WO2019129142A1 - 自分泌CD40抗体且靶向ErbB受体家族的CAR-T细胞及其用途 - Google Patents

自分泌CD40抗体且靶向ErbB受体家族的CAR-T细胞及其用途 Download PDF

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WO2019129142A1
WO2019129142A1 PCT/CN2018/124366 CN2018124366W WO2019129142A1 WO 2019129142 A1 WO2019129142 A1 WO 2019129142A1 CN 2018124366 W CN2018124366 W CN 2018124366W WO 2019129142 A1 WO2019129142 A1 WO 2019129142A1
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seq
amino acid
sequence
cancer
coding sequence
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French (fr)
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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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    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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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
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    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12N15/09—Recombinant DNA-technology
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • 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 pertains to genetic engineering and immunology, to CAR-T cells that secrete CD40 antibodies and target 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 wider targeting range.
  • the ErbB receptor family contains four members, the epidermal growth factor receptor ErbB1 (EGFR/Her2), ErbB2 (Her2), ErbB3 (Her3), and ErbB4 (Her4). In normal adults, the expression levels of these four receptors are at a low level. However, 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. Therefore, many of the 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. For example, 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.
  • Her2 is the best dimerization partner, especially when dimerized with EGFR or Her3 to enhance tyrosine kinase signal activation.
  • 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 invention relates to the EHCAR-EK-28TIZ chimeric antigen receptor, and the natural T1E and Herin fusion expression is used as an antigen recognition region of CAR, and the two can complement the ErbB receptor family to expand the target range.
  • CD40 antigen is a cell surface molecule belonging to the TNFR superfamily. It is a type I transmembrane glycoprotein widely expressed in T cells, antigen presenting cells (DC cells, monocytes, macrophages), hematopoietic cells, epithelial cells. Etc., also expressed in most B cell malignancies and solid tumors.
  • CD40L is a type II transmembrane glycoprotein belonging to the TNF superfamily.
  • CD40-CD40L is a pair of extremely important costimulatory molecules in the immune response with a wide range of biological effects.
  • the CD40 molecule mainly transmits signals by interacting with CD40 ligand (CD40L).
  • CD40-CD40L can also promote T cells. Secretion of a large number of cytokines, such as GM-CSF, TNF-a and IFN- ⁇ , enhances the CTL effect of CD8+ T cells and enhances the killing effect on tumors.
  • cytokines such as GM-CSF, TNF-a and IFN- ⁇
  • APX005M NCT024821678
  • Apexigen a key project developed by Apexigen
  • MSI-H high frequency microsatellite instability
  • the present invention provides a chimeric antigen receptor T cell that self-expresses a CD40 activating antibody and targets the ErbB receptor family, preferably, the T cell:
  • a coding sequence comprising a chimeric antigen receptor that targets a family of ErbB receptors and a coding sequence for a CD40 activating antibody;
  • the expression cassette of the CD40 activating antibody and the expression cassette of the chimeric antigen receptor that targets the ErbB receptor family are integrated into the genome of the T cell.
  • the chimeric antigen receptor comprises an optional signal peptide, a T1E peptide segment and a Herin peptide segment joined by a rigid linker, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and Intracellular signal domain.
  • the signal peptide is a CD8 signal peptide, a CD28 signal peptide or a CD4 signal peptide, preferably a CD8 signal peptide; more preferably, the amino acid sequence of the CD8 signal peptide is SEQ ID NO: 5 The amino acid residues 1-22 are shown.
  • the amino acid sequence of the T1E is represented by amino acid residues 23-77 of SEQ ID NO: 5; preferably, the coding sequence thereof is as set at bases 67-231 of SEQ ID NO: 6. The base sequence is shown.
  • the Herrin amino acid sequence is as set forth in amino acid residues 93-171 of SEQ ID NO: 5; preferably, the coding sequence is SEQ ID NO: 6 bases 277-513 The base sequence is shown.
  • the rigid linker is an EAAAK linker, preferably the amino acid sequence of the linker is as described in amino acid residues 78-92 of SEQ ID NO: 5; preferably, the coding sequence thereof is The nucleotide sequence of 232-276 of SEQ ID NO: 6 is shown.
  • the hinge region is selected from the group consisting of the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the CD28 extracellular hinge region, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD4.
  • 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 CD28 transmembrane region preferably having an amino acid sequence as shown in amino acid residues 400-427 of SEQ ID NO: 5, preferably having a coding sequence of SEQ ID NO: 6 base sequence 1198-1281 Shown.
  • 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 Inducible T cell costimulatory factor (ICOS) and the intracellular domain of DNAX activator protein 10; preferably, the intracellular domain of the costimulatory signaling molecule is a CD28 intracellular domain, the amino acid sequence of which is SEQ ID NO: 5 is shown in amino acid residues 428-468, and preferably the coding sequence thereof is shown in nucleotide sequence 1282-1404 of SEQ ID NO: 6.
  • a costimulatory signaling molecule including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase Inducible T cell costimulatory factor (ICOS) and the intracellular domain of DNAX activator protein 10; preferably, the intracellular domain of the costimulatory signaling molecule is
  • 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.
  • the ID NO: 5 is represented by amino acid residues at positions 469 to 580, and preferably the coding sequence thereof is represented by the nucleotide sequence of nucleotides 1505-1740 of SEQ ID NO: 6.
  • the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 23 to 580 of SEQ ID NO: 5, or as shown in SEQ ID NO: 5; preferably, The nucleotide sequence showing the chimeric antigen receptor is shown in the nucleotide sequence of 6717 to 4040 shown in SEQ ID NO: 6, or as shown in SEQ ID NO: 6.
  • the CD4 activating antibody comprises an anti-CD40 single-chain antibody and an IgG4 Fc; preferably, the amino acid sequence of the IgG4 Fc is represented by amino acid residues 269-497 of SEQ ID NO: 1, Preferably, the coding sequence thereof is shown as the base sequence of 805-1491 of SEQ ID NO: 2.
  • the antibody light chain variable region (VL region) amino acid sequence of the anti-CD40 single chain antibody (scFv) is set forth as amino acid residues 21 to 146 of SEQ ID NO: 1; The coding sequence thereof is shown in nucleotide sequence 64-438 of SEQ ID NO: 2.
  • the heavy chain variable region (VH region) amino acid sequence of the anti-CD40 single-chain antibody is set forth in amino acids at positions 161-168 of SEQ ID NO: 1; preferably, the coding thereof The sequence is shown in nucleotide sequence 481-804 of SEQ ID NO: 2.
  • the amino acid sequence of the anti-CD40 single-chain antibody is represented by amino acid residues 21 to 268 of SEQ ID NO: 1; preferably, the coding sequence thereof is SEQ ID NO: 2, 61.
  • the -804 base sequence is shown.
  • the CD40 activating antibody comprises, in order from the N-terminus to the C-terminus, a light chain signal peptide, an anti-CD40 single chain antibody, and an IgG4 Fc; preferably, the amino acid sequence of the light chain signal peptide is SEQ ID.
