WO2017032293A1 - 抗间皮素全人抗体以及靶向间皮素的免疫效应细胞 - Google Patents

抗间皮素全人抗体以及靶向间皮素的免疫效应细胞 Download PDF

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WO2017032293A1
WO2017032293A1 PCT/CN2016/096292 CN2016096292W WO2017032293A1 WO 2017032293 A1 WO2017032293 A1 WO 2017032293A1 CN 2016096292 W CN2016096292 W CN 2016096292W WO 2017032293 A1 WO2017032293 A1 WO 2017032293A1
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antibody
seq
amino acid
acid sequence
positions
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PCT/CN2016/096292
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English (en)
French (fr)
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王华茂
宋波
王鹏
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科济生物医药（上海）有限公司
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Priority to US15/754,076 priority Critical patent/US10793641B2/en
Priority to NZ740855A priority patent/NZ740855A/en
Priority to KR1020187007893A priority patent/KR20180055824A/ko
Priority to JP2018509805A priority patent/JP6994456B2/ja
Priority to SG11201801351RA priority patent/SG11201801351RA/en
Priority to CA2996060A priority patent/CA2996060A1/en
Application filed by 科济生物医药（上海）有限公司 filed Critical 科济生物医药（上海）有限公司
Priority to BR112018003339A priority patent/BR112018003339A8/pt
Priority to AU2016312015A priority patent/AU2016312015A1/en
Priority to EP16838552.4A priority patent/EP3339326A4/en
Priority to RU2018107802A priority patent/RU2748281C2/ru
Publication of WO2017032293A1 publication Critical patent/WO2017032293A1/zh
Priority to IL257563A priority patent/IL257563A/en

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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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
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    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention belongs to the field of tumor immunotherapy or diagnosis, and more particularly, the present invention relates to anti-mesothelin whole human antibodies and immune effector cells targeting mesothelin.
  • CTLs cytotoxic lymphocytes
  • TCR T Cell Receptor
  • CAR chimeric antigen receptor
  • MHC major Histocompatibility Complex
  • CAR-modified immune effector cells especially T cells
  • the antigenic gene targeted is actually a key choice.
  • CAR CAR-modified immune effector cells
  • the genes are very difficult.
  • tumor-specific antigens are difficult to find specific molecules for them that are suitable for the construction of CAR-modified immune effector cells.
  • Mesothelin is a cell surface glycoprotein with a molecular weight of 40-kDa. It is highly expressed in various tumors such as pancreatic cancer, ovarian cancer, and thymic mesothelioma. It is expressed in normal tissues only on normal mesothelial cells of the pleura, pericardium and peritoneum.
  • Mesothelin is synthesized as a 71 kDa precursor protein, and its mature portion is expressed on the cell surface. This precursor protein was proteolytically cleaved by a furin to a 31 kDa shedding moiety (called megakaryocyte chimeric factor, or MPF) and a 40 kDa mesothelin moiety). The latter component can remain bound to the cell surface via a GPI linkage and can also be shed via a proteolytic enzyme mechanism.
  • MPF megakaryocyte chimeric factor
  • Antibodies targeting mesothelin or other targeted therapies have been reported.
  • CAR-T also has reports of clinical research (Maus MV, Haas AR, Beatty GL, Albelda SM, Levine BL, Liu X, Zhao Y, Kalos M, June CH.
  • T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res. 2013;1(1):26-31; Beatty GL, Haas AR, Maus MV, Torigian DA, Soulen MC, Plesa G, Chew A, Zhao Y, Levine BL, Albelda SM, Kalos M, June CH.
  • a fully human antibody which specifically binds mesothelin, the whole human antibody being selected from the group consisting of:
  • an antibody comprising a heavy chain variable region having a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54 and a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55, comprising CDR3 of the amino acid sequence set forth in SEQ ID NO: 56;
  • an antibody comprising a light chain variable region having a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, and a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, comprising SEQ ID NO: 53 shows the CDR3 of the amino acid sequence;
  • an antibody comprising (a) a heavy chain variable region of said antibody and (b) a light chain variable region of said antibody;
  • an antibody comprising a heavy chain variable region having a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 60, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 61, comprising SEQ CDR3 of the amino acid sequence shown by ID NO: 62;
  • an antibody comprising a light chain variable region having a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58 comprising SEQ CDR3 of the amino acid sequence shown by ID NO: 59;
  • the fully human antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region of which is represented by positions 1-123 of SEQ ID NO: 6; The amino acid sequence of the variable region is as shown in positions 139-254 of SEQ ID NO: 6;
  • the fully human antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region thereof being represented by positions 1-124 of SEQ ID NO: 8; the amino acid sequence of the light chain variable region thereof As shown in SEQ ID NO: 8 in positions 140-247.
  • the fully human antibody that specifically binds mesothelin may be: single chain antibody (scFV), monoclonal antibody, domain antibody, Fab fragment, Fd fragment, Fv fragment, F(ab' 2 fragments and derivatives thereof, or other forms of antibodies; preferably single chain antibodies.
  • scFV single chain antibody
  • monoclonal antibody domain antibody
  • Fab fragment fragment
  • Fd fragment fragment
  • Fv fragment fragment
  • F(ab' 2 fragments and derivatives thereof or other forms of antibodies
  • other forms of antibodies preferably single chain antibodies.
  • nucleic acid encoding the antibody is provided.
  • an expression vector comprising the nucleic acid is provided.
  • a host cell comprising the nucleic acid to which the described expression vector or genome is integrated is provided.
  • a chimeric antigen receptor (Chimeric antigen receptor) of the antibody comprises a sequence-ligated antibody, a transmembrane region and an intracellular signal region of the invention.
  • the intracellular signal region is selected from the group consisting of CD3 ⁇ , Fc ⁇ RI ⁇ , CD27, CD28, CD137, CD134, MyD88, an intracellular signal sequence of CD40, or a combination thereof.
  • the transmembrane region comprises a transmembrane region of CD8 or CD28.
  • the chimeric antigen receptor comprises the following sequentially linked antibodies, a transmembrane region and an intracellular signal region:
  • the antibody the transmembrane region of the CD28 molecule, the intracellular signal region of the CD28 molecule, CD137 and CD3 ⁇ .
  • the antibody is a single chain antibody or a domain antibody.
  • the chimeric antigen receptor has:
  • SEQ ID NO: 41 or the amino acid sequence shown at positions 22-353;
  • SEQ ID NO: 42 or the amino acid sequence shown at positions 22-454;
  • SEQ ID NO: 43 or the amino acid sequence shown in positions 22-498;
  • SEQ ID NO: 44 or the amino acid sequence shown at positions 22-501;
  • SEQ ID NO: 45 or the amino acid sequence shown in positions 22-543;
  • SEQ ID NO: 46 or the amino acid sequence shown in positions 22-346;
  • SEQ ID NO: 47 or the amino acid sequence shown in positions 22-447;
  • SEQ ID NO: 48 or the amino acid sequence shown at positions 22-491;
  • SEQ ID NO: 49 or the amino acid sequence set forth at positions 22-494;
  • SEQ ID NO: 50 or the amino acid sequence shown at positions 22-536.
  • nucleic acid encoding the chimeric antigen receptor is provided.
  • nucleic acid encoding the chimeric antigen receptor has:
  • SEQ ID NO: 31 or the nucleotide sequence set forth in positions 473-1468;
  • SEQ ID NO: 32 or the nucleotide sequence set forth in positions 473-1771;
  • SEQ ID NO: 33 or the nucleotide sequence set forth in positions 473-1903;
  • SEQ ID NO: 34 or the nucleotide sequence set forth in positions 473-1912;
  • SEQ ID NO: 35 or the nucleotide sequence set forth in positions 473-2038;
  • SEQ ID NO: 36 or the nucleotide sequence set forth in positions 473-1447;
  • SEQ ID NO: 37 or the nucleotide sequence set forth in positions 4735 to 1550;
  • SEQ ID NO: 38 or the nucleotide sequence set forth in positions 473-1882;
  • SEQ ID NO: 39 or the nucleotide sequence set forth in positions 473-1891;
  • SEQ ID NO: 40 or the nucleotide sequence set forth in positions 473-2017.
  • an expression vector comprising the nucleic acid is provided.
  • the expression vector is derived from the lentiviral plasmid pWPT (or pWPT-eGFP).
  • a virus comprising the vector.
  • the use of the chimeric antigen receptor, or the nucleic acid, or the expression vector, or the virus, for the preparation of a tumor targeting mesothelin Genetically modified immune cells of cells for the preparation of a tumor targeting mesothelin Genetically modified immune cells of cells.
  • the mesothelin-expressing tumor includes, but is not limited to, pancreatic cancer, ovarian cancer, and thymic mesothelioma.
  • a genetically modified immune cell transduced with said nucleic acid, or said expression vector or said virus; or a surface thereof, said chimeric antigen receptor .
  • the immune cell further carries a coding sequence for a foreign cytokine; preferably, the cytokine comprises: IL-12, IL-15 or IL-21.
  • the immune cell further expresses another chimeric antigen receptor that does not contain CD3 ⁇ , but contains an intracellular signal domain of CD28, an intracellular signal domain of CD137, or both. Combination of people.
  • the immune cell further expresses a chemokine receptor; preferably, the chemokine receptor comprises: CCR2.
  • the immune cell further expresses an siRNA that reduces PD-1 expression or a protein that blocks PD-L1.
  • the immune cells further express a safety switch; preferably, the safety switch comprises: iCaspase-9, Truncated EGFR or RQR8.
  • the immune cells comprise: T lymphocytes, NK cells or NKT lymphocytes.
  • the use of the genetically modified immune cell for the preparation of a tumor suppressing drug, the tumor being a tumor expressing mesothelin.
  • a multifunctional immunoconjugate comprising: the antibody; and a linkage thereto (including covalent attachment, coupling, attachment, adsorption) a functional molecule; the functional molecule selected from the group consisting of: a molecule that targets a tumor surface marker, a molecule that inhibits tumors, a molecule that targets a surface marker of an immune cell, or a detectable label.
  • the molecule targeting the tumor surface marker is an antibody or a ligand that binds to a tumor surface marker; or the tumor suppressing molecule is resistant Tumor cytokines or anti-tumor toxins; preferably, the cytokines include, but are not limited to, IL-12, IL-15, IFN-beta, TNF-alpha.
  • the detectable label comprises: a fluorescent label, a chromogenic label.
  • the antibody that binds to a tumor surface marker refers to an antibody that recognizes other antigens other than mesothelin, and the other antigens include: EGFR , EGFRvIII, mesothelin, HER2, EphA2, Her3, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, folate receptor, Claudin 6, CD3, WT1, NY-ESO-1, MAGE 3, ASGPR1 or CDH16.
  • the molecule that targets a surface marker of an immune cell is an antibody that binds to a T cell surface marker, and forms a T cell with the antibody. Participating bispecific antibody (Bispecific T cell engager, BiTE).
  • the antibody that binds to a T cell surface marker is an anti-CD3 antibody.
  • the anti-CD3 antibody is a single chain antibody (scFV), a monoclonal antibody, a Fab fragment, an Fd fragment, an Fv fragment, a F(ab') 2 fragment and derivatives thereof, or other forms thereof.
  • An antibody preferably a single chain antibody.
  • the anti-CD3 antibody is humanized, chimeric, fully human or murine.
  • the multifunctional immunoconjugate is a fusion polypeptide, and a linker (linker) is further included between the antibody of the present invention and a functional molecule linked thereto.
  • the multifunctional immunoconjugate is administered by means of polypeptide administration or gene administration.
  • nucleic acid encoding the multifunctional immunoconjugate is provided.
  • the multifunctional immunoconjugate for the preparation of an antitumor drug, or for the preparation of a medicament for diagnosing a tumor, the tumor expressing mesothelin; or for preparing a chimeric
  • the antigen receptor-modified immune cell preferably, the immune cell comprises: T lymphocytes, NK cells or NKT lymphocytes.
  • composition comprising:
  • the genetically modified immune cell The genetically modified immune cell.
  • an antibody which is capable of competing with the antibodies of the invention for binding to mesothelin.
  • an antibody that is capable of binding to a mesothelin epitope as set forth in SEQ ID NO: 66 is provided.
  • an antibody that binds to the mesothelin epitope set forth in SEQ ID NO: 72 is also provided.
  • FIG. 1 Binding of antibodies P1A6E and P3F2 to human hu-mesothelin and control BSA in a single phage ELISA assay.
  • the values of the antibodies P1A6E and P3F2 for human mesothelin and the negative control BSA demonstrated that the two antibodies screened specifically bind to human mesothelin.
  • ELISA assay detects the binding of two different single-chain antibodies P1A6E and P3F2 to human mesothelin and BSA.
