WO2019129143A1 - 靶向ErbB受体家族的嵌合抗原受体修饰T细胞及其用途 - Google Patents

靶向ErbB受体家族的嵌合抗原受体修饰T细胞及其用途 Download PDF

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WO2019129143A1
WO2019129143A1 PCT/CN2018/124370 CN2018124370W WO2019129143A1 WO 2019129143 A1 WO2019129143 A1 WO 2019129143A1 CN 2018124370 W CN2018124370 W CN 2018124370W WO 2019129143 A1 WO2019129143 A1 WO 2019129143A1
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amino acid
sequence
cancer
cells
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钱其军
金华君
游术梅
江芏青
李林芳
王超
崔连振
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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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • 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)
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention belongs to genetic engineering and immunology, and relates to chimeric antigen receptor-modified T cells targeting the ErbB receptor family and uses 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 (CAR-T) therapy is one of the important branches of tumor immunotherapy. It has achieved very good curative effect in malignant hematological tumors. The complete remission rate of relapsed and refractory B cell leukemia is over 90. %.
  • 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
  • CAR-T cells After extensive expansion in vitro, CAR-T cells are returned to the patient and can exhibit potent anticancer effects in a non-MHC-restricted mode.
  • Point CART cells have better in vivo therapeutic effects and can reduce recurrence rates (J Clin Invest. 2016 Oct 3; 126(10)).
  • most of the antigen recognition regions of these CARs are artificially constructed single-chain antibodies, which have strong immunogenicity, and the in vivo treatment is easily recognized and eliminated, resulting in poor therapeutic effect. Therefore, the use of natural peptides to construct a multi-target antigen recognition region has the advantages of weak immunogenicity and better targeting range.
  • the ErbB receptor family contains four members, the epidermal growth factor receptor ErbB1 (EGFR/Her2), ErbB2 (Her2), ErbB3 (Her3), and ErbB4 (Her4).
  • ErbB1 epidermal growth factor receptor
  • ErbB2 ErbB2
  • ErbB3 ErbB3
  • ErbB4 ErbB4
  • the ligand activates the ErbB receptor tyrosine kinase, it can cause dimerization between the receptors, and autophosphorylation of tyrosine, thereby activating the downstream signaling pathway.
  • the expression levels of these four receptors are at a low level.
  • many malignant tumors are associated with overexpression of ErbB1 and/or ErbB2, including head and neck cancer, breast cancer, lung cancer, gastrointestinal cancer, prostate cancer, and pancreatic cancer.
  • anti-tumor drugs in clinical research are monoclonal antibodies and small-molecule tyrosine kinase inhibitors that target the extracellular domain of different receptors of ErbB.
  • HER2 humanized antibody Herceptin has been approved by the FDA. Clinical treatment of breast cancer.
  • ErbB receptors usually function in the form of tightly bound dimers.
  • the Her2 single receptor lacks a high-affinity ligand, but it is the best dimerization partner, especially when dimerized with EGFR or Her3 to enhance tyrosine kinase signaling.
  • Her3 has ligand binding ability but lacks tyrosine kinase activity.
  • the heterodimer formed with Her2 is the most powerful signal complex.
  • the most representative heterodimer in breast cancer is Her2/Her3. .
  • Anti-tumor drugs that target a certain receptor alone are likely to cause tumor recurrence because they cannot target other ErbB receptor dimers.
  • T1E is a chimeric polypeptide consisting of seven amino acids at the N-terminus of human transcriptional growth factor alpha (TGF ⁇ ) and 48 amino acids at the C-terminus of epidermal growth factor (EGF), which is based on ErbB1-based homodimers and isoforms.
  • the source dimer has a high affinity, and in addition, T1E can effectively bind ErbB2/3 heterodimer.
  • Herstatin is a Her2 truncated version of Her2 that is selectively cleaved, and its sequence includes 340 amino acids of the extracellular domain of Her2, I and II, and 79 amino acids encoded by the eighth intron. Soluble Her2 autoinhibitory factor. Herstatin binds to EGFR or Her2 with high affinity, the main function of which is the 79 amino acids encoded by the eighth intron (designated Herin).
  • the natural T1E and Herin fusion expression is used as an antigen recognition region of CAR, and the two can complement each other to recognize the ErbB receptor family and expand the targeting range.
  • the present invention provides a chimeric antigen receptor comprising, in order from the N-terminus to the C-terminus, an optional membrane protein signal peptide, a T1E peptide segment joined by a linker, and a Herin peptide segment, 50 amino acids in length. Hinge region, transmembrane region, intracellular costimulatory signal domain and intracellular signal domain above the residue.
  • the signal peptide is selected from the group consisting of a CD8 signal peptide, a CD28 signal peptide, and a CD4 signal peptide; preferably, the signal peptide is a CD8 signal peptide, preferably having an amino acid sequence of SEQ ID NO: 15 The amino acid residues 1-22 are shown.
  • amino acid sequence of the T1E is as shown in amino acid residues 23-77 of SEQ ID NO: 15.
  • amino acid sequence of Herin is set forth in amino acid sequence 93-171 of SEQ ID NO: 15.
  • the linker is selected from the group consisting of a flexible linker and a rigid linker; preferably, the flexible linker is a linker containing G and S, the rigid linker being a linker containing an EAAAK repeat sequence; preferably, The amino acid sequence of the linker is shown as amino acid residues 78-92 of SEQ ID NO: 15.
  • the hinge region longer than 50 amino acid residues is selected from the group consisting of a CD8 alpha hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region, and an IgG4 Fc CH2CH3 hinge region; preferably, the hinge region Is a CD8 ⁇ hinge region or an IgG4 Fc CH2CH3 hinge region; preferably, the amino acid sequence of the CD8 ⁇ hinge region is set forth in SEQ ID NO: 17, and the amino acid sequence of the IgG4 FcCH2CH3 hinge region is SEQ ID NO: 15 at 172-399 The amino acid sequence is shown.
  • the transmembrane region is selected from the group consisting 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.
  • the transmembrane region is a CD8 transmembrane region or a CD28 transmembrane region; preferably, the amino acid sequence of the CD8 transmembrane region is as set forth in SEQ ID NO: 18, The amino acid sequence of the CD28 transmembrane region is shown as amino acid residues 400-427 of SEQ ID NO: 15.
  • the intracellular costimulatory signal domain is an intracellular domain of a costimulatory signaling molecule selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, and DAP10 intracellular structures
  • a costimulatory signaling molecule selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, and DAP10 intracellular structures
  • the intracellular domain of the costimulatory signaling molecule is the CD137 intracellular domain, the amino acid sequence of which is set forth in SEQ ID NO: 19, or the costimulatory signal molecule
  • the intracellular domain is the CD28 intracellular domain and its amino acid sequence can be as shown in amino acid residues 428-468 of SEQ ID NO: 15.
  • the intracellular signal domain is an immunoreceptor tyrosine activation motif selected from the group consisting of a CD3 ⁇ and a tyrosine activation motif of Fc ⁇ RI ⁇ ; preferably, the immunoreceptor tyrosine
  • the activation motif is a CD3 ⁇ tyrosine activation motif, preferably, the amino acid sequence of which is shown in amino acid residues 469-580 of SEQ ID NO: 15.
  • the chimeric antigen receptor comprises, optionally from the N-terminus to the C-terminus, an optional CD8 signal peptide, a T1E, an EAAAK repeat, a Herin, an IgG4 Fc CH2CH3 hinge region, a CD28 transmembrane region, CD28 intracellular domain and CD3 ⁇ tyrosine activation motif.
  • the chimeric antigen receptor comprises, optionally from the N-terminus to the C-terminus, an optional CD8 signal peptide, Herin, EAAAK repeat, T1E, IgG4 Fc CH2CH3 hinge region, CD28 transmembrane region, CD28 intracellular domain and CD3 ⁇ tyrosine activation motif.
  • amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 15 or 16.
  • the invention also provides a polynucleotide sequence selected from the group consisting of:
  • the coding sequence of the chimeric antigen receptor has one or more of the characteristics:
  • the nucleotide sequence of the signal peptide is shown in SEQ ID NO:1;
  • the nucleotide sequence of the T1E is shown in SEQ ID NO: 2;
  • the nucleotide sequence of Herin is as shown in SEQ ID NO: 3;
  • the nucleotide sequence of the linker is shown in SEQ ID NO: 4;
  • the nucleotide sequence of the hinge region is as shown in SEQ ID NO: 5 or 6;
  • the nucleotide sequence of the transmembrane region is as shown in SEQ ID NO: 7 or 8;
  • nucleotide sequence of the intracellular domain of the costimulatory signal molecule is as set forth in SEQ ID NO: 9 or 10;
  • the nucleotide sequence of the immunoreceptor tyrosine activating motif is set forth in SEQ ID NO:11;
  • the polynucleotide sequence is selected from the polynucleotide sequence set forth in SEQ ID NO: 13 or 14, or a complement thereof.
  • the invention also provides a nucleic acid construct comprising a polynucleotide sequence as described herein.
  • the nucleic acid construct is an expression vector.
  • the expression vector is a eukaryotic expression vector, preferably comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 and Ty.
  • the invention also provides a recombinant host cell comprising a nucleic acid construct described herein, or a chimeric antigen receptor as described herein.
  • the host cell is a mammalian cell; more preferably, the host cell is a T cell; more preferably, the host cell is a primary cultured T cell.
  • the invention also provides the use of a chimeric antigen receptor and/or a coding sequence thereof and/or a nucleic acid construct described herein for the preparation of a recombinant host cell for use in the treatment or prevention of cancer; and a recombinant host cell as described herein Use in the preparation of a medicament for the treatment or prevention of cancer.