  • the NO:1 amino acid residue at positions 1-20 is shown; preferably, the coding sequence of the light chain signal peptide shown is shown in nucleotide sequence 1-60 of SEQ ID NO: 2.
  • the amino acid sequence of the CD40 activating antibody is set forth in amino acid sequence 21-497 of SEQ ID NO: 1, or as set forth in SEQ ID NO: 1; preferably, the coding sequence thereof
  • the nucleotide sequence of positions 61-1491 of SEQ ID NO: 2 is shown, or is preferably as shown in SEQ ID NO: 2.
  • the invention also provides a composition comprising:
  • a vector comprising an expression cassette of a chimeric antigen receptor as described herein, which vector is used to integrate the expression cassette into the genome of a host cell;
  • a vector comprising an expression cassette for a CD40 activating antibody described herein, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the invention also provides a kit, the kit comprising:
  • a vector comprising an expression cassette of a chimeric antigen receptor as described herein, which vector is used to integrate the expression cassette into the genome of a host cell;
  • a vector comprising an expression cassette for a CD40 activating antibody described herein, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the invention also provides a pharmaceutical composition comprising a T cell as described herein.
  • the present invention also provides the use of a T cell or a pharmaceutical composition thereof as described herein for the preparation of a medicament for the treatment or prevention of a malignant tumor; preferably, the cancer is a cancer whose surface of the cancer cell abnormally expresses at least one EGFR family member protein
  • the cancer is selected from the group consisting of liver cancer, adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, non-small cell carcinoma, gallbladder cancer, esophageal cancer, melanoma. , pancreatic cancer, urothelial cancer, head and neck cancer or prostate cancer.
  • Figure 1 Schematic diagram of the genetic structure of the recombinant plasmids pS328- ⁇ CD40 and pNB328-EHCAR-EK-28TIZ.
  • 2A-2B Positive rate and antibody expression level of chimeric antigen receptor-modified T cells constructed with different mass ratios of pNB328-EHCAR-EK-28TIZ and pS328- ⁇ CD40 plasmids.
  • Figure 4 Comparison of proliferation rates of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- ⁇ CD40 T cells.
  • Figures 5A-5D Cell phenotypic assay analysis of T cells of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- ⁇ CD40 T cells, 5A indicates senescence phenotype CD40, 5B and 5C indicate activation phenotypes CD69 and CD107 ⁇ , 5D, respectively Represents a memory phenotype.
  • FIG. 6 Comparison of killing of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- ⁇ CD40 T cells, including human liver cancer cell HCCLM3, human lung degenerative cancer cell Calu-6 and human non-small cell lung cancer H23.
  • Figure 7 Secretion of IL-2, IL-4, IL-6, IL-10, TNF- ⁇ and IFN- ⁇ cytokines by EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- ⁇ CD40 T cells stimulated by EGFR antigen Variety.
  • FIG. 8 EHCAR-EK-28TIZ T cells, EHCAR-EK-28TIZ- ⁇ CD40-wt T cells, EHCAR-EK-28TIZ- ⁇ CD40 T cells, Mock-T cells and PBS blank controls, respectively, after treatment of mice, different days The tumor cell fluorescence value changes.
  • 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 expresses CD40 activating antibodies on existing CAR-T cells, and found that CD40 activating antibodies can promote the activation and proliferation of CAR-T cells and increase cells. The amount of factor secreted increases the anti-tumor killing effect in the body.
  • the present invention mutates the CD40-activated antibody IgG4 Fc fragment to satisfy the T cell-expressing CD40-activating antibody. It can function well without causing ADCC reaction.
  • the present invention provides a CD40 activated antibody comprising an anti-CD40 single chain antibody and IgG4Fc.
  • the amino acid sequence of the IgG4 Fc is set forth in amino acid residues 269-497 of SEQ ID NO: 1; preferably, the coding sequence thereof is set at bases 805-1491 of SEQ ID NO: Shown.
  • the antibody light chain variable region (VL region) amino acid sequence of the anti-CD40 single-chain antibody is represented by amino acid residues 21 to 146 of SEQ ID NO: 1; preferably, Its coding sequence is shown in nucleotide sequence 64-438 of SEQ ID NO: 2.
  • the heavy chain variable region (VH region) amino acid sequence of the anti-CD40 single-chain antibody is set forth in amino acids at positions 161-168 of SEQ ID NO: 1; preferably, the coding sequence thereof is The nucleotide sequence at positions 481 to 804 of SEQ ID NO: 2 is shown.
  • the amino acid sequence of the anti-CD40 single-chain antibody is set forth as amino acid residues 21 to 268 of SEQ ID NO: 1; preferably, the coding sequence thereof is SEQ ID NO: 2, 61-804 The base sequence is shown.
  • the CD40 antibody further comprises a light chain signal peptide.
  • the CD40 antibody comprises, from the N-terminus to the C-terminus, a light chain signal peptide, an anti-CD40 single chain antibody, and an IgG4 Fc in sequence.
  • the amino acid sequence of the light chain signal peptide is represented by amino acid residues 1-20 of SEQ ID NO: 1; preferably, the coding sequence of the indicated light chain signal peptide is SEQ ID NO: 2 The base sequence of the 1-60th base is shown.
  • the amino acid sequence of the CD40 activating antibody is set forth in amino acid sequence 21-497 of SEQ ID NO: 1, or as set forth in SEQ ID NO: 1.
  • the invention also encompasses a coding sequence for the CD40 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 CD40 antibody comprises the sequence set forth in bases 61-1491 of SEQ ID NO: 2, preferably the sequence set forth in SEQ ID NO: 2.
  • the present invention also encompasses a nucleic acid construct comprising the coding sequence of a CD40 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 CD40 antibody, its coding sequence or complementary sequence, a nucleic acid construct, and a host cell for the preparation or treatment of a malignant tumor, particularly a CD40-associated tumor, including but not limited to Various malignant tumors.
  • the invention also provides T cells that self-express a CD40 activating antibody and target a chimeric antigen receptor of the ErbB receptor family.
  • a CD40 activating antibody a CD40 activating antibody
  • target a chimeric antigen receptor of the ErbB receptor family a CD40 activating antibody
  • the native 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 CAR of the present invention typically contains an optional signal peptide sequence, a fusion protein of T1E and Herin, 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; 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: 5; in certain embodiments, the coding sequence is set forth in bases 1-66 of SEQ ID NO: 6.
  • the natural T1E and Herin fusion expression is used as the antigen recognition region of CAR.
  • the 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).
  • TGF ⁇ human transcriptional growth factor alpha
  • EGF epidermal growth factor
  • the amino acid sequence of T1E is as shown in amino acid residues 23 to 77 of SEQ ID NO: 5; preferably, the coding sequence thereof is shown in nucleotide sequence 67-231 of SEQ ID NO: 6.
  • Herin refers to 79 amino acids encoded by the eighth intron of Herstatin.
  • the amino acid sequence thereof is shown as amino acid residues 93-171 of SEQ ID NO: 5.
  • the present invention optimizes codons encoding Herin amino acids. Therefore, the nucleotide sequence of the preferred Herin of the present invention is shown in the nucleotide sequence of 277-513 of SEQ ID NO: 6.