  • FIG. 7 Assays for specific binding of four single chain antibodies (P1A6E, P3F2 and control antibodies SS, C10) to PANC-1-MSLN cells by fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • FIG. 8 Assays for specific binding of four monoclonal antibodies (P1A6E, P3F2 and control antibodies SS, C10) to PANC-1-MSLN cells by fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • Figure 9 ELISA showing the binding of the antibodies scFv-P1A6E-Fc and scFv-P3F2-Fc to the regions R1, R2, R3.
  • Figure 10 ELISA showing the binding of the antibodies scFv-P1A6E-Fc and scFv-P3F2-Fc to the regions R1A, R1B, R1C, R1AB, R1BC.
  • the present inventors have previously investigated a large number of tumor-specific genes, and found that a considerable part of such genes are also expressed in normal cells of some tissues, and are difficult to apply to chimeric antigen receptor-modified immune effector cell technology; some tumors
  • the specific gene has better tumor-specific expression characteristics.
  • the CAR-modified immune effector cells based on its design have no tumor cell killing activity or low activity, which may be because the target can induce tumor cell secretion and immune effect.
  • the cell has an inhibitory factor such as PD-L1.
  • mesothelin As a target for designing CAR from a number of candidate molecules.
  • the present inventors have shown that CAR-modified T cells prepared based on mesothelin antibodies can selectively target mesothelin-positive tumor cells and have strong cytotoxicity against tumor cells.
  • the present inventors believe that the corresponding CAR-modified immune effector cells, particularly T cells, should be useful for the treatment of human tumors.
  • the present inventors screened a fully human-derived natural antibody library to obtain a specific antibody for mesothelin-binding ability and suitable for preparation of genetically modified immune effector cells, and found a key CDR region which exerts binding properties.
  • the antibody of the present invention may be an intact immunoglobulin molecule or an antigen-binding fragment, including but not limited to a Fab fragment, an Fd fragment, an Fv fragment, a F(ab') 2 fragment, a complementarity determining region (CDR) fragment, and a single Chain antibody (scFv), domain antibody, bivalent single chain antibody, single chain phage antibody, bispecific double chain antibody, triple chain antibody, four chain antibody.
  • the antigen binding properties of an antibody can be described by three specific regions located in the heavy and light chain variable regions, referred to as complementarity determining regions (CDRs), which divide the variable regions into four In the framework region (FR), the amino acid sequences of the four FRs are relatively conservative and are not directly involved in the binding reaction.
  • CDRs complementarity determining regions
  • These CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the CDR regions are sequences of immunologically interesting proteins, and the CDR regions of the antibodies of the invention are novel.
  • the antibody may comprise two, three, four, five or all six CDR regions as disclosed herein.
  • Another aspect of the invention includes a functional variant of an antibody as described herein, if the variant is capable of compete with the parent antibody for specific binding to mesothelin, and its ability to recognize mesothelin expressed by the tumor cell is similar to that provided in the embodiments of the invention Specific antibodies.
  • the functional variants can have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can include both natural as well as non-natural nucleotides and amino acids.
  • the modification of the sequence occurs in a region other than the CDR regions of the antibody on.
  • the antibodies of the present invention can be applied to the preparation of various targeted antitumor drugs as well as drugs for diagnosing tumors, in particular, for the preparation of immune effector cells targeting mesothelin.
  • the present invention provides a chimeric antigen receptor expressed on the surface of an immune effector cell (immune cell) comprising a sequence-linked extracellular binding region, a transmembrane region and an intracellular signal region, Wherein the extracellular binding region comprises an antibody of the invention.
  • an immune effector cell immunodeficiency virus
  • Expression of the chimeric antigen receptor on the surface of the immune effector cell allows the immune effector cell to have a highly specific cytotoxic effect on the mesothelin-bearing tumor cells.
  • immune cells are used interchangeably with “immune effector cells” and include: T lymphocytes, NK cells or NKT cells, and the like.
  • the chimeric antigen receptor comprises an antibody which is a single-chain antibody which is linked to the transmembrane region of CD8 or CD28 via a CD8 hinge region, and is followed by an intracellular signal in the transmembrane region. Area.
  • the invention also includes nucleic acids encoding the chimeric antigen receptors.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • the transmembrane region of the chimeric antigen receptor can be selected from the transmembrane region of a protein such as CD8 or CD28.
  • the human CD8 protein is a heterodimer composed of two chains, ⁇ or ⁇ .
  • the transmembrane region is selected from the transmembrane region of CD8 alpha or CD28.
  • the CD8 ⁇ hinge region is a flexible region, and therefore, CD8 or CD28 and a transmembrane region plus a hinge region are used to link the target recognition domain scFv of the chimeric antigen receptor CAR with the intracellular signal region. .
  • the intracellular signal region can be selected from the group consisting of CD3 ⁇ , Fc ⁇ RI ⁇ , CD27, CD28, CD137, CD134, MyD88, the intracellular signal region of the CD4 protein, and combinations thereof.
  • the CD3 molecule consists of five subunits, of which the CD3 ⁇ subunit (also known as CD3zeta, abbreviated as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex.
  • CD3 ⁇ Z is a truncated CD3 ⁇ sequence that does not have an ITAM motif and is generally constructed as a negative control in practice.
  • Fc ⁇ RI ⁇ is mainly distributed on the surface of mast cells and basophils, which contains an ITAM motif similar in structure, distribution and function to CD3 ⁇ .
  • CD28, CD137, and CD134 are costimulatory signal molecules, and the co-stimulatory effects of intracellular signal segments upon binding to their respective ligands cause sustained proliferation of immune effector cells (mainly T lymphocytes). It can also increase the levels of cytokines such as IL-2 and IFN- ⁇ secreted by immune effector cells, and increase the survival cycle and anti-tumor effect of CAR immune effector cells in vivo.
  • chimeric antigen receptors of the invention can be linked in the following manner:
  • the antibody of the present invention the transmembrane region of the CD28 molecule, the intracellular signal region of the CD28 molecule, and CD3 ⁇ ; or
  • the antibody of the present invention the transmembrane region of the CD28 molecule, the intracellular signal region of the CD28 molecule, CD137 and CD3 ⁇ .
  • CD28a in the relevant chimeric antigen receptor protein represents the transmembrane region of the CD28 molecule
  • CD28b represents the intracellular signal region of the CD28 molecule.
  • the various chimeric antigen receptors described above are collectively referred to as scFv (mesothelin)-CAR.
  • the present invention also provides a nucleic acid comprising the above chimeric antigen receptor protein encoding the surface of an immune effector cell.
  • Carrier a lentiviral plasmid vector pWPT-eGFP. This plasmid belongs to the third generation auto-inactivated lentiviral vector system.
  • the system has three plasmids, namely, the coding protein Gag/Pol, the packaging plasmid psPAX2 encoding Rev protein, and the envelope plasmid PMD2.G encoding VSV-G protein;
  • the vector pWPT-eGFP which can be used for recombinant introduction of a nucleic acid sequence of interest, ie a nucleic acid sequence encoding a CAR.
  • the expression of enhanced green fluorescent protein (eGFP) was regulated by the elongation factor-1 ⁇ (elongation factor-1 ⁇ , EF-1 ⁇ ) promoter in the empty vector pWPT-eGFP.
  • the recombinant expression vector pWPT-eGFP-F2A-CAR comprising the nucleic acid sequence of interest encoding CAR was obtained by ribosomal skipping sequence 2A (F2A). ) to achieve co-expression of eGFP and CAR. It should be understood that other expression vectors are also available.
  • the invention also includes viruses comprising the vectors described above.
  • the virus of the present invention includes a packaged infectious virus, and also includes a virus to be packaged containing components necessary for packaging as an infectious virus.
  • Other viruses known in the art that can be used to transduce foreign genes into immune effector cells and their corresponding plasmid vectors can also be used in the present invention.
  • the present invention also provides a genetically modified immune effector cell transduced with the nucleic acid of the present invention or the above-described recombinant plasmid comprising the nucleic acid of the present invention, or a virus comprising the same.
  • Conventional nucleic acid transduction methods including non-viral and viral transduction methods, can be used in the present invention.
  • Non-viral based transduction methods include electroporation and transposon methods.
  • the Nucleofector nuclear transfection device developed by Amaxa can directly introduce foreign genes into the nucleus to obtain efficient transduction of the target gene.
  • the transduction efficiency of the transposon system based on Sleeping Beauty system or PiggyBac transposon is much higher than that of ordinary electroporation, and the nucleofector transfection apparatus is combined with the Sleeping Beauty transposon system. It has been reported [Davies JK., et al. Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70(10): OF1-10.], The method has high transduction efficiency and can achieve targeted integration of the target gene.
  • the transduction method for the immune effector cells that effect the chimeric antigen receptor gene modification is based on a transduction method of a virus such as a retrovirus or a lentivirus.
  • the method has the advantages of high transduction efficiency, stable expression of the exogenous gene, and shortening the time for the cultured immune effector cells to reach the clinical level in vitro.
  • the transduced nucleic acid is expressed on its surface by transcription and translation.
  • the in vitro cytotoxicity assay of various cultured tumor cells demonstrates that the immune effector cells of the present invention have a highly specific tumor cell killing effect (also known as cytotoxicity).
  • the nucleic acid encoding the chimeric antigen receptor protein of the present invention, the plasmid comprising the nucleic acid, the virus comprising the plasmid, and the transgenic immune effector cell transduced with the nucleic acid, plasmid or virus can be effectively used for immunotherapy of tumors.
  • the immune cells of the present invention may also carry a coding sequence of a foreign cytokine; the cytokines include, but are not limited to, IL-12, IL-15 or IL-21 and the like. These cytokines have immunomodulatory or anti-tumor activity, enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects. Thus, those skilled in the art will appreciate that the use of these cytokines will help the immune cells function better.
  • the immune cells of the present invention may also express another chimeric antigen receptor other than the chimeric antigen receptor described above, which does not contain CD3 ⁇ , but contains an intracellular signal domain of CD28, an intracellular signal of CD137. A domain or a combination of the two.
  • the immune cells of the invention may also express a chemokine receptor; the chemokine receptors include, but are not limited to, CCR2. Those skilled in the art will appreciate that the CCR2 chemokine receptors may allow CCR2 binding in vivo to compete with it, which is advantageous for blocking tumor metastasis.
  • the immune cells of the present invention can also express siRNA that reduces PD-1 expression or a protein that blocks PD-L1.
  • siRNA that reduces PD-1 expression
  • a protein that blocks PD-L1 e.g., a protein that blocks PD-L1.
  • the immune cells of the present invention may also express a safety switch; preferably, the safety switch comprises: iCaspase-9, Truncated EGFR or RQR8.
  • the invention also provides a multifunctional immunoconjugate comprising an antibody described herein and further comprising at least one other type of functional molecule.
  • the functional molecule is selected from, but not limited to, a molecule that targets a tumor surface marker, a molecule that inhibits tumors, a molecule that targets a surface marker of an immune cell, or a detectable label.
  • the antibody and the functional molecule may constitute a composition by covalent attachment, coupling, attachment, crosslinking, or the like.
  • the immunoconjugate may comprise: an antibody of the invention and at least one molecule that targets a tumor surface marker or a tumor suppressor molecule.
  • the tumor suppressing molecule may be an anti-tumor cytokine or an anti-tumor toxin; preferably, the cytokine includes but is not limited to: IL-12, IL-15, IFN-beta, TNF -alpha.
  • the molecules that target tumor surface markers for example, can act synergistically with the antibodies of the invention to more accurately target tumor cells.
  • the immunoconjugate can comprise: an antibody of the invention and a detectable label.
  • the detectable label includes, but is not limited to, a fluorescent label, a chromogenic label; eg, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron emitting metal, and a non-radioactive paramagnetic Metal ion. More than one marker may also be included.
  • the label used to label the antibody for detection and/or analysis and/or diagnostic purposes depends on the particular detection/analysis/diagnostic technique and/or method used, such as immunohistochemical staining (tissue) samples, flow cytometry, and the like. Suitable labels for detection/analysis/diagnostic techniques and/or methods known in the art are well known to those skilled in the art.
  • the immunoconjugate may comprise: an antibody of the invention and a molecule that targets a surface marker of an immune cell.
  • the molecule targeting the surface marker of the immune cell can recognize the immune cell, which carries the antibody of the invention to the immune cell, and the antibody of the invention can target the immune cell to the tumor cell, thereby causing the immune cell to specifically kill Tumor.
  • the immunoconjugate can be produced as a fusion protein comprising an antibody of the invention and a suitable other protein.
  • the fusion protein can be produced by methods known in the art, for example by constructing a nucleic acid molecule comprising a nucleotide sequence encoding the antibody and encoding a suitable labeled nucleoside, and subsequently expressing the nucleic acid molecule. Acid sequence.