  • the cancer is a cancer whose surface of the cancer cell abnormally expresses at least one EGFR family member protein; preferably, the cancer is selected from the group consisting of: head and neck cancer, liver cancer, adenocarcinoma, lung cancer, colon cancer, Colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or prostate cancer.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant host cell as described herein and a pharmaceutically acceptable carrier.
  • the present invention also provides a polynucleotide sequence which is represented by SEQ ID NO: 3 or which is a complement of the sequence of SEQ ID NO: 3.
  • the present invention also provides a fusion protein which is formed by linking T1E and Herin through a linker sequence; wherein the T1E is composed of seven amino acids of the N-terminus of human transcriptional growth factor ⁇ and 48 amino acids of the C-terminus of epidermal growth factor; The 79th amino acid encoded by the eighth intron of Herstatin; the linker is selected from the group consisting of a flexible linker, a rigid linker and an in vivo cleavage linker; preferably a flexible linker comprising G and S, or a rigid linker comprising an EAAAK repeat sequence.
  • amino acid sequence of the fusion protein is set forth in amino acid sequence 23-171 of SEQ ID NO: 15 or 16.
  • the present invention also provides the coding sequence of the fusion protein or the complement thereof; preferably, the sequence is represented by the nucleotide sequence of positions 67-513 of SEQ ID NO: 13 or 14.
  • Figure 1 Schematic diagram of the structure of chimeric antigen receptor genes targeting various ErbB receptor families.
  • Figure 2 RT-PCR detection of copy number results for CAR gene expression in eCAR T cells and HerinCAR T cells.
  • Figure 3 Comparison of killing of eCAR T cells and HerinCAR T cells, including human non-small cell lung cancer H23-LUC and human lung cancer cells H292-LUC.
  • Figure 4 Copy number results for CAR gene expression in eCAR T cells, EHCAR-GS T cells, HECAR-GS T cells, EHCAR-EK T cells, and HECAR-EK T cells.
  • Figure 5 Killing comparison of eCAR T cells, EHCAR-GS T cells, HECAR-GS T cells, EHCAR-EK T cells and HECAR-EK T cells, including human ovarian cancer cells SKOV3-LUC and human liver cancer cells HCCLM3-LUC.
  • FIG. 6 Each set of columns shows left-to-right Mock T cells, HECAR-GS T cells, EHCAR-GS T cells, HECAR-EK T cells, EHCAR-EK T cells, and eCAR T cells under EGFR antigen stimulation -2, IL-4, IL-6, IL-10, TNF- ⁇ and IFN- ⁇ cytokine secretion changes.
  • Figure 7 Copy number results for CAR gene expression in eCAR T cells, EHCAR-EK-28 TIZ T cells, and HECAR-EK-28 TIZ T cells.
  • Figure 8 Comparison of killing of eCAR T cells, EHCAR-EK-28TIZ T cells and HECAR-EK-28TIZ T cells, including human ovarian cancer cells SKOV3-LUC, human hepatoma cells HCCLM3-LUC and human non-small cell lung cancer H23-LUC .
  • Figure 9 Each group of columns showed Mock T cells, eCAR T cells, EHCAR-EK-28TIZ T cells, and HECAR-EK-28TIZ T cells from left to right, respectively.
  • Figure 10 Changes in fluorescence values of tumor cells on different days after treatment of mice with eCAR T cells, EHCAR-EK-28 TIZ T cells, and HECAR-EK-28 TIZ T cells, Mock-T cells, and PBS blank controls, respectively.
  • expression cassette refers to the entire element required for expression of a gene, including a promoter and a gene coding 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 may be secreted or membrane anchored; preferably a membrane anchored.
  • the present invention devises a novel CAR-T cell targeting the ErbB receptor family.
  • the antigen recognition site of the CAR includes intact T1E and Herin structures, which are linked by a linker. Since the ErbB receptor usually functions in the form of a tightly bound dimer, the mechanism is complicated.
  • the inventors conducted various A series of CAR structures were designed, including CARs expressing T1E and Herin separately, CARs expressing T1E and Herin, and changing the positional order of the two fragments, different joints, and different transmembrane hinge regions.
  • the two CARs with the best in vitro were obtained, one containing the CD8 signal peptide, the T1E, the EAAAK linker, the Herin, the IgG4 FcCH2CH3 hinge region, and the CD28 transmembrane region from the N-terminus to the C-terminus.
  • EHCAR-EK-28TIZ CD28 intracellular domain and CD3 ⁇ tyrosine activation motif
  • HECAR-EK-28TIZ CD28 intracellular domain and CD3 ⁇ tyrosine activation motif
  • the present invention includes a chimeric antigen receptor (CAR) having the following structural features: from the N-terminus to the C-terminus, optionally containing an optional membrane protein signal peptide, a T1E peptide segment joined by a linker, and a Herin peptide segment (two peptides) The position is changeable, the hinge region, the transmembrane region, the intracellular costimulatory signal domain, and the intracellular signal domain are longer than 50 amino acid residues.
  • CAR chimeric antigen receptor
  • 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 herein is a membrane protein signal peptide, which may be selected from the group consisting of a CD8 signal peptide, a CD28 signal peptide, and a CD4 signal peptide. In a preferred embodiment, the invention employs a CD8 signal peptide.
  • the amino acid sequence of an exemplary CD8 signal peptide is shown as amino acid residues 1-22 of SEQ ID NO: 15; the nucleotide sequence of an exemplary CD8 signal peptide can be as set forth in SEQ ID NO: 1.
  • T1E is a chimeric polypeptide consisting of seven amino acids at the N-terminus of human transcriptional growth factor alpha (TGF ⁇ ) and 48 amino acids at the C-terminus of epidermal growth factor (EGF), which is based on ErbB1-based homodimers and isoforms. Source dimers have a high affinity. In addition, T1E is also effective in binding ErbB2/3 heterodimers.
  • the amino acid sequence of T1E of the present invention is shown in amino acid residues 23 to 77 of SEQ ID NO: 15; an exemplary nucleotide sequence can be shown as SEQ ID NO: 2.
  • Herstatin is a Her2 truncated version of Her2 that is selectively cleaved, and its sequence includes 340 amino acids of the extracellular domain of Her2, I and II, and 79 amino acids encoded by the eighth intron. Soluble Her2 autoinhibitory factor.
  • the Herin of the present invention is 79 amino acids encoded by the eighth intron of Herstatin, and its amino acid sequence is shown as amino acid residues 93-171 of SEQ ID NO: 15.
  • the invention optimizes codons encoding the amino acids thereof to obtain the nucleotide sequence set forth in SEQ ID NO:3.
  • the linker is used to link different amino acid sequences, which can stabilize the spatial structure of the protein and enhance its biological activity.
  • Suitable joints for use herein include flexible joints, rigid joints, and in vivo crack joints.
  • the flexible joints used herein are GS joints, ie, linkers that primarily contain G and S.
  • the nucleotide sequence of an exemplary GS linker is SEQ ID NO: 12; in certain embodiments, a rigid linker, such as a linker comprising an EAAAK repeat, herein also referred to as an EAAAK linker, is used herein.
  • the amino acid sequence of an exemplary EAAAK linker is amino acid residues 78-92 of SEQ ID NO: 15, and the exemplary nucleotide sequence is set forth in SEQ ID NO: 4.
  • 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 and antigenic epitope of the antibody. Complementary complementarity.
  • 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 plurality of hinge regions preferably having a length of 50 amino acid residues or more, more preferably 80 amino acid residues or more.
  • a CD8 alpha hinge region and an IgG4 Fc CH2CH3 hinge region are used herein.
  • An exemplary amino acid sequence of the CD8 alpha hinge region can be set forth in SEQ ID NO: 17, and an exemplary nucleotide sequence is set forth in SEQ ID NO: 5.
  • the amino acid sequence of the exemplary IgG4 FcCH2CH3 hinge region can be represented by amino acid sequence 172-399 of SEQ ID NO: 15, and an exemplary nucleotide sequence thereof is set forth in SEQ ID NO: 6.
  • the transmembrane region can be selected from one or more of the CD28 transmembrane region, the CD8 transmembrane region, the CD3 ⁇ transmembrane region, the CD134 transmembrane region, the CD137 transmembrane region, the ICOS transmembrane region, and the DAP10 transmembrane region.
  • the transmembrane region of a chimeric antigen receptor for use herein is a CD8 transmembrane region and a CD28 transmembrane region.
  • An exemplary amino acid sequence of the CD8 transmembrane region can be set forth in SEQ ID NO: 18, and an exemplary nucleotide sequence can be set forth in SEQ ID NO: 7.
  • the amino acid sequence of an exemplary CD28 transmembrane region can be represented by amino acid residues 400-427 of SEQ ID NO: 15, and an exemplary nucleotide sequence thereof can be set forth in SEQ ID NO: 8.
  • the intracellular costimulatory signal domain comprises an intracellular domain of a costimulatory signaling molecule, which may be selected from one or more of the intracellular domains of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, and DAP10.
  • the intracellular domain of CD28 or the intracellular domain of CD137 is used.
  • An exemplary amino acid sequence of the CD137 intracellular domain can be set forth in SEQ ID NO: 19, and an exemplary nucleotide sequence can be set forth in SEQ ID NO: 9; the exemplary amino acid sequence of the CD28 intracellular domain can be An exemplary nucleotide sequence can be set forth in SEQ ID NO: 10 as indicated by amino acid residues 428-468 of SEQ ID NO: 15.
  • the intracellular signal domain can be an immunoreceptor tyrosine activation motif and can be selected from the tyrosine activation motif of CD3 ⁇ and/or Fc ⁇ RI ⁇ .