  • T1E and Herin can be joined by a rigid linker sequence.
  • An illustrative example of a rigid linker sequence is two or more repeats of EAAAK as a unit, also referred to herein as an EAAAK linker.
  • An exemplary rigid linker sequence is set forth in amino acid residues 78-92 of SEQ ID NO: 5; exemplary coding sequences are set forth in base sequences 232-276 of SEQ ID NO: 6.
  • 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 172-399 of SEQ ID NO: 5, and the coding sequence for the exemplary IgG4 FcCH2CH3 hinge region is shown in SEQ ID NO: 6 at positions 514-1978. .
  • 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 CD28 transmembrane region
  • the amino acid sequence thereof is represented by amino acid residues 400-427 of SEQ ID NO: 5; in certain embodiments, the coding sequence is set forth at bases 1198-1281 of SEQ ID NO: 6.
  • 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 428-468 of SEQ ID NO: 5, exemplary The coding sequence is shown as bases 1282-1404 of SEQ ID NO: 6.
  • 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: 5, pp. 469-580; in certain embodiments, the coding sequence is set forth in bases 1405-1740 of SEQ ID NO: 6.
  • the chimeric antigen receptor further comprises a signal peptide, preferably the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO:5.
  • the invention also encompasses chimeric antibody receptors and coding sequences thereof as described herein.
  • the above-mentioned various parts forming the chimeric antigen receptor herein may be directly connected to each other or may be passed through
  • the linker sequence is linked.
  • 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: 6.
  • nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor described herein or a polynucleotide sequence encoding the CD40 antibody, and one or more operably linked to the sequences Regulatory sequence.
  • 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 CARs herein or the coding sequences for CD40 antibodies can be cloned into a wide variety 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 or a coding sequence for a CD40 antibody And optional optional markers.
  • 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 a chimeric antigen receptor described herein and the coding sequence of a CD40 antibody are separately cloned into a vector (also referred to as an integration vector) for integration of a nucleic acid sequence of interest into the genome of a host cell.
  • a vector also referred to as an integration 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.
  • 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 set forth 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 CD40 antibody and the signal peptide coding sequence (e.g., the coding sequence for a 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: 1, and the coding sequence of an exemplary light chain signal peptide is SEQ ID NO: 2 bases 1-60. Shown.
  • a T cell modified by a CAR gene and capable of expressing a CD40 antibody described herein can be transduced into: a transposase-containing coding sequence for integration into a chimeric antigen receptor coding sequence in a T cell genome Vector, and a vector containing no transposase coding sequence for integration into the coding sequence of the CD40 antibody described herein in 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.
  • a vector containing a CD40 antibody coding sequence is set forth in SEQ ID NO: 6; the coding sequence of the CD40 antibody is set forth in nucleotide sequence 61-1485 of SEQ ID NO: 2.
  • the signal peptide of the CD40 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 1-60 of SEQ ID NO:2. More specifically, in certain embodiments, the vector comprising a transposase coding sequence that incorporates a chimeric antigen receptor coding sequence in the T cell genome in turn contains a 5' LTR, a promoter, and a CD8 signal peptide encoding.
  • T1E, EAAAK linker, Herin coding sequence of IgG4 Fc CH2CH3 hinge region, coding sequence of CD28 transmembrane region, coding sequence of CD28 intracellular domain, coding sequence of CD3 intracellular signal domain, polyA tailing signal sequence, a coding sequence for a 3' LTR and a transposase and a promoter thereof; said vector comprising a transposase coding sequence encoding a coding sequence for a CD40 antibody as described herein in a T cell genome at 5' LTR and 3'
  • the LTR contains, in order, a promoter, a coding sequence for a light chain signal peptide, a coding sequence for a CD40 antibody, and a polyA tailing signal sequence.
  • the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the CD40 antibody coding sequence is from 1 to 7:1 to 7, preferably from 1:3 to 1:3, more preferably from 1 to 3:1, more preferably 1 to 1:1, still more preferably 5:3.
  • 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 coding sequence described herein and a vector comprising a coding sequence for a CD40 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 coding sequence described herein and a vector comprising a coding sequence for a CD40 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 cassette contains at least a suitable promoter and a PolyA tailing signal sequence in addition to the coding sequence comprising a chimeric antigen receptor or a CD40 activating antibody.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a T cell as described herein. 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 present invention also provides the use of a T cell or a pharmaceutical composition thereof as described herein for the preparation of a medicament for treating or preventing a malignant tumor (cancer); preferably, the malignant tumor is a tumor associated with CD40 and/or ErbB receptor More preferably, the cancer cell surface of the malignant tumor abnormally expresses at least one EGFR family member protein. More preferably, the malignant tumor is selected from the group consisting of liver cancer, adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, non-small cell carcinoma, gallbladder cancer, esophageal cancer, melanin. Tumor, pancreatic cancer, urothelial cancer, head and neck cancer or prostate cancer.
  • Example 1 Construction of recombinant plasmids pS328- ⁇ CD40-wt, pS328- ⁇ CD40 and pNB328-EHCAR-EK-28TIZ
  • Each gene was separately loaded into the pNB328 and pS328 vectors digested with EcoR1+SalI (the structure and sequence of pNB328 are described in CN 201510638974.7, the entire disclosure of which is incorporated herein by reference in its entirety in its entirety in in in In the subsequence, the other elements are identical to the pNB328 vector, plasmids were constructed and designated as pNB328-EHCAR-EK-28TIZ, pS328- ⁇ CD40 and pS328- ⁇ CD40-wt, respectively.
  • nucleotide sequence of the light chain signal peptide in the structural pattern diagram is shown in nucleotide sequence 1-60 of SEQ ID NO: 2; the encoding of Anti-CD40-wt is shown in SEQ ID NO: 4 (amino acid sequence such as SEQ ID NO: 3 is shown in amino acid residues 21-495); the nucleotide sequence of Anti-CD40 is shown in nucleotide sequence 61-1488 of SEQ ID NO: 2; the nucleotide sequence of CD8 signal peptide As shown in SEQ ID NO: 6 base sequence of 1-66; the nucleotide sequence of T1E is shown in nucleotide sequence 67-231 of SEQ ID NO: 6; the nucleotide sequence of Herin is SEQ ID NO :6 indicates the nucleotide sequence of 232-276; the nucleotide sequence of EAAAK-linker (EK-linker) is shown in the nucleotide sequence of 277-513 of SEQ ID NO: 6; the
  • CD28IC intracellular costimulatory signal structural region
  • CD28IC The nucleotide sequence of CD28 intracellular costimulatory signal structural region (CD28IC) is shown in nucleotide sequence 1282-1404 of SEQ ID NO: 6; the nucleotide sequence of CD3 intracellular signal domain is SEQ ID NO: 6
  • the nucleotide sequence of 1405-1740 is shown.
  • 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 Determination of the positive rate and antibody expression of chimeric antigen receptor-modified T cells constructed with pNB328-EHCAR-EK-28TIZ and pS328- ⁇ CD40 plasmids of different mass ratios
  • the amount of pNB328-EHCAR-EK-28TIZ and pS328- ⁇ CD40 plasmids were set to 1ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug, respectively.