  • Another aspect of the invention provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate thereof of the invention.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • the antibody of the present invention, the immunoconjugate comprising the antibody, and the genetically modified immune cell can be applied to the preparation of a pharmaceutical composition or a diagnostic reagent.
  • the composition may comprise a pharmaceutically acceptable carrier in addition to an effective amount of the antibody, immunoconjugate or immune cell.
  • pharmaceutically acceptable means that when the molecular body and composition are suitably administered to an animal or a human, they do not produce an adverse, allergic or other untoward reaction.
  • sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as carboxymethyl fibers Sodium, ethyl cellulose and methyl cellulose; western yellow gum powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Wetting agents, such as sodium lauryl sulfate; colorants; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers
  • composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician in accordance with factors such as patient type, age, body weight, and general disease condition, mode of administration, and the like.
  • the mode of administration can be, for example, injection or other treatment.
  • the vector system used in this embodiment belongs to the third generation self-inactivated lentiviral vector system, which has three plasmids, namely, the coding protein Gag/Pol, the packaging plasmid psPAX2 encoding the Rev protein, and the envelope plasmid PMD2 encoding the VSV-G protein. .G and recombinant plasmid pWPT-MSLN encoding the extracellular region and transmembrane region of the human mesothelin of the gene of interest based on the empty vector pWPT (purchased from Addgene).
  • a gene fragment containing the signal peptide, Flag tag, human mesothelin extracellular region and transmembrane region was synthesized according to Genbank accession number NM_005823 using a PCR-based gene synthesis method (SEQ ID NO: 1 (nucleotide), 2 (amino acid)), PCR amplification by primer pair pWmslnF (SEQ ID NO: 3, GCTTACGCGTCCTAGCGCTACCGGTCGCCACCATGAGGGCCTGGATC) and pWmslnR (SEQ ID NO: 4, CGAGGTCGAC CTAGGCCAGGGTGGAGGCTAGGAGCAGTGCCAGGACGG), the amplification conditions are pre-denaturation: 94 ° C, 4 min; Denaturation: 94 ° C, 30 s; annealing: 58 ° C, 30 s; extension: 68 ° C, 80 s; 30 cycles.
  • the theoretical size of the obtained fragment was 1113 bp, and the amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • the MluI and SalI restriction sites are introduced upstream and downstream of the open reading frame.
  • the target gene obtained above was digested with MluI and SalI, and ligated into the same double-digested pWPT vector to construct a successful lentiviral vector pWPT-MSLN, which was identified by MluI and SalI digestion and sequenced correctly. .
  • 293T cells (ATCC: CRL-11268) cultured to the 6th to 10th passages were inoculated at a density of 6 ⁇ 10 6 in a 10 cm culture dish, and cultured overnight at 37 ° C, 5% CO 2 for transfection.
  • the medium was DMEM (Invitrogen) containing 10% fetal bovine serum (Sigma), and the next day, the culture medium was changed to serum-free DMEM about 2 hours before transfection.
  • the human mesothelin gene fragment (SEQ ID NO: 1 at positions 88-942 (nucleotides), (SEQ ID NO: 12, positions 30-314) was synthesized according to Genbank accession number NM_005823 using a PCR-based gene synthesis method. Amino acid)), and PCR amplification.
  • the amplified product was inserted into the plasmid vector pCMV-V5 by NheI/BglII (the vector was expressed downstream of the multiple cloning site and expressed 6 ⁇ His tag, purchased from Shanghai Ruijin Biotechnology Co., Ltd.) And transformed into the host strain TOP10, picking the clone to identify the positive clone by PCR and confirming by sequencing to obtain the recombinant expression plasmid V5-MSLN.
  • the above expression plasmid was transfected into well-grown HEK-293F cells, cultured continuously for 7 days at 37 ° C, 5% CO 2 , 125 rpm shaker, centrifuged at 4000 rpm for 10 min, the precipitate was removed, and the supernatant was collected and filtered through a 0.45 ⁇ m filter.
  • a good sample was purified by HisTrap (purchased from GE) affinity chromatography column to obtain purified human mesothelin protein. The identification results are shown in Fig. 1.
  • Human mesothelin-specific antibodies were screened from a fully human-derived natural antibody library using phage display technology.
  • glycerol bacteria purchased from Shanghai Ruijin Biotechnology Co., Ltd.
  • Infection was carried out with 10 12 pfu of M13KO7 helper phage (purchased from Invitrogen), and cultured at 30 ° C and 50 rpm for 30 minutes.
  • the phage particles were precipitated by incubation in an ice bath for 1 hour. The pellet was then centrifuged (20 min, 8000 x g, 4 ° C), the supernatant was discarded, and the phage was suspended in 25 ml of pre-cooled PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ). Centrifugation (5 minutes, 20000 x g, 4 ° C). The supernatant was added to a 1/4 volume of 20% (w/v) PEG 8000, 2.5 M NaCl solution, and the phage particles were again precipitated by ice bath for 30 minutes.
  • the pellet was centrifuged (30 min, 20000 g, 4 °C), and the phage pellet was again resuspended in 2 ml of pre-cooled PBS, kept on ice for 30 minutes and centrifuged (30 min, 17000 x g, 4 °C).
  • the supernatant was mixed 1:1 with 4% (w/v) BSA in PBS, placed on a rotary mixer, incubated at room temperature for 30 minutes, and directly used for screening.
  • the screening protocol was as follows: the phage antibody library and biotinylated anti-human mesothelin were incubated at room temperature for 2 hours, and then Streptavidin magnetic beads MyOne C1 (purchased from Invitrogen) blocked with 2% (w/v) BSA (bovine serum albumin) in blocking solution were incubated at room temperature for 30 minutes. The magnetic beads were then washed with PBST (containing 0.1% Tween-20) buffer to remove non-specifically bound or weakly binding phage.
  • PBST containing 0.1% Tween-20
  • the phage with strong binding ability was eluted from the magnetic beads with glycine-hydrochloric acid (pH 2.2), and neutralized with Tris neutralizing solution (pH 9.1) to infect Escherichia coli ER2738 in the middle of logarithmic growth, and Used for the next round of screening.
  • the amount of magnetic beads was 50 ⁇ l, 20 ⁇ l, 10 ⁇ l and 10 ⁇ l, respectively.
  • the concentrations of biotin-labeled human mesothelin were 100nM, 10nM, 5nM and 1nM, respectively.
  • the washing times of PBST were 10 times and 10 times, respectively. 15 times and 20 times.
  • 96 clones were randomly selected from the fourth round of screening, and their ability to bind to human mesothelin was analyzed by single phage ELISA (enzyme-linked immunosorbent assay).
  • each single colony was inoculated with 300 ⁇ l of 2 ⁇ YT/ampicillin medium (containing 2% glucose) in a 96-well deep well culture plate, and cultured at 37° C. and 250 rpm for 16 hours with shaking. 20 ⁇ l of the culture was inoculated to 500 ⁇ l of 2 ⁇ YT/ampicillin medium (containing 0.1% glucose), and cultured at 37° C. and 250 rpm for 1.5 hours with shaking.
  • a helper phage solution was prepared, and 75 ⁇ l of M13KO7 (titer 3 ⁇ 10 12 pfu/ml) was mixed into 15 ml of 2 ⁇ YT medium, and 50 ⁇ l/well was added to the culture plate. Incubate for 30 minutes at 37 ° C and 150 rpm, then add 50 ⁇ l/well of the prepared kanamycin solution (take 180 ⁇ l of 50 mg/ml kanamycin and add to 15 ml of 2 ⁇ YT medium) at 37 ° C and 250 rpm. The culture was shaken for 16 hours. Finally, the cells were pelleted by centrifugation (30 min, 5000 x g, 4 ° C) and the supernatant was transferred to a new 96-well deep well plate.
  • anti-M13 antibody superoxide dismutase conjugate purchased from GE Healthcare
  • PBST peripheral blood mononuclear cell
  • 50 ⁇ l of TMB substrate was pipetted into the well and developed at room temperature for 10 minutes, followed by the addition of 50 ⁇ l of 2M H 2 SO 4 per well to terminate the color reaction.
  • the extinction value was measured at 450 nm using an enzyme-linked immunosorbent assay (Bio-Rad).
  • amino acid sequence of the heavy chain variable region is as shown in positions 1-123 of SEQ ID NO: 6; the amino acid sequence of the light chain variable region thereof is shown in positions 139-254 of SEQ ID NO: 6.
  • the light chain CDR1 amino acid sequence is TLRSGINVGIYRIY (SEQ ID NO: 51)
  • the light chain CDR2 amino acid sequence is YKSDSDKYQGS (SEQ ID NO: 52)
  • the light chain CDR3 amino acid sequence is MIWHSGGWV (SEQ ID NO: 53);
  • heavy chain CDR1 The amino acid sequence is GDTVSSDSAAWN (SEQ ID NO: 54)
  • the heavy chain CDR2 amino acid sequence is RTYYRSKWFNDYAVSVKG (SEQ ID NO: 55)
  • the heavy chain CDR3 amino acid sequence is SNSYYYYAMDV (SEQ ID NO: 56).
  • amino acid sequence of the heavy chain variable region is as shown in positions 1-124 of SEQ ID NO: 8; the amino acid sequence of the light chain variable region thereof is shown in positions 140-247 of SEQ ID NO: 8.
  • the light chain CDR1 amino acid sequence is RASQVISRALA (SEQ ID NO: 57)
  • the light chain CDR2 amino acid sequence is DASNLQS (SEQ ID NO: 58)
  • the light chain CDR3 amino acid sequence is QQFNSYPLT (SEQ ID NO: 59)
  • heavy chain CDR1 The amino acid sequence is GYTFTSYYMH (SEQ ID NO: 60)
  • the heavy chain CDR2 amino acid sequence is IINPSGGSTSYAQKFQG (SEQ ID NO: 61)
  • the heavy chain CDR3 amino acid sequence is SRSGTTVVNHDAFDI (SEQ ID NO: 62).
  • the scFv-P1A6E fragment was amplified from the resulting clone using primer pairs V5-P1A6E-F (SEQ ID NO: 9) and V5-P1A6E-R (SEQ ID NO: 10); using primer pair V5-P3F2-F (SEQ ID NO: 11) and V5-P3F2-R (SEQ ID NO: 12) amplified the scFv-P3F2 fragment, digested with NheI/BamHI, and digested the vector plasmid pCMV with NheI/BamHI by T4 DNA ligase.
  • V5-Fc this vector is fused to the Fc fragment expressing human antibody IgG1 downstream of the multiple cloning site, hereinafter referred to as V5-Fc, purchased from Shanghai Ruijin Biotechnology Co., Ltd.
  • V5-Fc Fc fragment expressing human antibody IgG1 downstream of the multiple cloning site
  • Positive clones were identified by PCR and confirmed by sequencing, and V5-scFv-P1A6E-Fc and V5-scFv-P3F2-Fc eukaryotic expression plasmids were obtained, respectively.
  • SEQ ID NO: 9 ACAGT GCTAGCA CAGGTACAGCTGGAACAG;
  • SEQ ID NO: 10 TTGTC GGATCC ACCTAGGACGGTGACC;
  • SEQ ID NO: 11 ACAGT GCTAGCA CAGATGCAGCTAGTGC;
  • SEQ ID NO: 12 TTGTC GGATCC ACGTTTGATCTCCAGC.
  • the above expression plasmids were transfected into well-preserved HEK-293F cells, cultured continuously for 7 days at 37 ° C, 5% CO 2 , 125 rpm shaker, centrifuged at 4000 rpm for 10 min, the precipitate was removed, and the supernatant was collected and filtered through a 0.45 ⁇ m filter.
  • the treated samples were affinity-purified with a protein A (purchased from GE) affinity column to finally obtain purified antibody-Fc fusion proteins scFv-P1A6E-Fc and scFv-P3F2-Fc, and the results of the identification are shown in FIG.
  • the expression of the monoclonal antibody uses a double plasmid system, and the antibody heavy chain variable region gene needs to be separately constructed into a pIH plasmid containing the human IgG1CH gene, and the antibody light chain variable region gene is constructed to include a human.
  • pIK plasmid of IgG CL gene (plasmid was purchased from Shanghai Ruijin Biotechnology Co., Ltd.).
  • VH-P1A6E fragment was amplified from the template plasmid V5-scFv-P1A6E-Fc using primer pairs P1A6E-HF (SEQ ID NO: 13, gcctttcctggtttcctgtctcaggtacagctag aacagtc) and P1A6E-HR (SEQ ID NO: 14, GATGGGCCCTTGGTGGAGGCACTCG AGACGGTGACCAG).
  • the HF1 fragment was amplified from the template plasmid pIH using primer pair HF1F (SEQ ID NO: 15, ggctaactagagaacccactgc) and HF1R (SEQ ID NO: 16, AGACAGGAAACCAGGAAAGGC); primer pair HF3F (SEQ ID NO: 17, gcctccaccaagggcccat) was used.