  • the amino acid sequence of an exemplary CD3 ⁇ tyrosine activating motif can be represented by amino acid residues 469-580 of SEQ ID NO: 15, and an exemplary nucleotide sequence can be set forth in SEQ ID NO: 11.
  • the above various components of the chimeric antigen receptors are formed, such as a CD8 signal peptide, a T1E fragment, a Herin fragment, a T1E fragment and a Herin fragment joined by a linker, a hinge region, a transmembrane region, a co-stimulatory signal molecule intracellular domain, and an immunity
  • the receptor tyrosine activating motifs and the like may be directly linked to each other or may be linked 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.
  • the chimeric antigen receptors herein comprise, in order from the N-terminus to the C-terminus, an optional CD8 signal peptide, a T1E, a CD8 hinge region, a CD8 transmembrane region, a CD137 intracellular domain, and a CD3 ⁇ tyrosine. Activation motif.
  • the chimeric antigen receptors herein comprise, in order from the N-terminus to the C-terminus, an optional CD8 signal peptide, Herin, IgG4 FcCH2CH3 hinge region, CD8 transmembrane region, CD137 intracellular domain, and CD3 ⁇ tyrosine Acid activation motif.
  • the chimeric antigen receptors herein comprise, in order from the N-terminus to the C-terminus, an optional CD8 signal peptide, Herin, GS linker, T1E, CD8 hinge region, CD8 transmembrane region, CD137 intracellular domain And CD3 ⁇ tyrosine activation motif.
  • the chimeric antigen receptors herein comprise, in order from the N-terminus to the C-terminus, an optional CD8 signal peptide, T1E, GS linker, Herin, CD8 hinge region, CD8 transmembrane region, CD137 intracellular domain And CD3 ⁇ tyrosine activation motif.
  • the chimeric antigen receptors herein comprise, in order from the N-terminus to the C-terminus, an optional CD8 signal peptide, Herin, EAAAK linker, T1E, CD8 hinge region, CD8 transmembrane region, CD137 intracellular domain And CD3 ⁇ tyrosine activation motif.
  • the chimeric antigen receptors herein comprise, in order from the N-terminus to the C-terminus, an optional CD8 signal peptide, T1E, EAAAK linker, Herin, CD8 hinge region, CD8 transmembrane region, CD137 intracellular domain And CD3 ⁇ tyrosine activation motif.
  • the chimeric antigen receptors herein comprise, in order from the N-terminus to the C-terminus, an optional CD8 signal peptide, Herin, EAAAK linker, T1E, IgG4 FcCH2CH3 hinge region, CD28 transmembrane region, CD28 intracellular structure Domain and CD3 ⁇ tyrosine activation motif.
  • the chimeric antigen receptors herein comprise, in order from the N-terminus to the C-terminus, an optional CD8 signal peptide, T1E, EAAAK linker, Herin, IgG4 FcCH2CH3 hinge region, CD28 transmembrane region, CD28 intracellular structure Domain and CD3 ⁇ tyrosine activation motif.
  • the chimeric antigen receptor comprises, optionally from the N-terminus to the C-terminus, an optional CD8 signal peptide, a T1E, an EAAAK linker, a Herin, an IgG4 FcCH2CH3 hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and a CD3 sputum. Amino acid activation motif.
  • amino acid sequence of an exemplary chimeric antigen receptor is shown in amino acid residues 23 to 580 of SEQ ID NO: 15, or as shown in SEQ ID NO: 15; preferably, the nucleotide sequence thereof is nucleus
  • the nucleotide sequence may be represented by the base sequence of SEQ ID NO: 13 at positions 67-1740, or as shown in SEQ ID NO: 13.
  • the chimeric antigen receptor comprises, optionally from the N-terminus to the C-terminus, an optional CD8 signal peptide, Herin, EAAAK linker, T1E, IgG4 FcCH2CH3 hinge region, CD28 transmembrane region, CD28 intracellular domain and CD3 sputum Amino acid activation motif.
  • An exemplary amino acid sequence of such a chimeric antigen receptor can be represented by amino acid residues 23-580 of SEQ ID NO: 16, or as set forth in SEQ ID NO: 16; preferably, the nucleotide sequence thereof is As shown in SEQ ID NO: 14, bases 67-1740, or as shown in SEQ ID NO: 14.
  • 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.
  • 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.
  • a template a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template.
  • nucleic acid constructs comprising a polynucleotide sequence encoding a chimeric antigen receptor as described herein, and one or more regulatory sequences operably linked to the sequences.
  • the nucleic acid constructs of the invention are expression cassettes.
  • 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 sequences for CAR herein can be cloned into a variety of 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 comprising a replication initiation site, a 3'LTR, a 5' LTR, a coding sequence for a CAR described herein, and optionally a Selected tag.
  • 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: 1 of the application.
  • the entire contents of this application are herein incorporated by reference.
  • 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.
  • an expression vector herein is an integration vector for integration of a coding sequence for a CAR of the invention into a host cell.
  • an expression vector herein is a eukaryotic expression vector, in particular a transposon vector.
  • 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.
  • Between the 5' LTR and the 3' LTR is the expression cassette for the CAR of the invention, including the corresponding promoter sequence, the coding sequence for the CAR, and sequences such as the polyA tailing signal.
  • a nucleic acid construct or expression vector herein comprises, in sequence from 5' to 3', a transposon 5' inverted terminal repeat (5'LTR), a promoter, a CD8 signal peptide coding sequence, T1E fragment and Herin fragment coding sequence, CD8 hinge region coding sequence or IgG4 Fc CH2CH3 hinge region coding sequence, CD8 transmembrane region coding sequence, CD28 intracellular domain coding sequence, CD3 ⁇ tyrosine activation motif linked by EAAAK-linker
  • the transposon vector may also contain a transposase coding sequence and a promoter that controls expression of the transposase coding sequence.
  • the eukaryotic expression vector is a pNB328 vector.
  • the vectors herein can be introduced into host cells by conventional methods, including microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like.
  • the nucleic acid constructs herein are introduced into a host cell using electroporation. Specifically, the recombinant plasmid is transferred to the host cell of interest by electrokinetic electrokinetics.
  • Host cells suitable for use herein can be mammalian cells well known in the art, preferably T cells, including various types of T cells from various sources.
  • T cells can be derived from PBMC in patients with B cell malignancies.
  • the T cell is a primary cultured T cell.
  • a recombinant host cell comprising a coding sequence for a chimeric antigen receptor described herein or a nucleic acid construct described herein; and/or the recombinant host cell expresses a Chimeric antigen receptor.
  • the recombinant host cell can be a host cell as described above that has been introduced into a vector described herein.
  • Herrin fragments for use in the chimeric antigen receptors herein and their optimized codon sequences are also included within the scope of this document. More specifically, the Herrin fragment whose nucleotide sequence is as shown in SEQ ID NO: 3 is included herein.
  • the IgG4 Fc CH2CH3 hinge region and its coding sequences for use in the chimeric antigen receptors herein are also included within the scope of the disclosure. More specifically, the IgG4 Fc CH2CH3 hinge region shown in SEQ ID NO: 6 and its coding sequence (including degenerate sequences) are included herein.
  • the use herein includes the IgG4 Fc CH2CH3 hinge region and/or its coding sequence for the preparation of a chimeric antigen receptor and/or a coding sequence thereof; the coding sequence of the chimeric antigen receptor is Use of a recombinant expression vector; use of the nucleic acid construct for the preparation of a recombinant host cell; and use of the recombinant host cell for the preparation of a medicament for the treatment or prevention of cancer.
  • the IgG4 Fc CH2CH3 hinge region and/or coding sequence thereof, the chimeric antigen receptor and/or coding sequence thereof, and the nucleic acid construct are prepared for use in the treatment or prevention of cancer The use of recombinant host cells.
  • Cancers suitable for treatment or prevention by the CAR or its expression cells described herein are preferably at least one EGFR family protein positive cancer, specifically a cancer having abnormal expression of ErbBl, ErbB2, ErbB3 and ErbB4 on the surface of cancer cells.
  • cancers may be selected from the group consisting of: head and neck cancer, liver cancer, adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer or Prostate cancer.
  • the chimeric antigen receptors herein have excellent killing effects on tumor cells that express both ErbB1 and ErbB2, such as ovarian cancer and lung cancer, and tumor cells that express ErbB1 alone, such as liver cancer.
  • Such CARs or their expressing cells are particularly useful for treating cancers that are positive for at least one EGFR family member.
  • kits comprising the recombinant expression vector described herein.
  • the kit may also contain reagents suitable for the transfer of the recombinant expression vector into a cell, and optionally instructions for those skilled in the art to transfer the recombinant expression vector into a cell.
  • composition comprising a recombinant host cell described herein and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a carrier well known in the art for cell administration, including but not limited to the pNB328 vector.
  • Methods of treating or preventing cancer are also included herein, the method comprising the step of administering a recombinant host cell or pharmaceutical composition described herein to a subject in need thereof.
  • the method of administration can be a method commonly used in cell therapy.
  • the dose administered can be considered based on factors such as the sex, age, disease, and physical condition of the patient.
  • Example 1 Recombinant plasmids pNB328-eCAR, pNB328-HerinCAR, pNB328-EHCAR-GS, pNB328-HECAR-GS, pNB328-EHCAR-EK, pNB328-HECAR-EK, pNB328-EHCAR-EK-28TIZ and pNB328-HECAR- Construction of EK-28TIZ
  • the nucleotide sequence of the CD8 signal peptide in the structural pattern diagram is shown in SEQ ID NO: 1; the nucleotide sequence of T1E is shown in SEQ ID NO: 2; the nucleotide sequence of Herin is shown in SEQ ID NO: 3.
