测这三种T细胞受间皮素抗原刺激后细胞因子的分泌情况，具体步骤如下：
(1)混合人的IL-2、IL-4、IL-6、IL-10、TNF-α、IFN-γ捕获磁珠，涡旋振荡混 匀捕获磁珠，每管加入50ul混匀后的捕获磁珠；
(2)加入50ul人的Th1/Th2细胞因子标准品(倍比稀释5000pg/ml、2500pg/ml、1250pg/ml、625pg/ml、312.5pg/ml、156pg/ml、80pg/ml、40pg/ml、20pg/ml、0pg/ml)和50ul的待测样品(经稀释液2倍稀释)；
(3)每管加入50ul的人的Th1/Th2-II-PE的检测抗体；
(4)室温避光孵育3h；
(5)每管加入1ml的洗涤缓冲液，200离心5min，弃上清；
(6)每管加入300ul的洗涤缓冲液重悬细胞，并转移至流式管中，用流式细胞仪检测荧光值。
结果如图6所示，自表达PD1抗体的meso3CAR-antiPD1 T细胞与单独的meso3CAR T细胞的细胞因子分泌量没有明显差异。
实施例8：meso3CAR与meso3CAR-antiPD1 T细胞对卵巢癌小鼠移植瘤模型的治疗效果
1、4～6周龄NSG完全免疫缺陷小鼠25只，平均重量22～27g，由百奥赛图生物公司提供，SPF级动物实验室饲养。
2、体外培育人卵巢癌细胞SK-OV-3-luc，取对数生长期贴壁生长细胞，0.25％胰酶消化，离心、收集细胞后用PBS重悬，1000rmp室温离心2分钟，弃上清，再用PBS液重悬后离心收集细胞，调整细胞悬液浓度至5×10 7个/ml。
3、于小鼠右肋背部皮下接种SK-OV-3-luc细胞，0.1ml/只。接种7天后，可通过活体成像仪观察荧光强度，将NSG免疫缺陷小鼠随机分为5组。每组注射相应的T细胞为1×10 7个/100ul，PBS组给予100ul PBS；给药途径为尾静脉注射。
4、每日观察小鼠的生活状态并每隔4天通过活体成像仪观察小鼠肿瘤变化。
结果如图7所示，在SK-OV-3卵巢癌小鼠移植瘤模型中，Meso3CAR-antiPD1 T细胞较Meso3CAR T细胞具有明显较好的治疗作用，而Meso3CAR-wt-antiPD1 T细胞基本没有效果。
尽管本发明的具体实施方式已经得到详细的描述。本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (10)

  1. 一种T细胞，所述T细胞自表达PD1抗体并靶向间皮素；优选地，所述T细胞：
    (1)含有识别间皮素的嵌合抗原受体的编码序列和PD-1抗体的编码序列；和/或
    (2)表达识别间皮素的嵌合抗原受体和PD-1抗体；
    优选地，所述T细胞的基因组中整合了PD1抗体的表达框以及识别间皮素的嵌合抗原受体的表达框。
  2. 如权利要求1所述的T细胞，其特征在于，所述识别间皮素的嵌合抗原受体从N端到C端依次含有任选的信号肽、抗间皮素III区单链抗体、铰链区、跨膜区、胞内共刺激信号域和胞内信号域。
  3. 如权利要求2所述的T细胞，其特征在于，
    所述信号肽为CD8信号肽、CD28信号肽、CD4信号肽或轻链信号肽；更优选地为CD8信号肽；优选地，所述CD8信号肽的氨基酸序列如SEQ ID NO:1第1-22位氨基酸残基所示；
    所述scFv的氨基酸序列如SEQ ID NO:1第23-272位氨基酸残基所示；
    所述铰链区为CD8α铰链区、IgD铰链区、IgG1 Fc CH2CH3铰链区或IgG4 Fc CH2CH3铰链区，优选为CD8α铰链区或IgG4 CH2CH3铰链区；优选地，所述IgG4 CH2CH3铰链区的氨基酸序列如SEQ ID NO:1第273-500位氨基酸残基所示；
    所述跨膜区为CD28跨膜区、CD8跨膜区、CD3ζ跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种；优选为CD8跨膜区，优选其氨基酸序列如SEQ ID NO:1第501-528位氨基酸残基所示；
    所述胞内共刺激信号域包括共刺激信号分子的胞内结构域，包括CD28、CD134/OX40、CD137/4-1BB、淋巴细胞特异性蛋白酪氨酸激酶、诱导性T细胞共刺激因子(ICOS)和DNAX激活蛋白10的胞内结构域；优选地，所述胞内共刺激信号域为CD28的胞内结构域；优选地，所述CD28的氨基酸序列如SEQ ID NO:1第529-569位氨基酸残基所示；和
    所述胞内信号域为CD3ζ胞内信号域或FcεRIγ胞内信号域；优选为CD3ζ胞内信号域，优选地所述CD3ζ胞内信号域的氨基酸序列如SEQ ID NO:1第570-681位氨基酸残基所示。
  4. 如权利要求2或3所述的T细胞，其特征在于，所述嵌合抗原受体具有以下一项或多项特征：
    所述信号肽的编码序列如SEQ ID NO:3第1-66位碱基所示；
    所述单链抗体的编码序列如SEQ ID NO:3第67-816位核苷酸序列所示；
    所述铰链区的编码序列如SEQ ID NO:3第817-1500位所示；
    所述跨膜区的编码序列如SEQ ID NO:3第1501-1584位碱基所示；
    所述胞内共刺激信号域的编码序列如SEQ ID NO:3第1585-1707位碱基所示；
    所述胞内信号域的编码序列如SEQ ID NO:3第1708-2043所示。