  • the HF3 fragment was amplified from the template plasmid pIH and HF3R (SEQ ID NO: 18, gacaatcttagcgcagaagtc). The three fragments were mixed in equimolar ratio and then spliced into PCR.
  • the fragments were recovered and digested with restriction endonuclease NheI/NotI, and ligated with T4 DNA ligase in the same NheI/NotI double-transgested vector plasmid pIH and transformed into host bacteria.
  • the clones were identified by PCR to identify positive clones and confirmed by sequencing to obtain the pIH-P1A6E eukaryotic expression plasmid.
  • the pIH-P3F2 eukaryotic expression plasmid was also obtained in the same manner.
  • the primer pair P1A6E-LF (SEQ ID NO: 19, ctttggtttccaggtgcaagatgtcaggctgtgctgactcag) and P1A6E-LR (SEQ ID NO: 20, GAAGACAGATGGTGCAGCCACCGTACCTAGGACGGTGACCTTG) were used to expand from the template plasmid V5-scFv-P1A6E-Fc.
  • the VL-P1A6E fragment was added; the LF1 fragment was amplified from the template plasmid pIK using the primer pair LF1F (SEQ ID NO: 21, ggctaactagagaacccactgc) and LF1R (SEQ ID NO: 22, ACATCTTGCACC TGGAAACCAAAG); primer pair LF3F (SEQ ID) was used. NO: 23, agggtggctgcaccatctgtcttc) and LF3R (SEQ ID NO: 24, GACAATCTTAGCGCAGAAGTC) amplified the LF3 fragment from the template plasmid pIK.
  • the three fragments were mixed in equimolar ratio and then spliced into PCR.
  • the fragments were recovered and digested with restriction endonuclease EcoRV/NotI, and ligated with T4 DNA ligase in EcoRV/NotI double-digested vector plasmid pIK and transformed into host bacteria.
  • TOP10 the clones were picked to identify positive clones by PCR and confirmed by sequencing.
  • the pIK-P3F2 eukaryotic expression plasmid was also obtained in the same manner.
  • the expression plasmids pIH-P1A6E and pIK-P1A6E were mixed in an equimolar amount, pIH-P3F2 and pIK-P3F2 were mixed in equal molars, and transfected into well-preserved HEK-293F cells, respectively, and cultured continuously at 37 ° C, 5% CO 2 , 125 rpm shaker 7 After centrifugation at 4000 rpm for 10 min, the precipitate was removed, and the supernatant was collected and filtered through a 0.45 ⁇ m filter. The treated sample was affinity-purified with a protein A (purchased from GE) affinity column to finally obtain a purified recombinant monoclonal antibody P1A6E. And P3F2, the identification results are shown in Figure 4.
  • the affinity and kinetic parameters of P1A6E and P3F2 single-chain antibodies and monoclonal antibodies were separately measured by capture using the Biacore T200 system (purchased from GE).
  • An anti-human IgG (Fc) antibody (purchased from GE) was coupled to the carboxymethyl dextran surface of sensor chip CM5 by primary amino group coupling with NHS/EDC according to the manufacturer's instructions. The measurement was carried out at 25 ° C, 30 ⁇ l / min, 1 ⁇ HBS-EP + working buffer, and the regeneration conditions were 3 M MgCl 2 and 10 ⁇ l/min for 30 seconds.
  • the antibody to be tested is first captured onto the chip, and a certain concentration of the analyte (human mesothelin) flows through the surface of the chip.
  • the interaction between human mesothelin and the captured antibody can be caused by the SPR signal.
  • the detection signal is defined as the resonance unit (RU), and the time (in seconds) is plotted to obtain the corresponding binding curve and dissociation curve.
  • the concentrations of human mesothelin were 10 nM, 20 nM, 40 nM, 80 nM and 160 nM, respectively, in different test cycles.
  • the resulting action curves were evaluated using Biacore T200evaluation software and the affinity KD values were calculated.
  • Figures 5 and 6 show the kinetic curves of monoclonal antibodies P1A6E and P3F2, respectively, in a Biacore affinity assay.
  • the binding data of the single-chain antibody and monoclonal antibody of P1A6E and P3F2 to human mesothelin, respectively, are summarized in Table 1.
  • the cells were digested with 10 mM EDTA, and the cells were collected by centrifugation at 200 g ⁇ 5 min.
  • the cells were resuspended in 1% of calf serum-containing phosphate buffer (NBS PBS) at a concentration of 1 ⁇ 10 6 to 1 ⁇ 10 7 /mL, and added to a flow-type dedicated tube in an amount of 100 ul / tube.
  • NBS PBS calf serum-containing phosphate buffer
  • the chimeric antigen receptor was constructed, and the joining sequence of each part of the chimeric antigen receptor exemplified in the present invention is shown in Table 2.
  • CD28a represents the transmembrane region of the CD28 molecule and CD28b represents the intracellular signal region of the CD28 molecule.
  • the lentiviral plasmid vector system used in this example belongs to the third generation lentivirus four-plasmid system, which has four plasmids, the envelope plasmid pCMV-VSV-G (purchased from addgene) encoding VSV-G protein, encoding Rev.
  • the protein packaging plasmid pRSV-Rev (purchased from addgene), pMDLg/pRRE (purchased from addgene) encoding Gal and Pol, and the coding gene of interest CAR based on the empty vector pRRLSIN.cPPT.PGK-GFP.WPRE (purchased from addgene) Recombinant expression vector.
  • All CAR gene vector promoters utilize the vector elongation factor-1 ⁇ (elongation factor-1 ⁇ , EF-1 ⁇ ) in the published patent 201310164725.X.
  • the specific method of construction is as follows:
  • the promoter fragment and the target CAR fragment obtained above are respectively associated with the consensus sequence fragment CD8-CD3 ⁇ zeta ( ⁇ Z), CD8-CD3zeta (Z), CD8-CD137-CD3zeta (BBZ), CD28a-CD28b-CD3zeta (28Z)
  • the primers pwpxlF and pwpxlR were used to amplify the fragments containing EF-1 ⁇ and the target gene CAT, respectively:
  • P1A6E-Z (SEQ ID NO: 32);
  • P1A6E-BBZ (SEQ ID NO: 33);
  • P1A6E-28Z (SEQ ID NO: 34);
  • P1A6E-28BBZ (SEQ ID NO: 35).
  • P3F2-Z (SEQ ID NO: 37);
  • P3F2-BBZ (SEQ ID NO: 38);
  • P3F2-28BBZ (SEQ ID NO: 40).
  • P1A6E- ⁇ Z (SEQ ID NO: 41);
  • P1A6E-Z (SEQ ID NO: 42);
  • P1A6E-BBZ (SEQ ID NO: 43);
  • P1A6E-28Z (SEQ ID NO: 44);
  • P1A6E-28BBZ (SEQ ID NO: 45).
  • P3F2-Z (SEQ ID NO: 47);
  • P3F2-BBZ (SEQ ID NO: 48);
  • P3F2-28Z (SEQ ID NO: 49);
  • P3F2-28BBZ (SEQ ID NO: 50).
  • HEK-293T cells (ATCC: CRL-11268) cultured to the 6th to 10th passages were inoculated at a density of 6 ⁇ 10 6 in a 10 cm culture dish, and cultured overnight at 37 ° C, 5% CO 2 for transfection.
  • the medium was DMEM containing 10% fetal bovine serum.
  • liquid A 10 ⁇ g of the target gene plasmid pRRLSIN-cPPT.EF-1 ⁇ -CAR (selected from pRRLSIN-EF1 ⁇ -P1A6E- ⁇ Z, pRRLSIN-EF1 ⁇ -P1A6E-Z, pRRLSIN-EF1 ⁇ -P1A6E-BBZ, pRRLSIN-EF1 ⁇ - P1A6E-28Z, pRRLSIN-EF1 ⁇ -P1A6E-28BBZ; pRRLSIN-EF1 ⁇ -P3F2-Z, pRRLSIN-EF1 ⁇ -P3F2-BBZ, pRRLSIN-EF1 ⁇ -P3F2-28Z, pRRLSIN-EF1 ⁇ -P3F2-28BBZ), respectively, with 7.5 ⁇ g package Plasmid pMDLg RRE and pRSV-REV, and 3 ⁇ g envelope plasmid pCMV-VSV-G, soluble Into 800
  • PEI polyethyleneimine, purchased from Polysciences
  • Formation of transfection complex Mix A solution into B solution, mix gently, mix immediately after vortexing or gently mix, incubate for 20 min at room temperature.
  • transfection complex 1.6 ml was added dropwise to HEK-293T cells, and after 4-5 hours, the transfected 293T cells were exchanged with 2% FBS in DMEM.
  • the virus was collected by filtration using a 0.45 ⁇ m filter, and then centrifuged at 28000 rpm in a Beckman Optima L-100XP ultracentrifuge for 2 hours at 4 ° C, the centrifuge supernatant was discarded, and the resulting precipitate was centrifuged for 1/10 to 1/50 of the original solution volume.
  • the AIM-V medium purchased from Invitrogen
  • Human peripheral blood mononuclear cells (provided by Shanghai Blood Center) were obtained from peripheral blood of healthy people by density gradient centrifugation. Peripheral blood mononuclear cells were obtained by CTL cell magnetic beads (purchased from Stem Cell Technologies) negative sorting method to obtain CTL. The sorted CTL cells were subjected to flow cytometry to detect the purity of CTL cells, and the positive rate of CTL cells was ⁇ 95%.
  • the Quantum 007 lymphocyte culture medium (purchased from PAA) was added at a density of about 1 ⁇ 10 6 /mL, and magnetic beads coated with anti-CD3 and CD28 antibodies were added at a cell:magnetic bead ratio of 1:1 (Invitrogen)
  • the company and the recombinant human IL-2 (purchased from Shanghai Huaxin Biotech Co., Ltd.) with a final concentration of 300 U/mL were stimulated for 24 h.
  • CTL cells were then infected with the above recombinant lentivirus at MOI ⁇ 5. The infected cells were passaged every other day at a density of 5 ⁇ 10 5 /mL, and a recombinant human IL-2 having a final concentration of 300 U/mL was supplemented in the lymphocyte culture solution.
  • Infected CTL cells were tested for expression of each of the different chimeric antigen receptors by flow cytometry on day 8 of culture.
  • the infected CAR T cells were incubated with biotinylated human mesothelin recombinant protein for 1 h at 37 ° C, washed twice with D-PBS, and then incubated with PE-labeled streptavidin for 40 min at 37 ° C.
  • the D-PBS was washed 3 times and the ratio of positive cells was measured by flow cytometry.
  • the positive rate of virus-infected CTL cells expressing different chimeric antigen receptors using uninfected T lymphocytes as a negative control is shown in Table 3. The positive rate results indicate that a certain positive rate of CAR + CTL cells can be obtained by a method of lentiviral infection.
  • CTL cells were subcultured, counted, and supplemented with IL-2 at a cell density of 5 ⁇ 10 5 /ml, respectively, after infection with a virus packed with different chimeric antigen receptors.
  • the final concentration was 300U/ml), about 20-40 times amplification on the 11th day of culture, indicating that CTL cells expressing different chimeric antigen receptors can be expanded in vitro, providing a follow-up in vitro toxicity test and in vivo test. Guarantee.
  • the mesothelin-negative pancreatic cancer cell line (PANC-1) shown in Table 4 and the PANC-1 (PANC-1-MSLN) cell line transfected with the mesothelin gene were used as target cells, and the effector cells were as in the examples.
  • 4 validated FACS detection of chimeric antigen receptor-positive positive cells in vitro for 12 days was recorded as chimeric antigen receptor-positive (CAR + ) CTL, and the target ratio was 3:1, 1:1 and 3, respectively. 1:3, the number of target cells is 10,000 cells/well, corresponding to effector cells according to different target-to-target ratios. Five replicate wells were set for each group, and the average of five replicate wells was taken. The detection time is 18h.
  • Each experimental group each target cell + CTL expressing different chimeric antigen receptors
  • Control group 1 The maximum release of LDH from target cells
  • Control group 2 The target cells spontaneously release LDH
  • Control group 3 Effector cells spontaneously released LDH.
  • CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used. This method is based on the colorimetric detection method and can replace the 51 Cr release method. CytoTox The assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme that is released when cells are lysed and released in much the same way as 51 Cr is released in radioactive analysis. The released LDH medium supernatant can be detected by a 30 minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is directly proportional to the number of cells lysed. Refer specifically to the instructions for the CytoTox 96 non-radioactive cytotoxicity test kit.