  • EAAAK linker EAAAK linker
  • GS linker GS-linker
  • CD8 alpha hinge transmembrane region
  • the nucleotide sequence of CD8EC is shown in SEQ ID NO: 5
  • the nucleotide sequence of the mutant IgG4 Fc CH2CH3 hinge transmembrane region is shown in SEQ ID NO: 6
  • the nucleotide sequence of CD8 transmembrane region CD8TM is SEQ.
  • CD28TM the nucleotide sequence of CD28 transmembrane region
  • CD137IC the nucleotide sequence of CD137 intracellular costimulatory signal structural region
  • SEQ ID NO: 10 The CD28 intracellular costimulatory signal structural region (CD28IC) nucleotide sequence is set forth in SEQ ID NO: 10; the CD3 sputum intracellular signal domain nucleotide sequence is set forth in SEQ ID NO:11.
  • 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 chimeric antigen receptor-modified T cells targeting the ErbB receptor family
  • 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.
  • pNB328-HerinCAR pNB328-EHCAR-GS, pNB328-HECAR-GS, pNB328-EHCAR-EK, pNB328-HECAR-EK, pNB328-EHCAR-EK-28TIZ and pNB328-HECAR-EK-28TIZ, resuspended and mixed cells, Add 6 ug of control plasmid (ie, pNB328 empty plasmid to construct Mock T cells) into the tube; transfer the mixture to an electric rotor, place the electrosurgical instrument, select the required procedure, and perform electric shock; use the micropipette in the kit to transfer the electricity well.
  • control plasmid ie, pNB328 empty plasmid to construct Mock T cells
  • the effector cells and target cells matched by MHC class I were selected, and the in vitro killing activity of eCAR-T and HerinCAR-T obtained in Example 2 was detected by using Essen's real-time label-free cell function analyzer (RTCA).
  • RTCA real-time label-free cell function analyzer
  • Example 5 Comparison of copy number of eCAR, EHCAR-GS, HECAR-GS, EHCAR-EK and HECAR-EK expression by RT-PCR
  • Mock T cells, eCAR T, EHCAR-GS, HECAR-GS, EHCAR-EK and HECAR-EK cells on day 10 after electroporation in Example 2 were collected, cell pellets were collected according to the number of 1 ⁇ 10 6 cells, and cell pellet was washed with PBS.
  • Example 6 Comparison of killing functions of eCAR, EHCAR-GS, HECAR-GS, EHCAR-EK and HECAR-EK
  • Example 2 Test the in vitro killing activity of eCAR T cells, EHCAR-GS T cells, HECAR-GS T cells, EHCAR-EK T cells and HECAR-EK T cells obtained in Example 2 according to the method of Example 4, using target cells They are human ovarian cancer cell SKOV3-LUC and human liver cancer cell HCCLM3-LUC.
  • Example 7 Comparison of cytokine release by eCAR T cells, EHCAR-GS T cells, HECAR-GS T cells, EHCAR-EK T cells and HECAR-EK T cells under specific stimulation of EGFR antigen
  • the 96-well plate was coated with 5 ug/ml of EGFR antigen, coated at 4 ° C overnight, washed 3 times with PBS, and 1 ⁇ 10 5 (100 ul volume) of the eCAR T cells prepared in Example 2, EHCAR-GS T cells, HECAR-GS T cells, EHCAR-EK T cells and HECAR-EK T cells and control Mock T cells (transferred into pNB328 empty vector) were cultured for 24 h to collect cell supernatants.
  • EHCAR-GS T cells, HECAR-GS T cells, EHCAR-EK T cells and HECAR-EK T cells and control Mock T cells (transferred into pNB328 empty vector) were cultured for 24 h to collect cell supernatants.
  • BD TM CBA Human Th1 / Th2 Cytokine Kit II detected by the four T cell secretion of the cytokines stimulated EGFR 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
  • 20 pg/ml, 0 pg/ml 50 ul of the sample to be tested
  • Example 8 Comparison of copy number expression of eCAR T cells, EHCAR-EK-28 TIZ T cells and HECAR-EK-28TIZ T cells by RT-PCR
  • Example 9 Comparison of killing function of eCAR T cells, EHCAR-EK-28TIZ T cells and HECAR-EK-28TIZ T cells
  • the in vitro killing activity of eCAR T cells, EHCAR-EK-28TIZ T cells and HECAR-EK-28TIZ T cells was tested according to the method of Example 4.
  • the target cells used were human ovarian cancer cells SKOV3-LUC and human hepatoma cells HCCLM3. - LUC and human non-small cell lung cancer cells.
  • Example 10 Comparison of cytokine release by eCAR T cells, EHCAR-EK-28 TIZ T cells and HECAR-EK-28 TIZ T cells under specific stimulation of EGFR antigen
  • the 96-well plate was coated with 5 ug/ml of EGFR antigen, coated at 4 ° C overnight, washed 3 times with PBS, and 1 ⁇ 10 5 (100 ul volume) of the eCAR T cells prepared in Example 2, EHCAR-EK-28TIZ T were added.
  • Cells and HECAR-EK-28 TIZT cells and control Mock T cells were cultured for 24 h and cell supernatants were collected. According to the method of Example 7, the secretion of cytokines after stimulation of the EGFR antigen by these four T cells was examined.
  • Example 11 In vivo anti-tumor effects of eCAR T cells, EHCAR-EK-28 TIZ T cells and HECAR-EK-28 TIZ T cells.
  • mice of 4-6 weeks old were purchased and divided into 5 groups, 4 rats in each group.
  • Hepatoma cell line HCCLM3-LUC was inoculated, each 1 ⁇ 10 7 , 10 days after tumor formation, PBS was injected into the tail vein respectively (100ul And Mock-T, eCAR T cells, EHCAR-EK-28 TIZ T cells and HECAR-EK-28 TIZ T cells (1 ⁇ 10 7 cells/cell) obtained in Example 2, and observed changes in tumor fluorescence in mice.