  • the construction method is as follows:
  • 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.
  • pS328- ⁇ CD40 h tube was added 6ug control plasmid (pBS328, used to construct Mock T cells); the mixture was transferred to an electric rotor, put into the electro-rotation instrument, select the required procedure, and electric shock; use the trace in the kit
  • the straw is transferred to the well-adjusted six-well plate (AIM-V solution containing 2% FBS), mixed, placed in a 37 ° C, 5% CO 2 incubator for six hours. After adding stimulating factors IL-2 and EGFR/anti-CD28, cultured at 37 ° C, 5% CO 2 for 3-4 days, observe the growth of T cells EHCAR-EK-28 TIZ T cells derived from the expressed CD40 antibody were obtained. The positive rate of CAR T cells and the amount of antibody secreted by 7 kinds of ratios were detected.
  • the above seven CAR-T and Mock-T cells were collected, each divided into two portions, each of which was 1 ⁇ 10 6 cells, washed twice with physiological saline, resuspended in 100 ul of physiological saline, and one ug of EGFR-biotin was added. The other one was not added and incubated at 4 ° 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 machine was tested, and only the secondary antibody was added as a control.
  • ELISA was used to detect the expression of EHCAR-EK-28TIZ- ⁇ CD40 T cell antibody.
  • the OD value was measured at 450 nm on a microplate reader, a standard curve was drawn, and the CD40 antibody concentration was calculated.
  • the amount of antibody secreted by the CAR-T cells constructed in the form of 5 ug + 3 ug of the pNB328-EHCAR-EK-28TIZ and pS328- ⁇ CD40 plasmids was the highest.
  • EHCAR-EK-28TIZ- ⁇ CD40 T cells were constructed with 5ug pNB328-EHCAR-EK-28TIZ and 3ug pS328- ⁇ CD40.
  • Example 3 Construction of EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ- ⁇ CD40 T cells and determination of positive rate and antibody expression
  • Example 3 cultured EHCAR-EK-28 TIZ T cells, EHCAR-EK-28 TIZ- ⁇ CD40 T cells and Mock-T cells of Example 2 were cultured, and placed in a 12-well plate, and cultured. The culture volume was 1 ml. A 96-well white opaque plate was prepared. From the three groups of cells, 80 ⁇ L of the cell-containing culture solution was added to each well, and 80 ⁇ L of the nutrient solution was added to the original 12-well plate. Add 80 ⁇ L of CellTiter-Glo reagent to the 96-well plate, mix it on the shaker for 2 min, and incubate for 10 min at room temperature. The microplate reader reads the Luc fluorescence value.
  • the CellTiter-Glo Luminescent Cell Viability Assay kit used was purchased from Promega. On the 9th, 10th, 11th, 12th, and 13th day of culture, samples were taken from the cells cultured in the 12-well plate every day, and detected according to the above procedure, and the cell proliferation curve was drawn according to the fluorescence value.
  • the proliferation rate of EHCAR-EK-28TIZ- ⁇ CD40 T cells was significantly higher than that of EHCAR-EK-28TIZ T cells, indicating that expression of CD40 antibody can promote the proliferation of CAR-T cells.
  • Example 5 Cell phenotypic analysis of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- ⁇ CD40 T cells
  • the two EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- ⁇ CD40 T cells obtained in Example 3 were collected, and the number of cells was added to 6 1.5 ml EP tubes in 1 ⁇ 10 6 cells/tube, and PBS was washed twice. The cells were centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. Two of the tubes were added with the flow antibodies anti-CD107 ⁇ -PE and anti-CD69-PE for detecting the activated T cell phenotype, and one tube was added to the flow cytometry for detecting the memory T cell phenotype.
  • Antibody anti-CD45RO-PECy5+anti-CD197-FITC+anti-CD62L-PE one tube was added with anti-PD1-PE to detect the inhibitory T cell phenotype, and the other two tubes were added to the isotype control flow.
  • CD62L L-selectin
  • CD197 was the marker of effector memory T cells
  • EHCAR- EK- The proportion of effector T cells in 28 TIZ- ⁇ CD40 T cells was significantly higher than that of EHCAR-EK-28 TIZ cells and Mock-T cells (Fig. 5D).
  • Example 6 Comparison of killing function of EHCAR-EK-28TIZ and EHCAR-EK-28TIZ- ⁇ CD40 T cells
  • the effector cells and target cells matched by MHC class I were selected, and two EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ obtained in Example 3 were detected by using Essen's real-time label-free cell function analyzer (RTCA).
  • RTCA real-time label-free cell function analyzer
  • Target cell plating human liver cancer cell HCCLM3, human lung degenerative cancer cell Calu-6 and human non-small cell lung cancer H23 (purchased from American Type Culture Collection ATCC) were plated at 10 4 cells/50 ⁇ l per well. In the plate of the detection electrode, place it for a few minutes, wait for the cell to stabilize, then put it into the instrument, and start step 2 to culture the cells;
  • Example 7 Comparison of cytokine release by EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ- ⁇ CD40 T cells stimulated specifically by 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 EHCAR-EK-28TIZ and EHCAR-EK prepared in Example 3 were added.
  • 28 TIZ- ⁇ CD40 T cells and control Mock T cells were cultured for 24 hours, and the cell supernatant was collected. According to the method of Example 7, the secretion of cytokines by these EGFR cells after stimulation with EGFR antigen was examined.
  • Example 8 In vivo anti-tumor effect of EHCAR-EK-28TIZ T cells, EHCAR-EK-28TIZ- ⁇ CD40-wt T cells and EHCAR-EK-28TIZ- ⁇ CD40 T cells
  • EHCAR-EK-28 TIZ- ⁇ CD40-wt T cells were constructed using the method described in Example 2 with 5 ug of pNB328-EHCAR-EK-28 TIZ and 3 ug of pS328- ⁇ CD40-wt.
  • mice Twenty NSC mice aged 4-6 weeks were randomly divided into 5 groups, 4 rats in each group. Hepatocellular carcinoma cell line HCCLM3-LUC was inoculated with 1 ⁇ 10 7 each. After 10 days of tumor formation, PBS was injected into the tail vein (100ul). , Mock-T obtained in Example 2 and EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ- ⁇ CD40 T cells obtained in Example 3 and EHCAR-EK-28TIZ- ⁇ CD40-wt T prepared in the present example Cells (1 ⁇ 10 7 cells / cell) were observed and recorded for changes in tumor fluorescence in mice.