  • INT tetrazolium salt
  • the cytotoxicity calculation formula is:
  • the CAR of the anti-mesothelin single-chain antibody (P1A6E, P3F2) of the present invention has an activity of killing mesothelin-positive pancreatic cancer cells, wherein the second and third generations are resistant.
  • the CAR T cells of mesothelin are slightly more potent than the first generation.
  • the mock group did not have obvious killing.
  • all CAR T cells were not cytotoxic to mesothelin-negative PANC-1 pancreatic cancer cells.
  • the first, second, and third generations of CAR T of the anti-mesothelin of the present invention exhibit a potent target-to-gradient-dependent target ratio of the mesothelin-positive tumor cells, and the higher the cytotoxic effect.
  • the human mesothelin gene fragment was amplified from SEQ ID NO: 1 by PCR, and the human mesothelin gene fragment was ligated into the eukaryotic expression vector pCMV-V5-muFc containing the murine Fc fragment by double digestion with NheI/BamHI.
  • the HEK-293F cells were transiently transfected according to Example 4, and the cell culture supernatant was processed and affinity-purified by a protein G (purchased from GE) affinity column to finally obtain a purified human mesothelin fragment-muFc fusion protein, which was then passed.
  • the binding of the antibody scFv-P1A6E-Fc and scFv-P3F2-Fc was identified by ELISA.
  • the mature human mesothelin is divided into three regions according to Genbank accession number NP_001170826.1 (SEQ ID NO: 65), region R1 (E296-T390, SEQ ID NO: 66), region R2 (S391-Q486, SEQ ID NO) :67), region R3 (N487-G581, SEQ ID NO: 68), ELISA results showed that both antibody scFv-P1A6E-Fc and scFv-P3F2-Fc bind only to region 1 (E296-T390).
  • Region 1 was further divided into 5 small fragments, which were respectively expressed in fusion with muFc.
  • Region R1A (296E-337D, SEQ ID NO: 69)
  • region R1B (328D-369I, SEQ ID NO: 70)
  • region R1C (360Y-405T, SEQ ID NO: 71)
  • region R1AB (296E-359L, SEQ) ID NO: 72
  • R1BC (328D-405T, SEQ ID NO: 73).
  • the ELISA results are shown in Figures 9 and 10, in which the antibody scFv-P1A6E-Fc and scFv-P3F2-Fc have significant binding to the region R1AB, and have a weak binding to the region R1A, and the region R1B does not bind. Therefore, the binding site of the antibody scFv-P1A6E-Fc and scFv-P3F2-Fc should be located near the position where R1A and R1B overlap.
  • This region contains 10 amino acids "DAALLATQMD", based on which the sequence is extended by 10 amino acids, or 5 amino acids, to form two peptides R1J10: "YKKWELEACVDAALLATQMDRVNAIPFTYE (SEQ ID NO: 74)" and R1J5: "LEACVDAALLATQMDRVNAI (SEQ ID NO: 75)” was purified and expressed in fusion with muFc, respectively.
  • the ELISA results showed that the antibodies scFv-P1A6E-Fc and scFv-P3F2-Fc did not bind to R1J10 and R1J5.
  • the epitopes of the antibodies scFv-P1A6E-Fc and scFv-P3F2-Fc should be the conformational epitopes located in the region R1AB (SEQ ID NO: 72).

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Abstract

本发明提供抗间皮素全人抗体以及靶向间皮素的免疫效应细胞。

Description

抗间皮素全人抗体以及靶向间皮素的免疫效应细胞 技术领域
本发明属于肿瘤免疫治疗或诊断领域，更具体地，本发明涉及抗间皮素全人抗体以及靶向间皮素的免疫效应细胞。
背景技术
免疫效应细胞在肿瘤免疫应答中的作用日益受到重视。基于免疫效应细胞的过继性免疫治疗在部分肿瘤中取得了一定的效果，并且该种免疫治疗方法可以克服抗体治疗的上述缺陷，但在大多数肿瘤的疗效仍不能令人满意[Grupp SA，et al.Adoptive cellular therapy.Curr Top Microbiol Immunol.，2011；344:149-72.]。近年来，根据细胞毒性T淋巴细胞(cytotoxic lymphocyte，CTL)对靶细胞的识别特异性依赖于T淋巴细胞受体(T Cell Receptor，TCR)的发现，将针对肿瘤细胞相关抗原的抗体的scFv与T淋巴细胞受体的CD3ζ或FcεRIγ等胞内信号激活基序融合成嵌合抗原受体(Chimeric antigen receptor，CAR)，并将其通过如慢病毒感染等方式基因修饰在T淋巴细胞表面。这种CAR T淋巴细胞能够以主要组织兼容性复合物(Major Histocompatibility Complex，MHC)非限制性方式选择性地将T淋巴细胞定向到肿瘤细胞并特异性地杀伤肿瘤。CAR T淋巴细胞是肿瘤免疫治疗领域的一个新的免疫治疗策略。CAR修饰的NK细胞或者NKT细胞也在临床前研究中展示出抗肿瘤活性。
设计CAR修饰的免疫效应细胞，特别T细胞时，所针对的抗原基因实际上是一种关键性的选择，鉴于体内基因表达的复杂性以及各种不可控因素，选择到一个合适的用于CAR的基因是非常困难的。并且，很多肿瘤特异性的抗原，很难找到针对其的且适合于构建CAR修饰的免疫效应细胞的特异性分子。
间皮素(mesothelin)是一个分子量为40-kDa的细胞表面糖蛋白。它在胰腺癌、卵巢癌以及胸腺间皮瘤等多种肿瘤中高表达。它在正常组织中，仅在胸膜、心包及腹膜的正常间皮细胞上表达。间皮素作为71kDa前体蛋白合成，其成熟部分在细胞表面上表达。该前体蛋白被弗林蛋白酶蛋白水解切割成31kDa脱落部分(称作巨核细胞嵌合因子，或MPF)和40kDa间皮素部分)。后一种组分可经GPI连接保持结合于细胞表面，也可经由蛋白水解酶机制脱落。
针对间皮素为靶点的抗体或者其它靶向治疗已经有报道。其中CAR-T也有临床研究的报道(Maus MV,Haas AR,Beatty GL,Albelda SM,Levine BL,Liu X,Zhao Y,Kalos M,June CH.T cells expressing chimeric antigen receptors can cause anaphylaxis in humans.Cancer Immunol Res.2013；1(1):26-31；Beatty GL,Haas AR,Maus MV,Torigian DA,Soulen MC,Plesa G,Chew A,Zhao Y,Levine BL,Albelda SM,Kalos M,June CH.Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies.Cancer Immunol Res.2014Feb；2(2):112-20)。但是，也发现用小鼠的抗人间皮素抗体所构建的CAR-T在临床中会出现抗鼠抗体及过敏等毒副作用。说明间皮素可能是一个潜在的治疗靶点，但是抗体本身的属性可能会影响其疗效和毒副作用。因此，本领域仍然需要找到能够克服由抗体所导致的疗效不理想或产生毒副作用的问题的方案。
发明内容
本发明的目的在于提供抗间皮素全人抗体以及靶向间皮素的免疫效应细胞。
在本发明的第一方面，提供可特异结合间皮素的全人抗体，该全人抗体选自：
(a)抗体，其包含重链可变区，所述重链可变区具有包含SEQ ID NO:54所示的氨基酸序列的CDR1、包含SEQ ID NO:55所示的氨基酸序列的CDR2、包含SEQ ID NO:56所示的氨基酸序列的CDR3；
(b)抗体，其包含轻链可变区，所述轻链可变区具有包含SEQ ID NO:51所示的氨基酸序列的CDR1、包含SEQ ID NO:52所示的氨基酸序列的CDR2、包含SEQ ID NO:53示氨基酸序列的CDR3；
(c)抗体，包含(a)所述抗体的重链可变区及(b)所述抗体的轻链可变区；
(d)抗体，包含重链可变区，所述重链可变区具有包含SEQ ID NO:60所示的氨基酸序列的CDR1、包含SEQ ID NO:61所示的氨基酸序列的CDR2、包含SEQ ID NO:62所示的氨基酸序列的CDR3；
(e)抗体，包含轻链可变区，所述轻链可变区具有包含SEQ ID NO:57所示的氨基酸序列的CDR1、包含SEQ ID NO:58所示的氨基酸序列的CDR2、包含SEQ ID NO:59所示的氨基酸序列的CDR3；
(f)包含(d)所述抗体的重链可变区及(e)所述抗体的轻链可变区；
(g)抗体，识别与(a)～(f)中任一项所述的抗体所识别的抗原决定部位相同的抗原决定部位。
在一个优选例中，该全人抗体包括重链可变区和轻链可变区，其重链可变区的氨基酸序列如SEQ ID NO:6中第1-123位所示；其轻链可变区的氨基酸序列如SEQ ID NO:6中第139-254位所示；或
该全人抗体包括重链可变区和轻链可变区，其重链可变区的氨基酸序列如SEQ ID NO:8中第1-124位所示；其轻链可变区的氨基酸序列如SEQ ID NO:8中第140-247位所示。
在另一优选例中，所述的特异结合间皮素的全人抗体可以是：单链抗体(scFV)，单克隆抗体，结构域抗体，Fab片段，Fd片段，Fv片段，F(ab’)2片段和其衍生物，或其它形式的抗体；较佳地为单链抗体。
在本发明的另一方面，提供编码所述的抗体的核酸。
在本发明的另一方面，提供一种表达载体，其包含所述的核酸。
在本发明的另一方面，提供一种宿主细胞，其包含所述的表达载体或基因组中整合有所述的核酸。
在本发明的另一方面，提供所述的抗体的用途，用于制备特异性靶向表达间皮素的肿瘤细胞的靶向性药物，抗体药物偶联物或多功能抗体；或用于制备诊断肿瘤的试剂，该肿瘤表达间皮素；或用于制备嵌合抗原受体修饰的免疫细胞。