  • PBS and Mock-T cells had no therapeutic effect on the tumor model, and eCAR T cells, EHCAR-EK-28TIZ T cells, and HECAR-EK-28TIZ T cells had excellent antitumor effects, and EHCAR.
  • eCAR T cells, EHCAR-EK-28TIZ T cells, and HECAR-EK-28TIZ T cells had excellent antitumor effects, and EHCAR.
  • - EK-28TIZ T cells and HECAR-EK-28TIZ T cells work better.

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Abstract

提供了一种嵌合抗原受体，该嵌合抗原受体从N端到C端依次含有膜蛋白信号肽、通过接头连接的T1E肽段和Herin肽段、长50个氨基酸残基以上的铰链区、跨膜区、共刺激信号分子胞内结构域和免疫受体酪氨酸活化基序。还提供了该嵌合抗原受体修饰的T细胞及其用途。

Description

靶向ErbB受体家族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，涉及靶向ErbB受体家族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。
嵌合抗原受体T细胞(CAR-T)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％。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，将这种疗法复制到实体瘤时却遇到了很多障碍。一是实体瘤缺乏肿瘤特异性抗原，无法找到完美的治疗靶点。二是实体瘤具有更强的肿瘤免疫抑制微环境。此外，肿瘤本身具有很强的异质性。这些因素导致肿瘤免疫治疗后容易复发。因此，制备多靶点、功能更强的CART细胞，是提高肿瘤治疗效果的关键。例如，采用CART19治疗B细胞急性淋巴细胞白血病，约有30％的病人治疗后会产生CD19抗原阴性复发，研究表明同时以CD19和IL3受体α链CD123作为靶点，构建的CART19/123双靶点的CART细胞，具有更好的体内治疗效果，可降低复发率(J Clin Invest.2016 Oct 3；126(10))。然而，这些CAR的抗原识别区大多是人工构建的单链抗体，具有较强的免疫原性，体内治疗容易被识别并清除，导致治疗效果不佳。因此，采用天然的肽段构建出多靶点的抗原识别区， 具有免疫原性弱，靶向范围更好的优点。
ErbB受体家族包含四个成员，分别是表皮生长因子受体ErbB1(EGFR/Her2)、ErbB2(Her2)、ErbB3(Her3)和ErbB4(Her4)。配体激活ErbB受体酪氨酸激酶后，可引起受体间的二聚化，酪氨酸的自身磷酸化，进而激活下游的信号通路。在正常成年人体内，这四种受体的表达量都处于较低水平。然而，许多恶性肿瘤的发生都与ErbB1和/或ErbB2的过表达有关，包括头颈癌、乳腺癌、肺癌、胃肠道癌、前列腺癌和胰腺癌等。因此，临床研究的许多抗肿瘤药物是靶向ErbB不同受体胞外区的单克隆抗体和小分子酪氨酸激酶抑制剂，如HER2人源化抗体赫赛汀(Herceptin)已被FDA批准用于乳腺癌的临床治疗。
然而，ErbB受体通常是以紧密结合的二聚体的形式发挥作用的。Her2单个受体缺乏高亲和力的配体，但它却是最佳的二聚化伙伴，尤其是与EGFR或Her3二聚化时能增强酪氨酸激酶信号活化。Her3具有配体结合能力但缺乏酪氨酸激酶活性，与Her2形成的异源二聚体是最强有力的信号复合体，在乳腺癌中最具有代表性的异源二聚体是Her2/Her3。单独靶向某一受体的抗肿瘤药物，由于不能靶向其他的ErbB受体二聚体，容易引起肿瘤的复发。
T1E是一种嵌合的多肽，它由人转录生长因子α(TGFα)N端的七个氨基酸和表皮生长因子(EGF)C端的48个氨基酸组成，它和基于ErbB1的同源二聚体和异源二聚体都有很高的亲和力，此外，T1E还能有效结合ErbB2/3异源二聚体。有研究表明，以T1E作为scFv的CAR转染T细胞，可有效治疗人头颈癌的小鼠肿瘤异种移植模型。但是，T1E不能结合单独的ErbB2或者ErbB3，在靶向ErbB受体家族的范围上还存在缺陷。
Herstatin是Her2被选择性地剪切而产生的Her2截短的版本,其序列包括Her2第Ⅰ及Ⅱ胞外区结构域的340个氨基酸和第八内含子编码的79个氨基酸,是一种可溶的Her2自身抑制因子。Herstatin能与单独的EGFR或Her2高亲和力结合，其中起主要作用的是第八内含子编码的79个氨基酸(命名为Herin)。本发明，将天然的T1E和Herin融合表达作为CAR的抗原识别区，二者可以互补识别ErbB受体家族，扩大靶向范围。
发明内容
本发明提供一种嵌合抗原受体，所述嵌合抗原受体从N端到C端依次包括任选的膜蛋白信号肽、通过接头连接的T1E肽段和Herin肽段、长50个氨基酸残基以上的铰链区、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，所述信号肽选自CD8信号肽、CD28信号肽和CD4信号肽；优选地，所述信号肽是CD8信号肽，优选其氨基酸序列如SEQ ID NO:15第1-22位氨基酸残基所示。
在一个或多个实施方案中，所述T1E的氨基酸序列如SEQ ID NO:15第23-77位氨基酸残基所示。
在一个或多个实施方案中，所述Herin的氨基酸序列如SEQ ID NO:15第93-171位氨基酸序列所示。
在一个或多个实施方案中，所述接头选自柔性接头和刚性接头；优选地，所述柔性接头为含有G和S的接头，所述刚性接头为含有EAAAK重复序列的接头；优选地，所述接头的氨基酸序列如SEQ ID NO:15第78-92位氨基酸残基所示。
在一个或多个实施方案中，所述长50个氨基酸残基以上的铰链区选自CD8α铰链区、IgD铰链区、IgG1 Fc CH2CH3铰链区和IgG4 Fc CH2CH3铰链区；优选地，所述铰链区是CD8α铰链区或IgG4 Fc CH2CH3铰链区；优选地，所述CD8α铰链区的氨基酸序列如SEQ ID NO:17所示，所述IgG4 FcCH2CH3铰链区的氨基酸序列如SEQ ID NO:15第172-399位氨基酸序列所示。
在一个或多个实施方案中，所述跨膜区选自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跨膜区中的一种或多种；优选地，所述跨膜区为CD8跨膜区或CD28跨膜区；优选地，所述CD8跨膜区的氨基酸序列如SEQ ID NO:18所示，所述CD28跨膜区的氨基酸序列如SEQ ID NO:15第400-427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为共刺激信号分子的胞内结构域，选自CD28、CD134/OX40、CD137/4-1BB、LCK、ICOS和DAP10胞内结构域中的一种或多种；优选地，所述共刺激信号分子的胞内结构域是CD137胞内结构域，其氨基酸序列如SEQ ID NO:19所示，或所述共刺激信号分子的胞内结构域是CD28胞内结构域，其氨基酸序列可如SEQ ID NO:15第428-468位氨基酸残基所示。
在一个或多个实施方案中，所述胞内信号域是免疫受体酪氨酸活化基序，选自CD3ζ和FcεRIγ的酪氨酸活化基序；优选地，所述免疫受体酪氨酸活化基序为CD3ζ酪氨酸活化基序，优选地，其氨基酸序列如SEQ ID NO:15第469-580位氨基酸残基所示。
在一个或多个实施方案中，所述嵌合抗原受体从N端到C端依次含有任选的CD8信号肽、T1E、EAAAK重复序列、Herin、IgG4 Fc CH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序。
在一个或多个实施方案中，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、Herin、EAAAK重复序列、T1E、IgG4 Fc CH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序。
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:15或16 所示。
本发明还提供一种多核苷酸序列，选自：
(1)编码本文所述嵌合抗原受体的多核苷酸序列；和
(2)(1)所述多核苷酸序列的互补序列；
在一个或多个实施方案中，所述嵌合抗原受体的编码序列具有特征中的一项或多项：
所述信号肽的核苷酸序列如SEQ ID NO:1所示；
所述T1E的核苷酸序列如SEQ ID NO:2所示；
所述Herin的核苷酸序列如SEQ ID NO：3所示；
所述接头的核苷酸序列如SEQ ID NO：4所示；
所述铰链区的核苷酸序列如SEQ ID NO:5或6所示；
所述跨膜区的核苷酸序列如SEQ ID NO:7或8所示；
所述共刺激信号分子胞内结构域的核苷酸序列如SEQ ID NO:9或10所示；和
所述免疫受体酪氨酸活化基序为的核苷酸序列如SEQ ID NO:11所示；
在一个或多个实施方案中，所述多核苷酸序列选自SEQ ID NO:13或14所示的多核苷酸序列或其互补序列。
本发明还提供一种核酸构建物，其含有本文所述的多核苷酸序列。
在一个或多个实施方案中，所述核酸构建物是表达载体。
在一个或多个实施方案中，所述表达载体是真核表达载体，优选含有选自piggybac、sleeping beauty、frog prince、Tn5和Ty的转座元件。
本发明还提供一种重组宿主细胞，所述重组宿主细胞含有本文所述的核酸构建物，或表达本文所述的嵌合抗原受体。
在一个或多个实施方案中，所述宿主细胞为哺乳动物细胞；更优选地，所述宿主细胞为T细胞；更优选地，所述宿主细胞为原代培养T细胞。
本发明还提供本文所述的嵌合抗原受体和/或其编码序列和/或的核酸构建物在制备用于治疗或预防癌症的重组宿主细胞中的应用；以及本文所述的重组宿主细胞在制备治疗或预防癌症用的药物中的应用。
在一个或多个实施方案中，所述癌症为其癌细胞表面异常表达至少一个EGFR家族成员蛋白的癌症；优选地，所述癌症选自：头颈癌、肝癌、腺癌、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、胰腺癌或前列腺癌。
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重组宿主细胞和药学上可接受的载体。
本发明还提供一种多核苷酸序列，所述多核苷酸序列如SEQ ID NO：3所示，或为SEQ ID NO:3所示序列的互补序列。
本发明还提供一种融合蛋白，其由T1E与Herin通过接头序列连接形成；其中，所述T1E由人转录生长因子αN端的七个氨基酸和表皮生长因子C端的48个氨基酸组成；所述Herin是Herstatin第八内含子编码的79个氨基酸；所述接头选自柔性接头、刚性接头和体内裂解接头；优选为含有G和S的柔性接头，或含有EAAAK重复序列的刚性接头。
在一个或多个实施方案中，所述融合蛋白的氨基酸序列如SEQ ID NO:15或16的第23-171位氨基酸序列所示。
本发明还提供所述融合蛋白的编码序列或其互补序列；优选地，所述序列如SEQ ID NO:13或14第67-513位碱基序列所示。
附图说明
图1：为各种靶向ErbB受体家族的嵌合抗原受体基因结构模式图。
图2：RT-PCR检测eCAR T细胞和HerinCAR T细胞中CAR基因表达的copy数结果。