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Abstract

一种自表达CD40激活性抗体并靶向ErbB受体家族的嵌合抗原受体T细胞及其用途。所述T细胞含有表达靶向ErbB受体家族的嵌合抗原受体的编码序列和CD40激活性抗体的编码序列；和/或(2)表达靶向ErbB受体家族的嵌合抗原受体和CD40激活性抗体。所述T细胞比单独的靶向ErbB受体家族的嵌合抗原受体T细胞具有更好的增殖活化能力和杀伤肿瘤细胞功能。

Description

自分泌CD40抗体且靶向ErbB受体家族的CAR-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，涉及自分泌CD40抗体且靶向ErbB受体家族的CAR-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，靶向范围更广的优点。
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)。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)。
本发明涉及的EHCAR-EK-28TIZ嵌合抗原受体，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。
CD40抗原是属于TNFR超家族的细胞表面分子,是一种I型跨膜糖蛋白，广泛表达于T细胞，抗原递呈细胞(DC细胞，单核细胞，巨噬细胞)，造血细胞、上皮细胞等，也表达于大部分B细胞恶性肿瘤及实体瘤等。CD40L是Ⅱ型跨膜糖蛋白，属于TNF超家族。CD40-CD40L是免疫应答中一对极其重要的共刺激分子，具有广泛的生物学效应。CD40分子主要通过与CD40配体(CD40L)相互作用传递信号，一方面，引起IL-12水平上调，活化DC细胞，增强APC对抗原的递呈能力，另一方面CD40-CD40L还可以促进T细胞分泌大量的细胞因子，如GM-CSF、TNF-a和IFN-γ，从而增强CD8+T细胞的CTL效应，增强对肿瘤的杀伤效应。哈德斯菲尔德大学的研究人员开发了一种利用CD40进行靶向治疗的方法，他们用CD40的配体来将其激活，再通过静脉注射进行靶向治疗，并进 行了专利申请(Oncogene,2017 May 4；36(18):2515-2528)。由于CD40分子广泛的生物学效应，因此在不同的领域，针对其研制了多株功能性抗体，例如美国生物药研发公司Apexigen开发的重点项目APX005M(NCT02482168)，对非小细胞癌，黑色素瘤，尿路上皮癌，高频微卫星不稳定性(MSI-H)，头颈癌等有良好的抑制效果。
发明内容
本发明提供一种自表达CD40激活性抗体并靶向ErbB受体家族的嵌合抗原受体T细胞，优选地，所述T细胞：
(1)含有表达靶向ErbB受体家族的嵌合抗原受体的编码序列和CD40激活性抗体的编码序列；和/或
(2)表达靶向ErbB受体家族的嵌合抗原受体和CD40激活性抗体。
在一个或多个实施方案中，所述T细胞的基因组中整合了CD40激活性抗体的表达框以及靶向ErbB受体家族的嵌合抗原受体的表达框。
在一个或多个实施方案中，所述嵌合抗原受体包含任选的信号肽、通过刚性接头连接的T1E肽段和Herin肽段、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信号肽或CD4信号肽，优选为CD8信号肽；更优选地，所述CD8信号肽的氨基酸序列如SEQ ID NO:5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:5第23-77位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:6第67-231位碱基序列所示。
在一个或多个实施方案中，所述Herin的氨基酸序列如SEQ ID NO:5第93-171位氨基酸残基所述；优选地，其编码序列如SEQ ID NO:6第277-513位碱基序列所示。
在一个或多个实施方案中，所述刚性接头为EAAAK接头，优选地，所述接头的氨基酸序列如SEQ ID NO:5第78-92位氨基酸残基所述；优选地，其编码序列如SEQ ID NO:6第232-276位碱基序列所示。
在一个或多个实施方案中，所述铰链区选自CD8的胞外铰链区、IgG1 Fc CH2CH3铰链区、IgD铰链区、CD28胞外铰链区、IgG4 Fc CH2CH3铰链区和CD4的胞外铰链区，优选为CD8铰链区或IgG4 CH2CH3铰链区；优选地，所述铰链区为IgG4 CH2CH3铰链区，其氨基酸序列如SEQ ID NO:5第172-399位氨基酸残基所示，优选地，其编码序列如SEQ ID NO:6第514-1197位碱基序列所示。
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CD28 跨膜区，优选其氨基酸序列如SEQ ID NO:5第400-427位氨基酸残基所示，优选其编码序列如SEQ ID NO:6第1198-1281位碱基序列所示。
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，所述共刺激信号分子胞内结构域是CD28胞内结构域，其氨基酸序列如SEQ ID NO:5第428-468位氨基酸残基所示，优选其编码序列如SEQ ID NO:6第1282-1404位碱基序列所示。
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:5第469-580位氨基酸残基所示，优选其编码序列如SEQ ID NO:6第1405-1740位碱基序列所示。
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:5第23-580位氨基酸残基所示，或如SEQ ID NO:5所示；优选地，所示嵌合抗原受体的核苷酸序列如SEQ ID NO:6所示第67-1740碱基序列所示，或如SEQ ID NO:6所示。
在一个或多个实施方案中，所述CD4激活性抗体含有抗CD40单链抗体和IgG4Fc；优选地，所述IgG4Fc的氨基酸序列如SEQ ID NO:1第269-497位氨基酸残基所示，优选地，其编码序列如SEQ ID NO:2第805-1491位碱基序列所示。
在一个或多个实施方案中，所述抗CD40单链抗体(scFv)中抗体轻链可变区(VL区)氨基酸序列如SEQ ID NO:1第21-146位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:2第64-438位碱基序列所示。
在一个或多个实施方案中，所述抗CD40单链抗体中的重链可变区(VH区)氨基酸序列如SEQ ID NO:1第161-268位氨基酸序列所示；优选地，其编码序列如SEQ ID NO:2第481-804位碱基序列所示。
在一个或多个实施方案中，所述抗CD40单链抗体的氨基酸序列如SEQ ID NO:1第21-268位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:2第61-804位碱基序列所示。
在一个或多个实施方案中，所述CD40激活性抗体从N端到C端依次含有轻链信号肽、抗CD40单链抗体和IgG4Fc；优选地，述轻链信号肽的氨基酸序列如SEQ ID NO:1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:2第1-60位碱基序列所示。
在一个或多个实施方案中，所述CD40激活性抗体的氨基酸序列如SEQ ID NO:1第21-497位氨基酸序列所示，或者如SEQ ID NO:1所示；优选地，其编码序列如SEQ ID NO:2 第61-1491位碱基序列所示，或者如优选如SEQ ID NO:2所示。
本发明还提供一种组合物，所述组合物含有：
(1)含本文所述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)含本文所述CD40激活性抗体的表达框的载体，所述载体用于将所述表达框整合到宿主细胞的基因组中。
本发明还提供一种试剂盒，所述试剂盒含有：
(1)含本文所述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)含本文所述CD40激活性抗体的表达框的载体，所述载体用于将所述表达框整合到宿主细胞的基因组中。
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。
本发明还提供本文所述的T细胞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；优选地，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：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、非小细胞癌、胆囊癌、食管癌、黑色素瘤、胰腺癌、尿路上皮癌、头颈癌或前列腺癌。
附图说明
图1：重组质粒pS328-αCD40和pNB328-EHCAR-EK-28TIZ的基因结构模式图。
图2A-2B：不同质量配比的pNB328-EHCAR-EK-28TIZ和pS328-αCD40质粒构建的嵌合抗原受体修饰T细胞的阳性率及抗体表达量。
图3A-3B：EHCAR-EK-28TIZ T细胞和EHCAR-EK-28TIZ-αCD40 T细胞并测定阳性率及抗体表达量。
图4：EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 T细胞的增殖速度对比。
图5A-5D：EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 T细胞的T细胞的细胞表型测定分析，5A表示衰老表型CD40，5B和5C分别表示活化表型CD69和CD107α，5D表示记忆表型。
图6：EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 T细胞的杀伤对比，包括人肝癌细胞HCCLM3、人肺退行性癌细胞Calu-6和人非小细胞肺癌H23。