在本发明的另一方面，提供所述的抗体的嵌合抗原受体(Chimeric antigen receptor， CAR)，所述的嵌合抗原受体包含顺序连接的：本发明所述的抗体，跨膜区和胞内信号区。
在一个优选例中，所述的胞内信号区选自：CD3ζ，FcεRIγ，CD27，CD28，CD137，CD134，MyD88，CD40的胞内信号区序列，或其组合。
在一个优选例中，所述的跨膜区包含CD8或CD28的跨膜区。
在另一优选例中，所述的嵌合抗原受体包括如下的顺序连接的抗体，跨膜区和胞内信号区：
所述的抗体、CD8和CD3ζ；
所述的抗体、CD8、CD137和CD3ζ；
所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
在另一优选例中，所述的抗体是单链抗体或结构域抗体。
在另一优选例中，所述的嵌合抗原受体具有：
SEQ ID NO:41或其中第22-353位所示的氨基酸序列；
SEQ ID NO:42或其中第22-454位所示的氨基酸序列；
SEQ ID NO:43或其中第22-498位所示的氨基酸序列；
SEQ ID NO:44或其中第22-501位所示的氨基酸序列；
SEQ ID NO:45或其中第22-543位所示的氨基酸序列；
SEQ ID NO:46或其中第22-346位所示的氨基酸序列；
SEQ ID NO:47或其中第22-447位所示的氨基酸序列；
SEQ ID NO:48或其中第22-491位所示的氨基酸序列；
SEQ ID NO:49或其中第22-494位所示的氨基酸序列；或
SEQ ID NO:50或其中第22-536位所示的氨基酸序列。
在本发明的另一方面，提供编码所述的嵌合抗原受体的核酸。
在另一优选例中，编码所述的嵌合抗原受体的核酸具有：
SEQ ID NO:31或其中第473-1468位所述的核苷酸序列；
SEQ ID NO:32或其中第473-1771位所述的核苷酸序列；
SEQ ID NO:33或其中第473-1903位所述的核苷酸序列；
SEQ ID NO:34或其中第473-1912位所述的核苷酸序列；
SEQ ID NO:35或其中第473-2038位所述的核苷酸序列；
SEQ ID NO:36或其中第473-1447位所述的核苷酸序列；
SEQ ID NO:37或其中第473-1750位所述的核苷酸序列；
SEQ ID NO:38或其中第473-1882位所述的核苷酸序列；
SEQ ID NO:39或其中第473-1891位所述的核苷酸序列；
SEQ ID NO:40或其中第473-2017位所述的核苷酸序列。
在本发明的另一方面，提供一种表达载体，其包含所述的核酸。
在另一优选例中，所述的表达载体来源于慢病毒质粒pWPT(或pWPT-eGFP)。
在本发明的另一方面，提供一种病毒，所述的病毒包含所述载体。
在本发明的另一方面，提供所述的嵌合抗原受体、或所述的核酸、或所述的表达载体、或所述的病毒的用途，用于制备靶向表达间皮素的肿瘤细胞的基因修饰的免疫细胞。
在一个优选例中，所述的表达间皮素的肿瘤包括(但不限于)：胰腺癌、卵巢癌，胸腺间皮瘤。
在本发明的另一方面，提供一种基因修饰的免疫细胞，其转导有所述的核酸，或所述的表达载体或所述的病毒；或其表面表达所述的嵌合抗原受体。
在一个优选例中，所述的免疫细胞还携带外源的细胞因子的编码序列；较佳地，所述的细胞因子包括：IL-12，IL-15或IL-21。
在另一优选例中，所述的免疫细胞还表达另一种嵌合抗原受体，该受体不含有CD3ζ，但含有CD28的胞内信号结构域、CD137的胞内信号结构域或者这两者的组合。
在另一优选例中，所述的免疫细胞还表达趋化因子受体；较佳地，所述的趋化因子受体包括：CCR2。
在另一优选例中，所述的免疫细胞还表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白。
在另一优选例中，所述的免疫细胞还表达安全开关；较佳地，所述的安全开关包括：iCaspase-9，Truancated EGFR或RQR8。
在另一优选例中，所述的免疫细胞包括：T淋巴细胞、NK细胞或者NKT淋巴细胞。
在本发明的另一方面，提供所述的基因修饰的免疫细胞的用途，用于制备抑制肿瘤的药物，所述的肿瘤是表达间皮素的肿瘤。
在本发明的另一方面，提供一种多功能免疫辍合物，所述的多功能免疫辍合物包括：所述的抗体；以及与之连接(包括共价连接、偶联、附着、吸附)的功能性分子；所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。
在一个优选例中，所述的多功能免疫辍合物中，所述的靶向肿瘤表面标志物的分子是结合肿瘤表面标志物的抗体或配体；或所述的抑制肿瘤的分子是抗肿瘤的细胞因子或抗肿瘤的毒素；较佳地，所述的细胞因子包括(但不限于)：IL-12、IL-15、IFN-beta、TNF-alpha。
在另一优选例中，所述的多功能免疫辍合物中，所述的可检测标记物包括：荧光标记物、显色标记物。
在另一优选例中，所述的多功能免疫辍合物中，所述的结合肿瘤表面标志物的抗体是指：识别间皮素以外的其它抗原的抗体，所述的其它抗原包括：EGFR，EGFRvIII，mesothelin，HER2，EphA2，Her3，EpCAM，MUC1，MUC16，CEA，Claudin 18.2，叶酸受体，Claudin6，CD3，WT1，NY-ESO-1，MAGE 3，ASGPR1或CDH16。
在另一优选例中，所述的多功能免疫辍合物中，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体，其与所述的抗体形成T细胞参与的双功能抗体(Bispecific T cell engager，BiTE)。
在另一优选例中，所述的多功能免疫辍合物中，所述的结合T细胞表面标志物的抗体是抗CD3抗体。
在另一优选例中，所述的抗CD3抗体是单链抗体(scFV)，单克隆抗体，Fab片段，Fd片段，Fv片段，F(ab’)2片段和其衍生物，或其它形式的抗体；较佳地为单链抗体。
在另一优选例中，所述的抗CD3抗体是人源化的，嵌合的，全人源的或鼠源的。
在另一优选例中，所述的多功能免疫辍合物是融合多肽，本发明所述的抗体以及与之连接的功能性分子之间，还包括连接肽(接头)。
在另一优选例中，所述的连接肽的序列为(GlyGlyGlyGlySer)n，其中n为1到5的整数；更佳地，n＝3。
在另一优选例中，所述的多功能免疫辍合物采用多肽给药或基因给药的方式。
在本发明的另一方面，提供编码所述的多功能免疫辍合物的核酸。
在本发明的另一方面，提供所述的多功能免疫辍合物的用途，用于制备抗肿瘤药物，或用于制备诊断肿瘤的试剂，该肿瘤表达间皮素；或用于制备嵌合抗原受体修饰的免疫细胞；较佳地，所述免疫细胞包括：T淋巴细胞、NK细胞或者NKT淋巴细胞。
在本发明的另一方面，提供药物组合物(包括药物或诊断试剂)，其包括：
所述的抗体或编码该抗体的核酸；或
所述的免疫辍合物或编码该辍合物的核酸；或
所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或
所述的基因修饰的免疫细胞。
在本发明的另一方面，提供了能够和本发明所述的抗体竞争结合间皮素的抗体。
在本发明的另一方面，提供了能够结合如SEQ ID NO:66所示的间皮素表位的抗体。在一优选例中，还提供了能结合SEQ ID NO：72所示的间皮素表位的抗体。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、在单噬菌体ELISA实验中抗体P1A6E和P3F2对人间皮素(hu-mesothelin)和对照BSA的结合情况。抗体P1A6E和P3F2对人间皮素和阴性对照BSA的值证明筛选到的两个抗体能特异性结合人间皮素。
图2、ELISA实验检测两个不同的单链抗体P1A6E和P3F2对人间皮素及BSA的结合情况。
图3、抗人间皮素的抗体的纯化SDS-PAGE电泳图。
图4、单克隆抗体P1A6E和P3F2的SDS-PAGE电泳图。
图5、在Biacore中单克隆抗体P1A6E对不同浓度的人间皮素的结合曲线。
图6、在Biacore中单克隆抗体P3F2对不同浓度的人间皮素的结合曲线。
图7、通过荧光激活细胞分选仪(FACS)显示的四种单链抗体(P1A6E，P3F2和对照抗体SS，C10)与PANC-1-MSLN细胞特异性结合的测定。
图8、通过荧光激活细胞分选仪(FACS)显示的四种单克隆抗体(P1A6E，P3F2和对照抗体SS，C10)与PANC-1-MSLN细胞特异性结合的测定。
图9、ELISA显示抗体scFv-P1A6E-Fc和scFv-P3F2-Fc和区域R1，R2，R3的结合情况。
图10、ELISA显示抗体scFv-P1A6E-Fc和scFv-P3F2-Fc和区域R1A，R1B，R1C，R1AB，R1BC的结合情况。
具体实施方式
本发明人前期考察了许多种肿瘤特异性基因，发现这类基因中相当大一部分也表达于部分组织的正常细胞中，较难应用于嵌合抗原受体修饰的免疫效应细胞技术；有的肿瘤特异性基因具有较好的肿瘤特异性表达特性，但是，基于其设计的CAR修饰的免疫效应细胞没有肿瘤细胞杀伤活性或者活性很低，这可能是因为该靶点可以引发肿瘤细胞分泌对免疫效应细胞起抑制作用的因子如PD-L1。
经过反复考察和筛选，本发明人从诸多的候选分子中找到了间皮素作为设计CAR的靶点。本发明人研究证明，基于间皮素抗体制备的CAR修饰的T细胞确实可以选择性地靶向间皮素阳性的肿瘤细胞，并且对于肿瘤细胞具有很强的细胞毒性。本发明人认为，相应的CAR修饰的免疫效应细胞，特别是T细胞应该可以用于人体肿瘤的治疗。
抗间皮素抗体
本发明人从全人源天然抗体库中筛选获得对于间皮素结合性能良好且适用于制备基因修饰的免疫效应细胞的特异性抗体，并且找到了其发挥结合性能的关键CDR区。
本发明的抗体可以是完整的免疫球蛋白分子，也可以是抗原结合片段，包括但不限于Fab片段，Fd片段，Fv片段，F(ab’)2片段、互补决定区(CDR)片段、单链抗体(scFv)、结构域抗体，二价单链抗体、单链噬菌体抗体、双特异双链抗体、三链抗体、四链抗体。
抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为互补决定区(complementarity determining region，CDR)，所述的CDR区将可变区间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。CDR区是免疫学感兴趣的蛋白质的序列，本发明的抗体的CDR区是全新的。所述抗体可包含本文揭示的二、三、四、五或者所有六个CDR区。
本发明的另一方面包括本文所述抗体的功能变体，如果变体能与亲代抗体竞争特异性结合间皮素，且其识别肿瘤细胞表达的间皮素的能力接近于本发明实施例中提供的具体的抗体。所述功能变体可以具有保守序列修饰，包括核苷酸和氨基酸取代、添加和缺失。这些修饰可以通过本领域己知的标准技术导入，例如定向诱变和随机PCR介导的诱变，并且可包含天然以及非天然核苷酸和氨基酸。较佳地，序列的修饰发生在所述抗体的CDR区以外的区域 上。
本发明的抗体可以被应用于制备各种靶向性抗肿瘤药物以及诊断肿瘤的药物，特别是应用于制备靶向间皮素的免疫效应细胞。
嵌合抗原受体及基因修饰的免疫细胞
本发明提供了一种表达于免疫效应细胞(免疫细胞)表面的嵌合抗原受体，所述的嵌合抗原受体包含顺序连接的：胞外结合区，跨膜区和胞内信号区，其中所述胞外结合区包含本发明的抗体。将该嵌合抗原受体表达于免疫效应细胞的表面，可使得免疫效应细胞对表间皮素的肿瘤细胞具有高度特异性的细胞毒性作用。
如本文所用，所述的“免疫细胞”与“免疫效应细胞”可互换使用，其包括：T淋巴细胞，NK细胞或NKT细胞等。
作为本发明的优选方式，所述的嵌合抗原受体中，包含的抗体为单链抗体，其通过CD8铰链区与CD8或者CD28的跨膜区相连接，跨膜区后紧接胞内信号区。
本发明也包括编码所述嵌合抗原受体的核酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。
嵌合抗原受体的跨膜区可以选自CD8或CD28等蛋白的跨膜区。人CD8蛋白是个异二聚体，由αβ或者γδ两条链组成。在本发明的一个实施方案中，跨膜区选自CD8α或者CD28的跨膜区。此外，CD8α铰链区(hinge)是一个柔性区域，因此，CD8或CD28和跨膜区加上铰链区被用于将嵌合抗原受体CAR的靶点识别结构域scFv和胞内信号区连接起来。
胞内信号区可以选自CD3ζ，FcεRIγ，CD27，CD28，CD137，CD134，MyD88，CD4蛋白的胞内信号区，及其组合。CD3分子由五个亚单位组成，其中CD3ζ亚单位(又称CD3zeta，简称Z)含有3个ITAM基序，该基序是TCR-CD3复合体中重要的信号转导区。CD3δZ是截短的不具有ITAM基序的CD3ζ序列，在实践中一般作为阴性对照的构建。FcεRIγ主要分布在肥大细胞和嗜碱性粒细胞表面，其含有一个ITAM基序，在结构、分布及功能上与CD3ζ类似。此外如前所述，CD28，CD137，CD134是共刺激信号分子，在与各自配体结合后其胞内信号区段产生的共刺激作用引起免疫效应细胞(主要是T淋巴细胞)的持续增殖，并能够提高免疫效应细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR免疫效应细胞在体内的存活周期和抗肿瘤效果。