图3：eCAR T细胞和HerinCAR T细胞的杀伤对比，包括人非小细胞肺癌H23-LUC和人肺癌细胞H292-LUC。
图4：eCAR T细胞、EHCAR-GS T细胞、HECAR-GS T细胞、EHCAR-EK T细胞和HECAR-EK T细胞中CAR基因表达的拷贝数结果。
图5：eCAR T细胞、EHCAR-GS T细胞、HECAR-GS T细胞、EHCAR-EK T细胞和HECAR-EK T细胞的杀伤对比，包括人卵巢癌细胞SKOV3-LUC和人肝癌细胞HCCLM3-LUC。
图6：每组柱从左到右分别显示Mock T细胞、、HECAR-GS T细胞、EHCAR-GS T细胞、HECAR-EK T细胞、EHCAR-EK T细胞和eCAR T细胞在EGFR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：eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞中CAR基因表达的拷贝数结果。
图8：eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞的杀伤对比，包括人卵巢癌细胞SKOV3-LUC、人肝癌细胞HCCLM3-LUC和人非小细胞肺癌H23-LUC。
图9：每组柱从左到右分别显示Mock T细胞、eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞在EGFR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10：eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞、Mock-T细胞和PBS空白对照，分别治疗小鼠后，不同天数的肿瘤细胞荧光值变化。
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，包括启动子和基因编码序列。
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；优选地为膜锚定型。
本发明设计了一种新型靶向ErbB受体家族的CAR-T细胞，该CAR的抗原识别部位包括完整的T1E和Herin两种结构，二者通过接头连接。由于ErbB受体通常是以紧密结合的二聚体的形式发挥作用，机制较复杂，在实验初期，为了获得功能最好的靶向ErbB受体家族的CAR-T细胞，本发明人进行了各种条件的摸索，设计了一系列的CAR结构，包括分别单独表达T1E和Herin的CAR、同时表达T1E和Herin的CAR且调换这两种片段的位置顺序进行比较、不同接头、不同跨膜铰链区和胞内共刺激区的比较，最终得到体外效果最好的两种CAR，一种从N端到C端依次含有CD8信号肽、T1E、EAAAK接头、Herin、IgG4 FcCH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序，命名为EHCAR-EK-28TIZ；另一种从N端到C端依次含有CD8信号肽、Herin、EAAAK接头、T1E、IgG4 FcCH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序，命名为HECAR-EK-28TIZ。体外实验表明，与其它结构的CAR-T细胞相比， EHCAR-EK-28TIZ和HECAR-EK-28TIZ能具有识别抗原和细胞杀伤作用。
本文包括具有如下结构特征的嵌合抗原受体(CAR)：从N端到C端，依次含有任选的膜蛋白信号肽、通过接头连接的T1E肽段和Herin肽段(两种肽段的位置可调换)、长50个氨基酸残基以上的铰链区、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。本文的信号肽为膜蛋白信号肽，可选自CD8信号肽、CD28信号肽和CD4信号肽。在优选的实施方案中，本发明使用CD8信号肽。示范性的CD8信号肽的氨基酸序列如SEQ ID NO:15第1-22位氨基酸残基所示；示范性的CD8信号肽的核苷酸序列可如SEQ ID NO：1所示。
T1E是一种嵌合的多肽，它由人转录生长因子α(TGFα)N端的七个氨基酸和表皮生长因子(EGF)C端的48个氨基酸组成，它和基于ErbB1的同源二聚体和异源二聚体都有很高的亲和力。此外，T1E还能有效结合ErbB2/3异源二聚体。本发明的T1E的氨基酸序列如SEQ ID NO:15第23-77位氨基酸残基所示；示例性的核苷酸序列可如SEQ ID NO：2所示。
Herstatin是Her2被选择性地剪切而产生的Her2截短的版本,其序列包括Her2第Ⅰ及Ⅱ胞外区结构域的340个氨基酸和第八内含子编码的79个氨基酸,是一种可溶的Her2自身抑制因子。本发明的Herin是Herstatin第八内含子编码的79个氨基酸，其氨基酸序列如SEQ ID NO:15第93-171位氨基酸残基所示。在本发明的某些实施方案中，本发明将编码其氨基酸的密码子优化，获得SEQ ID NO：3所示的核苷酸序列。
接头用于将不同的氨基酸序列连接起来，可起到稳定蛋白的空间结构、提高其生物学活性的作用。适用于本文的接头包括柔性接头、刚性接头和体内裂解接头。在某些实施方案中，本文使用的柔性接头为GS接头，即主要含有G和S的接头。示例性的GS接头的核苷酸序列如SEQ ID NO：12；在某些实施方案中，本文使用刚性接头，如含有EAAAK重复序列的接头，本文也称为EAAAK接头。示例性的EAAAK接头的氨基酸序列如SEQ ID NO:15第78-92位氨基酸残基，示例性的核苷酸序列如SEQ ID NO：4所示。
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，优选是长50个氨基酸残基以上、更优选长80个氨基酸残基以上的铰链区。在某些实施方案中，本文使用CD8α铰链区和IgG4 Fc CH2CH3铰链区。示例性的CD8α铰链区 的氨基酸序列可如SEQ ID NO:17所示，示例性的核苷酸序列如SEQ ID NO：5所示。示例性的IgG4 FcCH2CH3铰链区的氨基酸序列可如SEQ ID NO:15第172-399位氨基酸序列所示，其示例性的核苷酸序列如SEQ ID NO:6所示。
跨膜区可选自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跨膜区中的一种或多种。在某些实施方案中，本文使用的嵌合抗原受体的跨膜区为CD8跨膜区和CD28跨膜区。示例性的CD8跨膜区的氨基酸序列可如SEQ ID NO:18所示，示例性的核苷酸序列可如SEQ ID NO:7所示。示例性的CD28跨膜区的氨基酸序列可如SEQ ID NO:15第400-427位氨基酸残基所示，其示例性的核苷酸序列可如SEQ ID NO:8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、LCK、ICOS和DAP10的胞内结构域中的一种或多种。在某些实施方案中，使用CD28的胞内结构域或CD137的胞内结构域。示例性的CD137胞内结构域的氨基酸序列可如SEQ ID NO:19所示，示例性的核苷酸序列可如SEQ ID NO:9所示；示例性的CD28胞内结构域的氨基酸序列可如SEQ ID NO:15第428-468位氨基酸残基所示，示例性的核苷酸序列可如SEQ ID NO:10所示。
胞内信号域可以是免疫受体酪氨酸活化基序，可选自CD3ζ和/或FcεRIγ的酪氨酸活化基序。示例性的CD3ζ酪氨酸活化基序的氨基酸序列可如SEQ ID NO:15第469-580位氨基酸残基所示，示例性的核苷酸序列可如SEQ ID NO:11所示。
形成本文嵌合抗原受体的上述各部分，如CD8信号肽、T1E片段、Herin片段、通过接头连接的T1E片段和Herin片段、铰链区、跨膜区、共刺激信号分子胞内结构域以及免疫受体酪氨酸活化基序等，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。
在某些实施方案中，本文的嵌合抗原受体从N端到C端依次含有任选的CD8信号肽、T1E、CD8铰链区、CD8跨膜区、CD137胞内结构域和CD3ζ酪氨酸活化基序。
在某些实施方案中，本文的嵌合抗原受体从N端到C端依次含有任选的CD8信号肽、Herin、IgG4 FcCH2CH3铰链区、CD8跨膜区、CD137胞内结构域和CD3ζ酪氨酸活化基序。
在某些实施方案中，本文的嵌合抗原受体从N端到C端依次含有任选的CD8信号肽、Herin、GS接头、T1E、CD8铰链区、CD8跨膜区、CD137胞内结构域和CD3ζ酪氨酸活化基序。
在某些实施方案中，本文的嵌合抗原受体从N端到C端依次含有任选的CD8信号肽、T1E、GS接头、Herin、CD8铰链区、CD8跨膜区、CD137胞内结构域和CD3ζ酪氨酸活化基序。
在某些实施方案中，本文的嵌合抗原受体从N端到C端依次含有任选的CD8信号肽、Herin、EAAAK接头、T1E、CD8铰链区、CD8跨膜区、CD137胞内结构域和CD3ζ酪氨酸活化基序。
在某些实施方案中，本文的嵌合抗原受体从N端到C端依次含有任选的CD8信号肽、T1E、EAAAK接头、Herin、CD8铰链区、CD8跨膜区、CD137胞内结构域和CD3ζ酪氨酸活化基序。
在某些实施方案中，本文的嵌合抗原受体从N端到C端依次含有任选的CD8信号肽、Herin、EAAAK接头、T1E、IgG4 FcCH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序。
在某些实施方案中，本文的嵌合抗原受体从N端到C端依次含有任选的CD8信号肽、T1E、EAAAK接头、Herin、IgG4 FcCH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序。
优选地，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、T1E、EAAAK接头、Herin、IgG4 FcCH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序。示例性的嵌合抗原受体的氨基酸序列如SEQ ID NO:15所示第23-580位氨基酸残基所示，或者如SEQ ID NO:15所示；优选地，其核苷酸序列可核苷酸序列可如SEQ ID NO:13第67-1740位碱基序列所示，或者如SEQ ID NO:13所示。
优选地，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、Herin、EAAAK接头、T1E、IgG4 FcCH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序。这类嵌合抗原受体的示例性氨基酸序列可SEQ ID NO:16所示第23-580位氨基酸残基所示，或者如SEQ ID NO:16所示；优选地，其核苷酸序列可如SEQ ID NO:14第67-1740位碱基序列所示，或者如SEQ ID NO:14所示。
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。