  5. 如权利要求2或3所述的T细胞，其特征在于，所述嵌合抗原受体含有CD8信号肽、抗间皮素III区单链抗体、IgG4CH2CH3铰链区、CD8跨膜区、CD28的胞内结构域和CD3ζ胞内信号域；
    优选地，所述嵌合抗原受体的氨基酸序列如SEQ ID NO:1第23-681位氨基酸残基所示，或如SEQ ID NO:1所示；优选地，所述嵌合抗原受体的编码序列如SEQ ID NO:3第67-2043位碱基所示，或如SEQ ID NO:3所示。
  6. 如权利要求1-5中任一项所述的T细胞，其特征在于，所述PD-1抗体含有抗PD-1单链抗体和IgG4Fc；其中，所述IgG4Fc的氨基酸序列如SEQ ID NO:2第267-495位氨基酸残基所示；
    优选地，所述抗PD-1单链抗体中抗体轻链可变区氨基酸序列如SEQ ID NO:2第21-131位氨基酸残基所示；优选地，所述抗PD-1单链抗体中的重链可变区氨基酸序列如SEQ ID NO:2第147-266位氨基酸序列所示；
    优选地，所述PD-1抗体还含有轻链信号肽；优选地，所述轻链信号肽的氨基酸序列如SEQ ID NO:2第1-20位氨基酸残基所示。
  7. 如权利要求6所述的T细胞，其特征在于，所述抗PD-1单链抗体的氨基酸序列如SEQ ID NO:2第21-266位氨基酸残基所示，或如SEQ ID NO:2所示；优选地，其编码序列如SEQ ID NO:4第61-798位碱基序列所示，或如SEQ ID NO:4所示；
    优选地，所述PD1抗体的氨基酸序列如SEQ ID NO:2第21-495位氨基酸序列所示，或者如SEQ ID NO:2所示；优选地，所述PD1抗体的编码序列如SEQ ID NO:4第61-1485位氨基酸残基所示，或如SEQ ID NO:4所示。
  8. 一种组合物或试剂盒，所述组合物含有：
    (1)含权利要求2-5中任一项所限定的嵌合抗原受体的表达框的载体，所述载体用于将所述表达框整合到宿主细胞的基因组中；和
    (2)含权利要求6-7所限定的PD1抗体的编码序列或其互补序列的载体，所述载体用于将所述表达框整合到宿主细胞的基因组中。
  9. 一种药物组合物，所述药物组合物含有权利要求1-7中任一项所述的T细胞或所述T细胞及其表达的PD1抗体。
  10. 权利要求1-7中任一项所述的T细胞或所述T细胞及其表达的PD1抗体在制备 治疗或预防恶性肿瘤的药物中的用途；优选地，所述恶性肿瘤为其癌细胞表面异常表达间皮素的癌症；优选地，所述癌症选自：腺癌、间皮瘤、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、黑色素瘤、非小细胞肺癌、肾细胞癌、头颈鳞状上皮细胞癌、直肠癌、霍奇金淋巴瘤、胰腺癌或前列腺癌；更优选地，所述癌症是间皮素和CA125/MUC16同时高表达的癌症。
PCT/CN2018/123560 2017-12-28 2018-12-25 自表达pd-1抗体并靶向间皮素的嵌合抗原受体修饰t细胞及其用途 WO2019128998A1 (zh)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017011580A2 (en) * 2015-07-13 2017-01-19 Cytomx Therapeutics, Inc. Anti-pd-1 antibodies, activatable anti-pd-1 antibodies, and methods of use thereof
CN106459989A (zh) * 2013-12-19 2017-02-22 诺华股份有限公司 人间皮素嵌合抗原受体及其用途

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CN105026429B (zh) * 2012-08-21 2018-09-25 美国政府(由卫生和人类服务部的部长所代表) 间皮素结构域-特异性单克隆抗体及其用途
EP3094653B1 (en) * 2014-01-13 2019-10-23 Stephen J. Forman Chimeric antigen receptors (cars) having mutations in the fc spacer region and methods for their use
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HUE056201T2 (hu) * 2015-07-30 2022-02-28 Macrogenics Inc PD-1-hez kötõdõ molekulák és alkalmazásukra szolgáló eljárások
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Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459989A (zh) * 2013-12-19 2017-02-22 诺华股份有限公司 人间皮素嵌合抗原受体及其用途
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