图7：EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 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8：EHCAR-EK-28TIZ T细胞、EHCAR-EK-28TIZ-αCD40-wt T细胞、EHCAR-EK-28TIZ-αCD40 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。
为了提高EHCAR-EK-28TIZ T细胞的免疫治疗效果，本发明在已有的CAR-T细胞上表达CD40激活性抗体，结果发现CD40激活性抗体可以促进CAR-T细胞的活化和增殖，提高细胞因子的分泌量，增加其体内的抗肿瘤杀伤效应。
有研究表明CD40激活性抗体的IgG4 Fc片段容易被单核/巨噬细胞识别而被吞噬，本发明对CD40激活性抗体IgG4 Fc片段进行碱基突变改造以满足T细胞自身表达的CD40激活性抗体既可以很好的发挥功能又不引起ADCC反应。
因此，本发明提供一种CD40激活型抗体，其含有抗CD40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:1第269-497位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:2第805-1491位碱基序列所示。
在某些实施方案中，所述抗CD40单链抗体(scFv)中抗体轻链可变区(VL区)氨基酸序列如SEQ ID NO:1第21-146位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:2第64-438位碱基序列所示。在某些实施方案中，所述抗CD40单链抗体中的重链可变区(VH区)氨基酸序列如SEQ ID NO:1第161-268位氨基酸序列所示；优选地，其编 码序列如SEQ ID NO:2第481-804位碱基序列所示。在某些实施方案中，所述抗CD40单链抗体的氨基酸序列如SEQ ID NO:1第21-268位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:2第61-804位碱基序列所示。
在某些实施方案中，所述CD40抗体还含有轻链信号肽。在某些实施方案中，所述CD40抗体从N端到C端，依次含有轻链信号肽、抗CD40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:1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:2第1-60位碱基序列所示。
在某些实施方案中，所述CD40激活性抗体的氨基酸序列如SEQ ID NO:1第21-497位氨基酸序列所示，或者如SEQ ID NO:1所示。
本发明还包括所述CD40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CD40抗体的编码序列含有SEQ ID NO:2第61-1491位碱基序列所示的序列，优选含有SEQ ID NO:2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CD40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CD40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CD40相关的肿瘤，包括但不限于本文所述的各种恶性肿瘤。
本发明还提供自表达CD40激活性抗体并靶向ErbB受体家族的嵌合抗原受体的T细胞。所述嵌合抗原受体(CAR)中，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。
本发明的CAR通常含有任选的信号肽序列、T1E与Herin的融合蛋白、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；更优选地为CD8信号肽。CD8信号肽的氨基酸序列可如SEQ ID NO:5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:6第1-66位碱基所示。
本文将天然的T1E和Herin融合表达作为CAR的抗原识别区。所述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。优选地，T1E的氨基酸序列如SEQ ID NO:5第23-77位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:6第67-231位碱基序列所示。
本发明中，Herin指Herstatin第八内含子编码的79个氨基酸。优选地，其氨基酸序列如SEQ ID NO:5第93-171位氨基酸残基所示。本发明将编码Herin氨基酸的密码子进行优化。因此，本发明优选的Herin的核苷酸序列如SEQ ID NO：6第277-513位碱基序列所示。
通常，T1E和Herin可通过刚性接头序列连接。刚性接头序列的一个示例性的例子是以EAAAK为一个单元的2个或多个重复序列，本文也称为EAAAK接头。示例性的刚性接头序列如SEQ ID NO:5第78-92位氨基酸残基所示；示例性的编码序列如SEQ ID NO:6第232-276位碱基序列所示。
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如SEQ ID NO:5第172-399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:6第514-1197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CD28跨膜区，优选其氨基酸序列如SEQ ID NO:5第400-427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:6第1198-1281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:5第428-468位氨基酸残基所示，示例性的编码序列如SEQ ID NO:6第1282-1404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:5第469-580所述；在某些实施方案中，其编码序列如SEQ ID NO:6第1405-1740 位碱基所示。
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CD信号肽、T1E、EAAAK接头、Herin、IgG4 Fc CH2CH3铰链区、CD28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:5第23-580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信号肽，优选地，该嵌合抗原受体的氨基酸序列如SEQ ID NO:5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、T1E、Herin、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:6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CD40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，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的编码序列或CD40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CD40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CD40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 201510638974.7 SEQ 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 201510638974.7 SEQ 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CD40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：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：2第1-60位碱基所示。
在某些实施方案中，本文所述的经CAR基因修饰并能表达CD40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编码序列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CD40抗体的编码序列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CD40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:6所示；所述CD40抗体的编码序列如SEQ ID NO:2第61-1485位碱基序列。在某些实施方案中，所述含CD40抗体的编码序列的载体中，CD40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:2第1-60位氨基酸残基所示。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、CD8信号肽编码序列、T1E、EAAAK接头、Herin、IgG4 Fc CH2CH3铰链区的编码序列、CD28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以及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CD40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、CD40抗体的编码序列以及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CD40抗体编码序列的载体的质量比为1～7：1～7，优选1：3～1：3，更优选1～3：1，更优选1～：1，更优选5：3。