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，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，本发明的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，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:1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:2所示的含CD3e增强子和EF1α启动子的TEF启动子；SEQ ID NO:3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:4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:5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:5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，本文的表达载体是整合载体，用于将本发明的CAR的编码序列整合入宿主细胞中。在某些实施方案中，本文的表达载体是真核表达载体，具体是转座子载体。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)。在5’LTR和3’LTR之间是本发明的CAR的表达框，包括相应的启动子序列、CAR的编码序列以及如polyA加尾信号序列。
例如，在某些实施方案中，本文的核酸构建物或表达载体从5’到3’依次含有转座子5’反向末端重复序列(5’LTR)、启动子、CD8信号肽编码序列、通过EAAAK-linker连接的T1E片段和Herin片段编码序列、CD8铰链区编码序列或IgG4 Fc CH2CH3铰链区编码序列、CD8跨膜区编码序列、CD28胞内结构域编码序列、CD3ζ酪氨酸活化基序编码序 列、polyA加尾信号序列以及转座子3’反向末端重复序列(3’LTR)。所述转座子载体还可含有转座酶编码序列和控制转座酶编码序列表达的启动子。在某些实施方案中，所述真核表达载体是pNB328载体。
可采用常规的方法将本文的载体导入宿主细胞中，这些方法包括显微注射法、基因枪法、电穿孔法、病毒介导的转化法、电子轰击法、磷酸钙沉淀法等。在某些实施方案中，本文采用电穿孔法将本文的核酸构建物导入宿主细胞中。具体地，将重组的质粒经电转仪高压电的作用转到感兴趣的宿主细胞中。
适用于本文的宿主细胞可以是本领域周知的哺乳动物细胞，优选是T细胞，包括各种来源的各种类型的T细胞。例如，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。
因此，本文也包括一种重组宿主细胞，所述重组宿主细胞含有本文所述嵌合抗原受体的编码序列或本文所述的核酸构建物；和/或所述重组宿主细胞表达本文所述的嵌合抗原受体。该重组宿主细胞可以是前文所述的转入了本文所述载体的宿主细胞。
用于本文嵌合抗原受体中的Herin片段及其优化的密码子序列也包括在本文的范围之内。更具体而言，本文包括其核苷酸序列如SEQ ID NO:3所示的Herin片段。
用于本文嵌合抗原受体中的IgG4 Fc CH2CH3铰链区及其编码序列也包括在本文的范围之内。更具体而言，本文包括其核苷酸序列如SEQ ID NO:6所示的IgG4 Fc CH2CH3铰链区及其编码序列(包括简并序列)。
本文还包括前文提及的各种氨基酸序列、核酸序列、重组宿主细胞等的用途。具体而言，本文包括所述IgG4 Fc CH2CH3铰链区和/或其编码序列在制备本文所述嵌合抗原受体和/或其编码序列中的用途；所述嵌合抗原受体的编码序列在制备重组表达载体中的用途；所述核酸构建物在制备重组宿主细胞中的用途；以及所述重组宿主细胞在制备治疗或预防癌症用的药物中的用途。在某些实施方案中，本文包括所述IgG4 Fc CH2CH3铰链区和/或其编码序列、所述嵌合抗原受体和/或其编码序列以及所述核酸构建物在制备用于治疗或预防癌症的重组宿主细胞中的应用。
适用于本文所述CAR或其表达细胞进行治疗或预防的癌症优选至少一个EGFR家族蛋白阳性的癌症，具体包括癌细胞表面异常表达ErbB1、ErbB2、ErbB3和ErbB4的癌症。具体而言，这类癌症可选自：头颈癌、肝癌、腺癌、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、胰腺癌或前列腺癌。
在某些实施方案中，本文的嵌合抗原受体对同时表达ErbB1和ErbB2的肿瘤细胞如卵巢癌和肺癌，以及单独表达ErbB1的肿瘤细胞如肝癌，均具有优异的杀伤效果，因此，本文的这类CAR或其表达细胞可特别用于治疗至少一个EGFR家族成员阳性的癌症。
本文还提供一种试剂盒，所述试剂盒含有本文所述的重组表达载体。试剂盒还可含有适用于将所述重组表达载体转入细胞中的试剂，以及任选的指导本领域技术人员将所述重组表达载体转入细胞的说明书。
本文还提供一种药物组合物，所述药物组合物含有本文所述的重组宿主细胞和药学上可接受的载体。所述药学上可接受的载体可以是本领域周知的适用于细胞给药的载体，包括但不限于pNB328载体。
治疗或预防癌症的方法也包括在本文的范围之内，所述方法包括将本文所述的重组宿主细胞或药物组合物给予有需要的个体的步骤。给予的方法可以是细胞治疗中常用的方法。给予的剂量可根据病患性别、年龄、所患疾病、身体状况等因素加以考虑。
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。
实施例1：重组质粒pNB328-eCAR、pNB328-HerinCAR、pNB328-EHCAR-GS、pNB328-HECAR-GS、pNB328-EHCAR-EK、pNB328-HECAR-EK、pNB328-EHCAR-EK-28TIZ和pNB328-HECAR-EK-28TIZ的构建
委托上海捷瑞生物公司合成eCAR基因、HerinCAR基因、EHCAR-GS基因、HECAR-GS基因、EHCAR-EK基因、HECAR-EK基因、EHCAR-EK-28TIZ基因和HECAR-EK-28TIZ基因，结构模式如图A所示。将各基因装入用EcoR1+SalI双酶切的pNB328载体中(pNB328载体见CN 201510812654.9，含EF1α启动子)，构建质粒，分别命名为pNB328-eCAR、pNB328-HerinCAR、pNB328-EHCAR-GS、pNB328-HECAR-GS、pNB328-EHCAR-EK、pNB328-HECAR-EK、pNB328-EHCAR-EK-28TIZ和pNB328-HECAR-EK-28TIZ。
结构模式图中的CD8信号肽的核苷酸序列如SEQ ID NO:1所示；T1E的核苷酸序列如SEQ ID NO:2所示；Herin的核苷酸序列如SEQ ID NO:3所示；EAAAK接头(EK-linker)的核苷酸序列如SEQ ID NO:4所示；GS接头(GS-linker)的核苷酸序列如SEQ ID NO:12所示；CD8α铰链跨膜区(CD8EC)核苷酸序列如SEQ ID NO:5所示；突变型IgG4Fc CH2CH3铰链跨膜区核苷酸序列如SEQ ID NO:6所示；CD8跨膜区(CD8TM)的核苷酸序列如SEQ ID NO:7所示；CD28跨膜区(CD28TM)的核苷酸序列如SEQ ID NO:8所示； CD137胞内共刺激信号结构区域(CD137IC)核苷酸序列如SEQ ID NO:9所示；CD28胞内共刺激信号结构区域(CD28IC)核苷酸序列如SEQ ID NO:10所示；CD3ζ胞内信号域核苷酸序列如SEQ ID NO:11所示。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：靶向ErbB受体家族的嵌合抗原受体修饰T细胞构建
外周血单核细胞(PBMCs)由上海细胞治疗生产中心分离获得。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9个1.5ml离心管，每管加入5×10 6个细胞，编号a、b、c、d、e、f、g、h和i，1200rmp离心3min，弃上清，取电转试剂盒(来自Lonza公司)，各管按比例加入电转试剂共100ul，a、b、c、d、e、f、g和h管分别加入6ug构建好的重组质粒pNB328-eCAR、pNB328-HerinCAR、pNB328-EHCAR-GS、pNB328-HECAR-GS、pNB328-EHCAR-EK、pNB328-HECAR-EK、pNB328-EHCAR-EK-28TIZ和pNB328-HECAR-EK-28TIZ，重悬混匀细胞，i管加入6ug对照质粒(即pNB328空质粒，构建Mock T细胞)；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anti-CD3/anti-CD28，37℃，5％CO2培养3～4天，观察T细胞的生长情况，获得表达eCAR基因、HerinCAR基因、EHCAR-GS基因、HECAR-GS基因、EHCAR-EK基因、HECAR-EK基因、EHCAR-EK-28TIZ基因和HECAR-EK-28TIZ基因的T细胞。
实施例3：RT-PCR检测eCAR和HerinCAR表达的copy数比较
将实施例2中电转后第10天的Mock T细胞、eCAR T细胞和HerinCAR T细胞，按照1×10 6细胞数目收集细胞沉淀，PBS清洗细胞沉淀1遍，使用gDNA提取试剂盒(9765,Takara)抽提细胞基因组DNA(gDNA)。实验步骤参照试剂盒内附带的说明书，使用实时荧光定量PCR法(qRT-PCR)检测基因组DNA中CAR基因(检测CD137)插入的拷贝数。反应程序为：50℃，2min→95℃,10min→95℃,15s→60℃,1min，40个循环。得到的eCAR和HerinCAR基因组的CT值及Actin的CT值根据相应的公式计算出绝对拷贝数含量。结果如图2所示。
实施例4：eCAR和HerinCAR的杀伤功能对比
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)检测实施例2获得的eCAR-T和HerinCAR-T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，选择步骤1，调零；
(2)靶细胞铺板：人非小细胞肺癌H23-LUC和人肺癌细胞H292-LUC(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，开始步骤2，培养细胞；
(3)加入效应细胞：靶细胞培养24h后，暂停步骤2，加入效应细胞，每孔50μl，效靶比分别设置为4:1，以转入pNB328空载体的Mock T细胞作为对照，开始步骤3，继续共培养24h后，观察细胞增殖曲线；
结果如图3所示。单独表达T1E片段的CAR-T细胞对肿瘤细胞的杀伤作用明显强于单独表达Herin的CAR-T细胞以及对照T细胞。
实施例5：RT-PCR检测eCAR、EHCAR-GS、HECAR-GS、EHCAR-EK和HECAR-EK表达的拷贝数比较
将实施例2中电转后第10天的Mock T细胞、eCAR T、EHCAR-GS、HECAR-GS、EHCAR-EK和HECAR-EK细胞，按照1×10 6细胞数目收集细胞沉淀，PBS清洗细胞沉淀1遍，按照实施例3的方法，检测检测基因组DNA中CAR基因(检测CD137)插入的拷贝数。结果如图4所示。
实施例6：eCAR、EHCAR-GS、HECAR-GS、EHCAR-EK和HECAR-EK的杀伤功能对比
按照实施例4的方法，测试实施例2获得的eCAR T细胞、EHCAR-GS T细胞、HECAR-GS T细胞、EHCAR-EK T细胞和HECAR-EK T细胞体外杀伤活性的测试，所用的靶细胞分别为人卵巢癌细胞SKOV3-LUC和人肝癌细胞HCCLM3-LUC。
结果如图5所示。在使用相同的CD8α铰链跨膜区、CD8跨膜区和CD137胞内共刺激信号结构区域的情况下，单独表达T1E片段的eCAR-T细胞对肿瘤细胞的杀伤作用明显强于同时表达T1E片段和Herin片段的HECAR-GS和HECAR-EK细胞(Herin片段在前，T1E片段在后)以及EHCAR-GS和EHCAR-EK细胞(T1E片段在前，Herin片段在后)以及对照T细胞；此外，使用EAAAK接头的HECAR-EK和EHCAR-EK细胞的杀伤作用明显强于使用GS接头的HECAR-GS和EHCAR-GS细胞，说明用EAAAK接头连接T1E和Herin，能更好的提高融合蛋白的生物活性，增强其功能。