本发明的嵌合抗原受体可以按如下方式顺序连接：
本发明的抗体、CD8和CD3ζ；
本发明的抗体、CD8、CD137和CD3ζ；
本发明的抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
本发明的抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
及其组合，其中相关嵌合抗原受体蛋白中CD28a代表CD28分子的跨膜区，CD28b代表CD28分子的胞内信号区。上述各种嵌合抗原受体统称为scFv(间皮素)-CAR。
本发明还提供了包含上述编码表达于免疫效应细胞表面的嵌合抗原受体蛋白的核酸的 载体。在一个具体实施方案中，本发明使用的载体是一种慢病毒质粒载体pWPT-eGFP。该质粒属于第三代自灭活慢病毒载体系统，该系统共有三个质粒即编码蛋白Gag/Pol、编码Rev蛋白的包装质粒psPAX2；编码VSV-G蛋白的包膜质粒PMD2.G；及空载体pWPT-eGFP，其可以用于重组引入目的核酸序列，即编码CAR的核酸序列。空载体pWPT-eGFP中由延长因子-1α(elongation factor-1α，EF-1α)启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的表达。而包含编码CAR的目的核酸序列的重组表达载体pWPT-eGFP-F2A-CAR是通过由来自口蹄疫病毒(food-and-mouth disease virus，FMDV)的核糖体跳跃序列(ribosomal skipping sequence 2A)(简称F2A)实现eGFP与CAR的共表达的。应理解，其它表达载体也是可用的。
本发明还包括包含上述载体的病毒。本发明的病毒包括包装后的具有感染力的病毒，也包括包含包装为具有感染力的病毒所必需成分的待包装的病毒。本领域内已知的其它可用于将外源基因转导入免疫效应细胞的病毒及其对应的质粒载体也可用于本发明。
本发明还提供了基因修饰的免疫效应细胞，其被转导有本发明的核酸或被转导有本发明的上述包含所述含有该核酸的重组质粒，或包含该质粒的病毒。本领域常规的核酸转导方法，包括非病毒和病毒的转导方法都可以用于本发明。基于非病毒的转导方法包括电穿孔法和转座子法。近期Amaxa公司研发的Nucleofector核转染仪能够直接将外源基因导入细胞核获得目的基因的高效转导。另外，基于睡美人转座子(Sleeping Beauty system)或PiggyBac转座子等转座子系统的转导效率较普通电穿孔有较大提高，将nucleofector转染仪与睡美人转座子系统联合应用已有报道[Davies JK.，et al.Combining CD19redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res，2010，70(10):OF1-10.]，该方法既具有较高的转导效率又能够实现目的基因的定点整合。在本发明的一个实施方案中，实现嵌合抗原受体基因修饰的免疫效应细胞的转导方法是基于病毒如逆转录病毒或慢病毒的转导方法。该方法具有转导效率高，外源基因能够稳定表达，且可以缩短体外培养免疫效应细胞到达临床级数量的时间等优点。在该转基因免疫效应细胞表面，转导的核酸通过转录、翻译表达在其表面。通过对各种不同的培养的肿瘤细胞进行体外细胞毒实验证明，本发明的免疫效应细胞具有高度特异性的肿瘤细胞杀伤效果(亦称细胞毒性)。因此本发明的编码嵌合抗原受体蛋白的核酸，包含该核酸的质粒，包含该质粒的病毒和转导有上述核酸，质粒或病毒的转基因免疫效应细胞可以有效地用于肿瘤的免疫治疗。
本发明所述的免疫细胞还可以携带外源的细胞因子的编码序列；所述的细胞因子包括但不限于：IL-12，IL-15或IL-21等。这些细胞因子具有免疫调节或抗肿瘤的活性，能增强效应T细胞及活化的NK细胞的功能，或直接发挥抗肿瘤作用。因此，本领域技术人员可以理解，这些细胞因子的运用有助于所述的免疫细胞更好地发挥作用。
本发明所述的免疫细胞还可以表达除了上述嵌合抗原受体以外的另一种嵌合抗原受体，该受体不含有CD3ζ，但含有CD28的胞内信号结构域、CD137的胞内信号结构域或者这两者的组合。
本发明所述的免疫细胞还可以表达趋化因子受体；所述的趋化因子受体包括但不限于CCR2。本领域技术人员可以理解，所述的CCR2趋化因子受体可以使得体内的CCR2与之竞争性结合，对于阻断肿瘤的转移是有利的。
本发明所述的免疫细胞还可以表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白。本领域技术人员可以理解，竞争性阻断PD-L1与其受体PD-1的相互作用，有利于恢复抗肿瘤T细胞反应，从而抑制肿瘤生长。
本发明所述的免疫细胞还可以表达安全开关；较佳地，所述的安全开关包括：iCaspase-9，Truancated EGFR或RQR8。
免疫辍合物
本发明还提供了多功能免疫缀合物，其包含本文所述抗体以及进一步包含至少一种其它类型的功能性分子。所述的功能性分子选自但不限于：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。所述抗体与所述功能性分子可以通过共价连接、偶联、附着、交联等方式构成辍合物。
作为一种优选方式，所述免疫缀合物可包含：本发明的抗体以及至少一种靶向肿瘤表面标志物的分子或抑制肿瘤的分子。所述的抑制肿瘤的分子可以是抗肿瘤的细胞因子，或抗肿瘤的毒素；较佳地，所述的细胞因子包括(但不限于)：IL-12、IL-15、IFN-beta、TNF-alpha。所述的靶向肿瘤表面标志物的分子例如可以与本发明的抗体协同作用，更精准地靶向肿瘤细胞。
作为一种优选方式，所述免疫缀合物可包含：本发明的抗体以及可检测标记物。所述的可检测标记物包括但不限于：荧光标记物、显色标记物；如：酶、辅基、荧光材料、发光材料，生物发光材料、放射性材料、正电子发射金属以及非放射性顺磁性金属离子。也可包含一个以上的标记物。为了检测和/或分析和/或诊断目的用于标记抗体的标记依赖于使用的特定检测/分析/诊断技术和/或方法例如免疫组织化学染色(组织)样品、流式细胞计量术等。对于本领域已知的检测/分析/诊断技术和/或方法合适的标记为本领域技术人员所熟知。
作为一种优选方式，所述免疫缀合物可包含：本发明的抗体以及靶向免疫细胞的表面标志物的分子。所述靶向免疫细胞的表面标志物的分子可识别免疫细胞，其携带本发明的抗体达到免疫细胞，同时本发明的抗体可将免疫细胞靶向于肿瘤细胞，从而引发免疫细胞特异性地杀伤肿瘤。
作为通过直接或间接(例如通过接头)缀合而化学产生免疫缀合物的一种方式，所述免疫缀合物可以作为融合蛋白而产生，所述融合蛋白包含本发明的抗体及合适的其它蛋白。融合蛋白可以通过本领域已知方法产生，例如通过构建核酸分子以及随后表达所述核酸分子而重组产生，所述核酸分子包含符合读框的编码抗体的核苷酸序列以及编码合适标记的核苷酸序列。
本发明另一方面提供了编码本发明的至少一种抗体、其功能变体或者免疫缀合物的核酸分子。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
药物组合物
本发明的抗体、包含该抗体的免疫辍合物以及基因修饰的免疫细胞可以应用于制备药物组合物或诊断试剂。所述的组合物除了包括有效量的所述抗体、免疫辍合物或免疫细胞，还可包含药学上可接受的载体。术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如
Figure PCTCN2016096292-appb-000001
润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用注射或其它治疗方式。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、间皮素稳定表达细胞系的构建
1.1 质粒载体的构建
本实施例使用的载体系统属于第三代自灭活慢病毒载体系统，该系统共有三个质粒即编码蛋白Gag/Pol、编码Rev蛋白的包装质粒psPAX2；编码VSV-G蛋白的包膜质粒PMD2.G及基于空载体pWPT(购自Addgene公司)的编码目的基因人间皮素胞外区域和跨膜区域的重组质粒pWPT-MSLN。
根据Genbank登录号NM_005823，使用基于PCR搭桥的基因合成方法，合成包含信号肽、Flag tag、人间皮素胞外区域和跨膜区域的目的基因片段(SEQ ID NO:1(核苷酸)，2(氨基酸))，通过引物对pWmslnF(SEQ ID NO:3，GCTTACGCGTCCTAGCGCTACCGGTCGCCACCATGAGGGCCTGGATC)和pWmslnR(SEQ ID NO:4，CGAGGTCGAC CTAGGCCAGGGTGGAGGCTAGGAGCAGTGCCAGGACGG)进行PCR扩增，扩增条件为预变性：94℃，4min； 变性：94℃，30s；退火：58℃，30s；延伸：68℃，80s；30个循环。获得的片段理论大小为1113bp，扩增产物经琼脂糖电泳确认与理论大小一致。其中在开放阅读框的上下游引入MluI和SalI酶切位点。上述获得的目的基因由MluI和SalI双酶切，连入同样双酶切的pWPT载体中，构建成功的慢病毒载体pWPT-MSLN，经MluI和SalI酶切鉴定及序列测定正确后进行慢病毒包装。
1.2 质粒转染293T细胞包装慢病毒
以6×106的密度接种培养至第6～10代的293T细胞(ATCC：CRL-11268)于10cm培养皿中，37℃，5％CO2培养过夜准备用于转染。培养基为含10％胎牛血清(Sigma公司)的DMEM(Invitrogen公司)，次日，在转染前约2小时更换培养液为无血清DMEM。
转染的步骤如下：
1)将5μg目的基因质粒pWPT-MSLN，分别与7.5μg包装质粒PAX2和2.5μg包膜质粒pMD2.G，溶入500μL MillQ水中，混匀，
2)逐滴加入62μL 2.5M CaCl2(Sigma公司)，以1200rpm/min vortex混匀，
3)最后逐滴加入500μL 2×HBS(280mM NaCl,10mM KCl，1.5mM Na2HPO4-,12mM葡萄糖，50mM Hepes(Sigma公司)，pH7.05,0.22μM过滤除菌)，1200rpm/min振荡混匀10s，
4)立即逐滴加入培养皿中，轻轻摇匀，37℃，5％CO2，培养4～6h后，更换为含10％胎牛血清的DMEM。
在转染48h或72h后，离心除去细胞碎片，然后用0.45μm滤器(Millipore公司)过滤收集病毒。
1.3 重组慢病毒感染PANC-1细胞
将上述收集的病毒液经浓缩滴定后，分别感染铺于6cm平皿内的细胞PANC-1(购自ATCC)。感染三天后收集细胞，取部分混合克隆，使用细胞裂解液裂解后，取40μg细胞蛋白进行SDS-PAGE凝胶电泳，随后对凝胶电泳进行免疫印迹，并用小鼠抗Flag-tag抗体染色。PBS洗涤后，使用辣根过氧化物酶标记的羊抗小鼠抗体孵育，洗涤后使用ECL试剂显色，最后进行显影。Western blot结果显示，在感染了人间皮素MSLN的PANC-1细胞(即PANC-1-MSLN)中可检测到分子量大小约38kDa的条带，而未感染的空细胞中未检测到相应的条带。其余的细胞扩大培养后冻存一部分，并用于下一步实验。
实施例2、人间皮素抗原的制备
根据Genbank登录号NM_005823，使用基于PCR搭桥的基因合成方法，合成人间皮素基因片段(SEQ ID NO:1第88-942位(核苷酸)，(SEQ ID NO:12第30-314位(氨基酸))，并进行PCR扩增。扩增产物通过NheI/BglII插入到质粒载体pCMV-V5(该载体在多克隆位点下游融合表达6×His标签，购自上海锐劲生物技术有限公司)，并转化于宿主菌TOP10中，挑取克隆通过PCR鉴定阳性克隆并通过测序确认，获得重组表达质粒V5-MSLN。
将上述表达质粒转染生长良好的HEK-293F细胞，37℃，5％CO2，125rpm摇床连续培养7天，4000rpm离心10min，去除沉淀，收集上清，并用0.45μm滤膜过滤，将处理好的 样品以HisTrap(购自GE)亲和层析柱进行纯化，最终获得纯化的人间皮素蛋白，鉴定结果如图1所示。
实施例3、人间皮素单链抗体的筛选
3.1 基于噬菌体展示的人间皮素特异性结合抗体的筛选
利用噬菌体展示技术，从全人源天然抗体库中筛选人间皮素特异性抗体。为此目的，在400ml 2×YT/氨苄青霉素培养基接种噬菌体展示全人源单链抗体天然库的甘油菌(购自上海锐劲生物技术有限公司)，使细胞密度达到OD600＝0.1，在37℃和200rpm条件下振荡培养直至细胞密度达到OD600＝0.5。用1012pfu的M13KO7辅助噬菌体(购自Invitrogen)感染，在30℃和50rpm条件下培养30分钟。加入50mg/l卡那霉素后在37℃和200rpm条件下振荡培养30分钟后，通过离心(15分钟，1600×g，4℃)分离沉淀，重悬于400ml 2×YT/氨苄青霉素/卡那霉素培养基，在37℃和200rpm条件下振荡培养16小时。最后细胞通过离心(20分钟，5000×g，4℃)分离沉淀并丢弃，上清用0.45μm规格滤膜过滤后，加入1/4体积20％(w/v)PEG8000、2.5M NaCl溶液并在冰浴中保温1小时沉淀噬菌体颗粒。随后离心沉淀(20分钟，8000×g，4℃)，弃上清，将噬菌体重悬于25ml预冷PBS(137mM NaCl，2.7mM KCl，8mM Na2HPO4，2mM KH2PO4)中，离心(5分钟，20000×g，4℃)。向上清液加入1/4体积20％(w/v)PEG8000、2.5M NaCl溶液，并冰浴30分钟再次沉淀噬菌体颗粒。离心沉淀(30分钟，20000×g，4℃)，再次将噬菌体沉淀重悬于2ml预冷PBS中，在冰上保持30分钟并离心(30分钟，17000×g，4℃)。上清液与含4％(w/v)BSA的PBS溶液以1:1混合，置于旋转混合器上，室温下保温30分钟，然后直接用于筛选。