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编码序列的载体和含本文所述CD40抗体的编码序列的载体。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编码序列的载体和含本文所述CD40抗体的编码序列的载体，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。
如本文所述，所述表达框中除含有嵌合抗原受体或CD40激活性抗体的编码序列外，至少还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。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细胞或其药物组合物在制备治疗或预防恶性肿瘤(癌症)的药物中的用途；优选地，所述恶性肿瘤是与CD40和/或ErbB受体相关的肿瘤；更优选地，所述恶性肿瘤的癌细胞表面异常表达至少一个EGFR家族成员蛋白。更优选地，所述恶性肿瘤选自：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、非小细胞癌、胆囊癌、食管癌、黑色素瘤、胰腺癌、尿路上皮癌、头颈癌或前列腺癌。
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pS328-αCD40-wt、pS328-αCD40和pNB328-EHCAR-EK-28TIZ的构建
1、委托上海捷瑞生物公司合成EHCAR-EK-28TIZ基因(核苷酸序列如SEQ ID NO：6所示，氨基酸序列如SEQ ID NO:5所示)、anti-CD40基因(核苷酸序列如SEQ ID NO：2所示，氨基酸序列如SEQ ID NO:1所示)和anti-CD40-wt基因(核苷酸序列如SEQ ID NO：4所示，氨基酸序列如SEQ ID NO:3所示)，结构模式如图1所示。将各基因分别装入用EcoR1+SalI双酶切的pNB328和pS328载体(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转座子序列，其它元件与pNB328载体相同)中，构建质粒，分别命名为pNB328-EHCAR-EK-28TIZ、pS328-αCD40和pS328-αCD40-wt。
结构模式图中的轻链信号肽的核苷酸序列如SEQ ID NO:2第1-60位碱基序列所示；Anti-CD40-wt的编码如SEQ ID NO:4所示(氨基酸序列如SEQ ID NO:3第21-495位氨基酸残基所示)；Anti-CD40的核苷酸序列如SEQ ID NO:2第61-1488位碱基序列所示；CD8信号肽的核苷酸序列如SEQ ID NO:6第1-66位碱基序列所示；T1E的核苷酸序列如SEQ ID NO:6第67-231位碱基序列所示；Herin的核苷酸序列如SEQ ID NO:6第232-276位碱基序列所示；EAAAK-linker(EK-linker)的核苷酸序列如SEQ ID NO:6第277-513位碱基序列所示；mIgG4Fc CH2CH3铰链跨膜区核苷酸序列如SEQ ID NO:6第514-1197位碱基序列所示；CD28跨膜区(CD28TM)的核苷酸序列如SEQ ID NO:6第1-66位碱基序列1198-1281所示；CD28胞内共刺激信号结构区域(CD28IC)核苷酸序列如SEQ ID NO:6第1282-1404位碱基序列所示；CD3ζ胞内信号域核苷酸序列如SEQ ID NO:6第1405-1740位碱基序列所示。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：不同质量配比的pNB328-EHCAR-EK-28TIZ和pS328-αCD40质粒构建的嵌合抗原受体修饰T细胞的阳性率及抗体表达量测定
分别将pNB328-EHCAR-EK-28TIZ和pS328-αCD40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。构建方法如下：
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；取八个1.5ml离心管，每管加入5×10 6个细胞，编号a、b、c、d、e、f、g和h，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和g管分别加入不同质量配比的重组质粒pNB328-EHCAR-EK-28TIZ和pS328-αCD40，h管加入6ug对照质粒(pBS328，用于构建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％CO 2培养箱培养，六小时后加入刺激因子IL-2和EGFR/anti-CD28，37℃，5％CO 2培养3～4天，观察T细胞的生长情况，获得自表达CD40抗体的EHCAR-EK-28TIZ T细胞。 分别检测7种配比下构建的CAR T细胞阳性率及抗体分泌量。
1.流式检测CAR T细胞阳性率
收集上述七种CAR-T和Mock-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EGFR-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所示，pNB328-EHCAR-EK-28TIZ和pS328-αCD40质粒的量，以5ug+3ug的形式构建的CAR-T细胞阳性率最高。
2.ELISA检测EHCAR-EK-28TIZ-αCD40 T细胞抗体表达量。
①用包被液将CD40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CD40抗体浓度。
结果如图2B所示，pNB328-EHCAR-EK-28TIZ和pS328-αCD40质粒以5ug+3ug的形式构建的CAR-T细胞分泌的抗体量最高。
综合阳性率和抗体分泌量结果，选用5ug pNB328-EHCAR-EK-28TIZ和3ug pS328-αCD40构建EHCAR-EK-28TIZ-αCD40 T细胞，效果最好。
实施例3：构建EHCAR-EK-28TIZ T细胞和EHCAR-EK-28TIZ-αCD40 T细胞并测定阳性率及抗体表达量
取6ug pNB328-EHCAR-EK-28TIZ质粒用于构建EHCAR-EK-28TIZ T细胞，分别取5ug pNB328-EHCAR-EK-28TIZ和3ug pS328-αCD40质粒用于构建EHCAR-EK-28TIZ-αCD40 T细胞，构建方法同实施例2。
流式检测实施例2的Mock-T细胞以及本实施例构建的EHCAR-EK-28TIZ T细胞和 EHCAR-EK-28TIZ-αCD40 T细胞的阳性率，方法同实施例2。结果见图3A，自表达CD40抗体不会降低CART细胞的阳性率。
ELISA检测Mock-T细胞、EHCAR-EK-28TIZ T细胞以及EHCAR-EK-28TIZ-αCD40 T细胞抗体表达量，方法同实施例2，结果见图3B。
实施例4：EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 T细胞增殖速率的对比
各取3×10 5个实施例3培养第8天的EHCAR-EK-28TIZ T细胞、EHCAR-EK-28TIZ-αCD40 T细胞和实施例2的Mock-T细胞，放置到12孔板中培养，培养体积均为1ml。准备96孔白色不透光板，从三组细胞中，各取80μL含细胞的培养液加入到不同的孔中，同时补80μL营养液至原来的12孔板中。再向96孔板中加入80μL CellTiter-Glo试剂，在震荡仪上混匀2min，并在室温下孵育10min，酶标仪读Luc荧光值。所用的CellTiter-Glo Luminescent Cell Viability Assay试剂盒购自Promega公司。培养的第9，10，11，12，13天，每天都从12孔板中培养的细胞中，取样，按以上步骤检测，根据荧光值，绘制细胞增殖曲线。
结果如图4所示，EHCAR-EK-28TIZ-αCD40 T细胞的增殖速度明显高于EHCAR-EK-28TIZ T细胞，说明表达CD40抗体可以促进CAR-T细胞的增殖。
实施例5：EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 T细胞的细胞表型测定分析
收集实施例3获得的两种EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 T细胞，细胞数后以1×10 6个细胞/管分别加入6个1.5ml的EP管中，PBS洗涤两次，1200rpm离心5min，弃上清；其中2管分别加入检测活化T细胞表型的流式抗体anti-CD107α-PE和anti-CD69-PE，有1管加入检测记忆T细胞表型的流式抗体anti-CD45RO-PECy5+anti-CD197-FITC+anti-CD62L-PE，有1管分别加入检测抑制性T细胞表型的流式抗体anti-PD1-PE，另外2管分别加入同型对照流式抗体IgG1-PE和IgG1-PE+IgG2a-PECy5+IgG2a-PE，每种抗体2μl(均购自Jackson ImmunoResearch公司)，轻弹沉淀使其混合均匀；室温避光孵育30min后，PBS清洗一遍，1200rpm离心5min，弃上清加入400μl的生理盐水，将细胞转移至流式管中，上机检测。