实施例7：eCAR T细胞、EHCAR-GS T细胞、HECAR-GS T细胞、EHCAR-EK T细胞和HECAR-EK T细胞在EGFR抗原的特异性刺激下细胞因子释放对比
用5ug/ml的EGFR抗原包被96孔板，4℃包被过夜，PBS清洗3遍，加入1×10 5(100ul体积)的实施例2制备得到的eCAR T细胞、EHCAR-GS T细胞、HECAR-GS T细胞、EHCAR-EK T细胞和HECAR-EK T细胞以及对照的Mock T细胞(转入pNB328空载体)，培养24h后收集细胞上清。用BD TMCBA Human Th1/Th2 Cytokine Kit II检测这四种T细胞受EGFR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、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，eCAR-T细胞的IL-2和IFN-γ分泌量显著高于EHCAR-GS T细胞、HECAR-GS T细胞、EHCAR-EK T细胞和HECAR-EK T细胞；EHCAR-EK T细胞和HECAR-EK T细胞的IL-2和IFN-γ分泌量显著高于EHCAR-GS T细胞和HECAR-GS T细胞。
实施例8：RT-PCR检测eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞表达的拷贝数比较
将实施例2中电转后第11天的Mock T细胞、eCAR-T细胞、EHCAR-EK-28TIZT细胞和HECAR-EK-28TIZ T细胞，按照1×10 6细胞数目收集细胞沉淀，PBS清洗细胞沉淀1遍，按照实施例3的方法检测检测基因组DNA中CAR基因插入的拷贝数，eCAR-T细胞检测CD137，EHCAR-EK-28TIZ细胞和HECAR-EK-28TIZ细胞检测CD28。结果如图7所示。
实施例9：eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞 的杀伤功能对比
按照实施例4的方法，进行eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞体外杀伤活性的测试，所用的靶细胞分别为人卵巢癌细胞SKOV3-LUC、人肝癌细胞HCCLM3-LUC和人非小细胞肺癌细胞。
结果如图8所示，将EHCAR-EK和HECAR-EK的CD8α铰链跨膜区替换为突变型IgG4Fc CH2CH3，CD8跨膜区替换为CD28，CD137胞内共刺激信号结构区域替换为CD28胞内共刺激信号结构区域后，即改造成为EHCAR-EK-28TIZ和HECAR-EK-28TIZ，它们的杀伤能力明显高于单独表达T1E片段的eCAR-T细胞。
实施例10：eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞在EGFR抗原的特异性刺激下细胞因子释放对比
用5ug/ml的EGFR抗原包被96孔板，4℃包被过夜，PBS清洗3遍，加入1×10 5(100ul体积)的实施例2制备得到的eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZT细胞以及对照的Mock T细胞(转入pNB328空载体)，培养24h后收集细胞上清。按照实施例7的方法，检测这四种T细胞受EGFR抗原刺激后细胞因子的分泌情况。
结果如图9所示，eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZT细胞的IL-2和IFN-γ分泌量相较于Mock T细胞，都有明显提高，但EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞显著高于eCAR T细胞。
实施例11：eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞体内抗肿瘤作用。
购买4～6周龄NSG小鼠20只，平均分为5组，每组4只，接种肝癌细胞株HCCLM3-LUC，每只1×10 7，成瘤10天后，尾静脉分别注射PBS(100ul)和实施例2获得的Mock-T、eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞(1×10 7个细胞/只)，观察记录小鼠体内肿瘤荧光变化。
结果如图10所示，PBS和Mock-T细胞对肿瘤模型没有治疗效果，eCAR T细胞、EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞都有很好的抗肿瘤效果，且EHCAR-EK-28TIZ T细胞和HECAR-EK-28TIZ T细胞效果更好。
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 (14)

  1. 一种嵌合抗原受体，其特征在于，从N端到C端，依次包括任选的膜蛋白信号肽、通过接头连接的T1E肽段和Herin肽段、长50个氨基酸残基以上的铰链区、跨膜区、胞内共刺激信号域和胞内信号域。
  2. 如权利要求1所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体具有以下一个或多个特征：
    所述信号肽选自CD8信号肽、CD28信号肽和CD4信号肽；优选地，所述信号肽是CD8信号肽，优选其氨基酸序列如SEQ ID NO:15第1-22位氨基酸残基所示；
    所述T1E的氨基酸序列如SEQ ID NO:15第23-77位氨基酸残基所示；
    所述Herin的氨基酸序列如SEQ ID NO:15第93-171位氨基酸序列所示；
    所述接头选自柔性接头和刚性接头；优选地，所述柔性接头为含有G和S的接头，所述刚性接头为含有EAAAK重复序列的接头；优选地，所述接头的氨基酸序列如SEQ ID NO:15第78-92位氨基酸残基所示，
    所述长50个氨基酸残基以上的铰链区选自CD8α铰链区、IgD铰链区、IgG1 Fc CH2CH3铰链区和IgG4 Fc CH2CH3铰链区；优选地，所述铰链区是CD8α铰链区或IgG4 Fc CH2CH3铰链区；优选地，所述CD8α铰链区的氨基酸序列如SEQ ID NO:17所示，所述IgG4 FcCH2CH3铰链区的氨基酸序列如SEQ ID NO:15第172-399位氨基酸序列所示；
    所述跨膜区选自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跨膜区中的一种或多种；优选地，所述跨膜区为CD8跨膜区或CD28跨膜区；优选地，所述CD8跨膜区的氨基酸序列如SEQ ID NO:18所示，所述CD28跨膜区的氨基酸序列如SEQ ID NO:15第400-427位氨基酸残基所示；
    所述胞内共刺激信号域为共刺激信号分子的胞内结构域，选自CD28、CD134/OX40、CD137/4-1BB、LCK、ICOS和DAP10胞内结构域中的一种或多种；优选地，所述共刺激信号分子的胞内结构域是CD137胞内结构域，其氨基酸序列如SEQ ID NO:19所示，或所述共刺激信号分子的胞内结构域是CD28胞内结构域，其氨基酸序列可如SEQ ID NO:15第428-468位氨基酸残基所示；和
    所述胞内信号域是免疫受体酪氨酸活化基序，选自CD3ζ和FcεRIγ的酪氨酸活化基序；优选地，所述免疫受体酪氨酸活化基序为CD3ζ酪氨酸活化基序，优选地，其氨基酸序列如SEQ ID NO:15第469-580位氨基酸残基所示。
  3. 如权利要求1－2中任一项所述的嵌合抗原受体，其特征在于，
    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、T1E、EAAAK重复序列、Herin、IgG4 Fc CH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序，或者
    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、Herin、EAAAK重复序列、T1E、IgG4 Fc CH2CH3铰链区、CD28跨膜区、CD28胞内结构域和CD3ζ酪氨酸活化基序；
    优选地，所述嵌合抗原受体的氨基酸序列如SEQ ID NO:15或16所示。
  4. 一种多核苷酸序列，选自：
    (1)编码权利要求1－3中任一项所述嵌合抗原受体的多核苷酸序列；和
    (2)(1)所述多核苷酸序列的互补序列；
  5. 如权利要求4所述的多核苷酸序列，其特征在于，所述嵌合抗原受体的编码序列具有特征中的一项或多项：
    所述信号肽的核苷酸序列如SEQ ID NO:1所示；
    所述T1E的核苷酸序列如SEQ ID NO:2所示；
    所述Herin的核苷酸序列如SEQ ID NO：3所示；
    所述接头的核苷酸序列如SEQ ID NO：4所示；
    所述铰链区的核苷酸序列如SEQ ID NO:5或6所示；
    所述跨膜区的核苷酸序列如SEQ ID NO:7或8所示；
    所述共刺激信号分子胞内结构域的核苷酸序列如SEQ ID NO:9或10所示；和
    所述免疫受体酪氨酸活化基序为的核苷酸序列如SEQ ID NO:11所示；
    优选地，所述多核苷酸序列选自SEQ ID NO:13或14所示的多核苷酸序列或其互补序列。
  6. 一种核酸构建物，其含有权利要求4或5所述的多核苷酸序列；
    优选地，所述核酸构建物是表达载体；
    更优选地，所述表达载体是真核表达载体，优选含有选自piggybac、sleeping beauty、frog prince、Tn5和Ty的转座元件。
  7. 一种重组宿主细胞，所述重组宿主细胞含有权利要求6所述的核酸构建物，或表达权利要求1－3中任一项所述的嵌合抗原受体；
    优选地，所述宿主细胞为哺乳动物细胞；更优选地，所述宿主细胞为T细胞；更优选地，所述宿主细胞为原代培养T细胞。
  8. 权利要求1－3中任一项所述的嵌合抗原受体和/或其编码序列和/或权利要求6所述的核酸构建物在制备用于治疗或预防癌症的重组宿主细胞中的应用；以及权利要求7 所述的重组宿主细胞在制备治疗或预防癌症用的药物中的应用。
  9. 如权利要求8所述的应用，其特征在于，所述癌症为其癌细胞表面异常表达至少一个EGFR家族成员蛋白的癌症；优选地，所述癌症选自：头颈癌、肝癌、腺癌、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、胰腺癌或前列腺癌。
  10. 一种药物组合物，其特征在于，所述药物组合物含有权利要求7所述的重组宿主细胞和药学上可接受的载体。
  11. 一种多核苷酸序列，所述多核苷酸序列如SEQ ID NO：3所示，或为SEQ ID NO:3所示序列的互补序列。
  12. 一种融合蛋白，其由T1E与Herin通过接头序列连接形成；
    其中，所述T1E由人转录生长因子αN端的七个氨基酸和表皮生长因子C端的48个氨基酸组成；
    所述Herin是Herstatin第八内含子编码的79个氨基酸；
    所述接头选自柔性接头、刚性接头和体内裂解接头；优选为含有G和S的柔性接头，或含有EAAAK重复序列的刚性接头。
  13. 如权利要求12所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO:15或16的第23-171位氨基酸序列所示。
  14. 权利要求12或13所述融合蛋白的编码序列或其互补序列；优选地，所述序列如SEQ ID NO:13或14第67-513位碱基序列所示。
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