实验结果发现，流式检测EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 T细胞的衰老表型PD1的表达量差不多(图5A)；EHCAR-EK-28TIZ-αCD40 T细胞的活化表型CD69和CD107α的表达量高于EHCAR-EK-28TIZ细胞(图5B和5C)；同时，CD62L(L-选择素)为中央记忆T细胞的标志，CD197为效应记忆T细胞的标志，EHCAR- EK-28TIZ-αCD40 T细胞中效应T细胞的比例明显高于EHCAR-EK-28TIZ细胞和Mock-T细胞(图5D)。这些结果说明，表达CD40抗体可以促进CAR-T细胞的活化，增强其免疫杀伤功能。
实施例6：EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 T细胞杀伤功能对比
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3获得的两种EHCAR-EK-28TIZ T细胞和EHCAR-EK-28TIZ-αCD40 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，选择步骤1，调零；
(2)靶细胞铺板：人肝癌细胞HCCLM3、人肺退行性癌细胞Calu-6和人非小细胞肺癌H23(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6所示。自表达CD40抗体的EHCAR-EK-28TIZ-αCD40 T细胞对多种肿瘤细胞的杀伤作用明显强于EHCAR-EK-28TIZ T细胞以及对照T细胞。
实施例7：EHCAR-EK-28TIZ T细胞和EHCAR-EK-28TIZ-αCD40 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3制备得到的EHCAR-EK-28TIZ和EHCAR-EK-28TIZ-αCD40 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。按照实施例7的方法，检测这三种T细胞受EGFR抗原刺激后细胞因子的分泌情况。
结果如图7所示，EHCAR-EK-28TIZ-αCD40的IL-2和IFN-γ分泌量相显著高于EHCAR-EK-28TIZ T细胞和Mock-T，说明自表达CD40激活性抗体可以促进CAR-T细胞分泌细胞因子。
实施例8：EHCAR-EK-28TIZ T细胞、EHCAR-EK-28TIZ-αCD40-wt T细胞和EHCAR-EK-28TIZ-αCD40 T细胞体内抗肿瘤作用
采用实施例2所述的方法，用5ug pNB328-EHCAR-EK-28TIZ和3ug pS328-αCD40-wt构建EHCAR-EK-28TIZ-αCD40-wt T细胞。
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，分别尾静脉注射PBS(100ul)、实施例2获得的Mock-T和实施例3获得的EHCAR-EK-28TIZ T细胞和EHCAR-EK-28TIZ-αCD40 T细胞以及本实施例制备得到的EHCAR-EK-28TIZ-αCD40-wt T细胞(1×10 7个细胞/只)，观察记录小鼠体内肿瘤荧光变化。
结果显示，PBS、Mock-T、EHCAR-EK-28TIZ-αCD40-wt T细胞对肿瘤模型没有治疗效果，EHCAR-EK-28TIZ T细胞、EHCAR-EK-28TIZ-αCD40 T细胞有抗肿瘤效果，但EHCAR-EK-28TIZ-αCD40 T细胞效果明显更好。具体如图8所示。
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，其特征在于，所述T细胞：
    (1)含有表达靶向ErbB受体家族的嵌合抗原受体的编码序列和CD40激活性抗体的编码序列；和/或
    (2)表达靶向ErbB受体家族的嵌合抗原受体和CD40激活性抗体；
    优选地，所述T细胞的基因组中整合了CD40激活性抗体的表达框以及靶向ErbB受体家族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，所述嵌合抗原受体包含任选的信号肽、通过刚性接头连接的T1E肽段和Herin肽段、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，所述嵌合抗原受体具有以下一项或多项特征：
    所述信号肽为CD8信号肽、CD28信号肽或CD4信号肽，优选为CD8信号肽；更优选地，所述CD8信号肽的氨基酸序列如SEQ ID NO:5第1-22位氨基酸残基所示；
    所述T1E的氨基酸序列如SEQ ID NO:5第23-77位氨基酸残基所示；优选地，其编码序列如SEQ ID NO:6第67-231位碱基序列所示；
    所述Herin的氨基酸序列如SEQ ID NO:5第93-171位氨基酸残基所述；优选地，其编码序列如SEQ ID NO:6第277-513位碱基序列所示；
    所述刚性接头是EAAAK接头，优选其氨基酸序列如SEQ ID NO:5第78-92位氨基酸残基所述；优选地，其编码序列如SEQ ID NO:6第232-276位碱基序列所示；
    所述铰链区选自CD8的胞外铰链区、IgG1 Fc CH2CH3铰链区、IgD铰链区、CD28胞外铰链区、IgG4 Fc CH2CH3铰链区和CD4的胞外铰链区；优选为CD8铰链区或IgG4 CH2CH3铰链区；优选地，所述铰链区为IgG4 CH2CH3铰链区，其氨基酸序列如SEQ ID NO:5第172-399位氨基酸残基所示，优选地，其编码序列如SEQ ID NO:6第514-1197位碱基序列所示；
    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CD28跨膜区，优选其氨基酸序列如SEQ ID NO:5第400-427位氨基酸残基所示，优选其编码序列如SEQ ID NO:6第1198-1281位碱基序列所示；
    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，所述共刺激信号分子胞内结构域是CD28胞内结构域，其氨基酸序列如SEQ ID NO:5第428-468位氨基酸残基所示，优选其编码序列如SEQ ID NO:6第1282-1404位碱基序列所示；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:5第469-580位氨基酸残基所示，优选其编码序列如SEQ ID NO:6第1405-1740位碱基序列所示。
  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：
    信号肽的编码序列如SEQ ID NO:6第1-66位碱基序列所示；
    T1E的编码序列如SEQ ID NO:6第67-231位碱基序列所示；
    Herin的编码序列如SEQ ID NO:6第277-513位碱基序列所示；
    刚性接头的编码序列如SEQ ID NO:6第232-276位碱基序列所示；
    铰链区的编码序列如SEQ ID NO:6第514-1197位碱基序列所示；
    跨膜区的编码序列如SEQ ID NO:6第1198-1281位碱基序列所示；
    胞内共刺激信号域的编码序列如SEQ ID NO:6第1282-1404位碱基序列所示；和
    胞内信号域的编码序列如SEQ ID NO:6第1405-1740位碱基序列所示。
  5. 如权利要求1或2所述的T细胞，其特征在于，所述嵌合抗原受体的氨基酸序列如SEQ ID NO:5第23-580位氨基酸残基所示，或如SEQ ID NO:5所示；优选地，所示嵌合抗原受体的核苷酸序列如SEQ ID NO:6所示第67-1740碱基序列所示，或如SEQ ID NO:6所示。
  6. 如权利要求1-5所述的T细胞，其特征在于，所述CD4激活性抗体含有抗CD40单链抗体和IgG4Fc；其中，所述IgG4Fc的氨基酸序列如SEQ ID NO:1第269-497位氨基酸残基所示；
    优选地，所述抗CD40单链抗体中抗体轻链可变区氨基酸序列如SEQ ID NO:1第21-146位氨基酸残基所示；优选地，所述抗CD40单链抗体中的重链可变区氨基酸序列如SEQ ID NO:1第161-268位氨基酸序列所示；
    优选地，所述CD40激活性抗体还含有轻链信号肽；优选地，述轻链信号肽的氨基酸序列如SEQ ID NO:1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CD40激活性抗体的氨基酸序列如SEQ ID NO:1第21-497位氨基酸序列所示，或者如SEQ ID NO:1所示；优选地，其编码序列如SEQ ID NO:2第61-1491位碱基序列所示，或者如优选如SEQ ID NO:2所示。
  8. 一种组合物或试剂盒，所述组合物或试剂盒含有：
    (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中任一项所限定的CD40激活性抗体的表达框的载体，所述载体用于将所述表达框整合到宿主细胞的基因组中。
  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。
  10. 权利要求1－7中任一项所述的T细胞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；优选地，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：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、非小细胞癌、胆囊癌、食管癌、黑色素瘤、胰腺癌、尿路上皮癌、头颈癌或前列腺癌。
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