利用上述噬菌体抗体库，针对生物素标记的人间皮素重组蛋白实施了四轮定向筛选，筛选方案如下：该噬菌体抗体库与生物素标记的抗人间皮素，在室温下保温2小时，然后与经封闭液2％(w/v)BSA(牛血清白蛋白)封闭过的链霉亲和素磁珠MyOne C1(购自Invitrogen)在室温下保温30分钟。随后用PBST(含0.1％吐温-20)缓冲液洗涤磁珠,除去非特异性结合或结合能力较弱的噬菌体。结合能力强的噬菌体，则用甘氨酸-盐酸(pH 2.2)从磁珠上洗脱下来，用Tris中和液(pH 9.1)中和后，用于感染处于对数生长中期的大肠杆菌ER2738，并被用于下一轮筛选。在四轮筛选中，磁珠的用量分别为50μl、20μl、10μl和10μl，生物素标记的人间皮素浓度分别为100nM、10nM、5nM和1nM,PBST的洗涤次数分别为10次、10次、15次和20次。
3.2 人间皮素特异性结合抗体的鉴定
从第四轮筛选所得的克隆中随机挑选96个，并用单噬菌体ELISA(酶联免疫吸附实验)分析其与人间皮素的结合能力。为此目的，每个单菌落接种300μl 2×YT/氨苄青霉素培养基(含2％葡萄糖)于96孔深孔培养板，并在37℃和250rpm下振荡培养16小时。用20μl培养物接种到500μl 2×YT/氨苄青霉素培养基(含0.1％葡萄糖)，在37℃和250rpm下振荡培养1.5小时。准备辅助噬菌体溶液，取75μl的M13KO7(滴度为3×1012pfu/ml)混入到15ml 2×YT培养基中，50μl/孔加到培养板中。在37℃和150rpm条件培养30分钟，然后加入准备好的卡那霉素溶液50μl/孔(取180μl的50mg/ml卡那霉素，加入到15ml 2×YT培养基)，在37℃ 和250rpm下振荡培养16小时。最后离心沉淀细胞(30分钟，5000×g，4℃)，上清转移到新的96孔深孔培养板。
为进行单噬菌体ELISA，在96孔MediSorp ELISA板(购自Nunc)上分别使用100ng/孔抗原人间皮素以及阴性对照蛋白BSA(100μl/孔)，在4℃包被过夜。每个孔用含2％BSA(w/v)的PBST封闭。随后用PBST清洗孔三次并排净。然后加入100μl/孔上面制备的每种噬菌体溶液到板上各孔中。37℃保温2小时后，用PBST洗涤三次。为了检测结合的噬菌体，将抗M13抗体过氧化物歧化酶偶联物(购自GE Healthcare)以1:5000稀释于PBST中，并取100μl加到每个孔中。37℃保温1小时后用PBST漂洗孔三次，然后用PBS漂洗三次。最后吸取50μl TMB底物加入到孔中，并在室温下显色10分钟，随后加入每孔50μl的2M H2SO4终止显色反应。用酶联免疫检测仪(Bio-Rad)在450nm测量消光值。结合测序分析，观察到两个不同的单链抗体P1A6E(SEQ ID NO:5(核苷酸)，6(氨基酸))和P3F2(SEQ ID NO:7(核苷酸)，8(氨基酸))，在ELISA实验中对人间皮素(hu-mesothelin)结合信号较强，对BSA无结合(图2)。
SEQ ID NO:5(核苷酸)
Figure PCTCN2016096292-appb-000002
SEQ ID NO:6(氨基酸)
Figure PCTCN2016096292-appb-000003
其中，重链可变区的氨基酸序列如SEQ ID NO:6中第1-123位所示；其轻链可变区的氨基酸序列如SEQ ID NO:6中第139-254位所示。
其中，轻链CDR1氨基酸序列为TLRSGINVGIYRIY(SEQ ID NO:51)，轻链CDR2氨基酸序列为YKSDSDKYQGS(SEQ ID NO:52)，轻链CDR3氨基酸序列为MIWHSGGWV(SEQ ID NO:53)；重链CDR1氨基酸序列为GDTVSSDSAAWN(SEQ ID NO:54)，重链CDR2氨基酸序列为RTYYRSKWFNDYAVSVKG(SEQ ID NO:55)，重链CDR3氨基酸序列为SNSYYYYAMDV(SEQ ID NO:56)。
SEQ ID NO:7(核苷酸)
Figure PCTCN2016096292-appb-000004
SEQ ID NO:8(氨基酸)
Figure PCTCN2016096292-appb-000005
Figure PCTCN2016096292-appb-000006
其中，重链可变区的氨基酸序列如SEQ ID NO:8中第1-124位所示；其轻链可变区的氨基酸序列如SEQ ID NO:8中第140-247位所示。
其中，轻链CDR1氨基酸序列为RASQVISRALA(SEQ ID NO:57)，轻链CDR2氨基酸序列为DASNLQS(SEQ ID NO:58)，轻链CDR3氨基酸序列为QQFNSYPLT(SEQ ID NO:59)；重链CDR1氨基酸序列为GYTFTSYYMH(SEQ ID NO:60)，重链CDR2氨基酸序列为IINPSGGSTSYAQKFQG(SEQ ID NO:61)，重链CDR3氨基酸序列为SRSGTTVVNHDAFDI(SEQ ID NO:62)。
实施例4、单链抗体和单克隆抗体的制备
4.1 人间皮素单链抗体的制备
使用引物对V5-P1A6E-F(SEQ ID NO:9)和V5-P1A6E-R(SEQ ID NO:10)从所得克隆中扩增出scFv-P1A6E片段；使用引物对V5-P3F2-F(SEQ ID NO:11)和V5-P3F2-R(SEQ ID NO:12)扩增出scFv-P3F2片段，通过NheI/BamHI双酶切，以T4DNA连接酶于同样以NheI/BamHI双酶切载体质粒pCMV-V5-Fc(该载体在多克隆位点下游融合表达人抗体IgG1的Fc片段，以下简称V5-Fc，购自上海锐劲生物技术有限公司)连接并转化于宿主菌TOP10中，挑取克隆通过PCR鉴定阳性克隆并通过测序确认，分别获得V5-scFv-P1A6E-Fc和V5-scFv-P3F2-Fc真核表达质粒。
SEQ ID NO:9：ACAGTGCTAGCACAGGTACAGCTGGAACAG；
SEQ ID NO:10：TTGTCGGATCCACCTAGGACGGTGACC；
SEQ ID NO:11：ACAGTGCTAGCACAGATGCAGCTAGTGC；
SEQ ID NO:12：TTGTCGGATCCACGTTTGATCTCCAGC。
将上述表达质粒分别转染生长良好的HEK-293F细胞，37℃，5％CO2，125rpm摇床连续培养7天，4000rpm离心10min，去除沉淀，收集上清，并用0.45μm滤膜过滤，将处理好的样品以protein A(购自GE)亲和柱进行亲和纯化，最终获得纯化的抗体-Fc融合蛋白scFv-P1A6E-Fc和scFv-P3F2-Fc，鉴定结果如图3所示。
4.2 人间皮素单克隆抗体的制备
在本实施例中，单克隆抗体的表达使用双质粒系统，需要分别把抗体重链可变区基因构建到包含了人IgG1CH基因的pIH质粒，把抗体轻链可变区基因构建到包含了人IgG CL基因的pIK质粒(质粒购自上海锐劲生物技术有限公司)。
使用引物对P1A6E-HF(SEQ ID NO:13，gcctttcctggtttcctgtctcaggtacagctgg aacagtc)和P1A6E-HR(SEQ ID NO:14，GATGGGCCCTTGGTGGAGGCACTCG AGACGGTGACCAG)从模板质粒V5-scFv-P1A6E-Fc中扩增出VH-P1A6E片段。使用引物对HF1F(SEQ ID NO:15，ggctaactagagaacccactgc)和HF1R(SEQ ID NO:16，AGACAGGAAACCAGGAAAGGC)从模板质粒pIH中扩增出HF1片段；使用引物对HF3F(SEQ ID NO:17，gcctccaccaagggcccatc) 和HF3R(SEQ ID NO:18，gacaatcttagcgcagaagtc)从模板质粒pIH中扩增出HF3片段。三个片段等摩尔比混合后进行拼接PCR，片段回收后经限制性内切酶NheI/NotI双酶切，以T4DNA连接酶于同样以NheI/NotI双酶切载体质粒pIH连接并转化于宿主菌TOP10中，挑取克隆通过PCR鉴定阳性克隆并通过测序确认，获得pIH-P1A6E真核表达质粒。以同样的方式也获得了pIH-P3F2真核表达质粒。
为获得pIK-P1A6E真核表达质粒，使用引物对P1A6E-LF(SEQ ID NO:19，ctttggtttccaggtgcaagatgtcaggctgtgctgactcag)和P1A6E-LR(SEQ ID NO:20，GAAGACAGATGGTGCAGCCACCGTACCTAGGACGGTGACCTTG)从模板质粒V5-scFv-P1A6E-Fc中扩增出VL-P1A6E片段；使用引物对LF1F(SEQ ID NO:21，ggctaactagagaacccactgc)和LF1R(SEQ ID NO:22，ACATCTTGCACC TGGAAACCAAAG)从模板质粒pIK中扩增出LF1片段；使用引物对LF3F(SEQ ID NO:23，acggtggctgcaccatctgtcttc)和LF3R(SEQ ID NO:24，GACAATCTTAGCGCAGAAGTC)从模板质粒pIK中扩增出LF3片段。三个片段等摩尔比混合后进行拼接PCR，片段回收后经限制性内切酶EcoRV/NotI双酶切，以T4DNA连接酶于同样以EcoRV/NotI双酶切载体质粒pIK连接并转化于宿主菌TOP10中，挑取克隆通过PCR鉴定阳性克隆并通过测序确认。以同样的方式也获得了pIK-P3F2真核表达质粒。
将表达质粒pIH-P1A6E和pIK-P1A6E等摩尔混合，pIH-P3F2和pIK-P3F2等摩尔混合，分别转染生长良好的HEK-293F细胞，37℃，5％CO2，125rpm摇床连续培养7天，4000rpm离心10min，去除沉淀，收集上清，并用0.45μm滤膜过滤，将处理好的样品以protein A(购自GE)亲和柱进行亲和纯化，最终获得纯化的重组单克隆抗体P1A6E和P3F2，鉴定结果如图4所示。
实施例5、人间皮素抗体的亲和力
为定量分析抗体与人间皮素的结合，使用Biacore T200系统(购自GE)通过捕获法分别测量了P1A6E和P3F2单链抗体和单克隆抗体的亲和力和动力学参数。根据制造商的说明，用NHS/EDC偶联通过伯氨基将抗人IgG(Fc)的抗体(购自GE)偶联到传感器芯片CM5的羧甲基葡聚糖表面。测量在25℃、30μl/min、1×HBS-EP+工作缓冲液中进行，再生条件为3M MgCl2、10μl/min作用30秒。在每一轮测试循环中，待测试抗体首先被捕获到芯片上，一定浓度的分析物(人间皮素)流过芯片表面，由于产生SPR信号，人间皮素与被捕获的抗体的相互作用可以被检测。检测信号被定义为共振单位(RU)，对时间(单位为秒)作图可以得到相应的结合曲线和解离曲线。在不同的测试循环中，人间皮素的浓度分别为10nM、20nM、40nM、80nM和160nM。用Biacore T200evaluation software评估所得作用曲线，并计算亲和力KD值。图5和图6分别显示了单克隆抗体P1A6E和P3F2在Biacore亲和力测定实验中的动力学曲线。P1A6E和P3F2的单链抗体和单克隆抗体分别对人间皮素的结合数据总结在表1中。
表1、P1A6E和P3F2的单链抗体和单克隆抗体对人间皮素的亲和力参数
Figure PCTCN2016096292-appb-000007
Figure PCTCN2016096292-appb-000008
实施例6、人间皮素抗体的细胞结合特性(单链抗体及单克隆抗体)
通过荧光激活细胞分选仪(FACS)(Guava 8HT，由Millipore公司提供)分析抗体scFv-P1A6E-Fc和scFv-P3F2-Fc各自与细胞表面的mesothelin的结合能力。
具体方法如下：
1)取对数生长期的细胞PANC-1-MSLN和PANC-1分别接种到6cm平皿中，接种细胞密度约为90％，37℃孵箱过夜培养。
2)使用10mM的EDTA消化细胞，200g×5min离心收集细胞。以1×106～1×107/mL的浓度重悬于1％含小牛血清的磷酸盐缓冲液(NBS PBS)中，按100ul/管的量加入流式专用管中。
3)200g×5min离心，弃上清。
4)两个实验组分别加入待测抗体P1A6E和P3F2，同时两个阳性对照组加入抗体ss和C10(购自上海锐劲生物技术有限公司)作为阳性对照，另一个对照组为不加抗体的PBS空白对照。各抗体的终浓度均为20μg/ml，每管加入100ul。冰浴，45分钟。
5)每管加入2ml 1％NBS PBS，以200g×5min离心，共二遍。
6)弃上清，加入1:100稀释的羊抗人抗体-FITC(来自上海业力生物科技有限公司)，每管加入100ul。冰浴，45分钟。
7)每管加入2ml 1％NBS PBS，以200g×5min离心，共二遍。
8)弃上清，重悬于300ul 1％NBS PBS中，流式细胞仪检测。
9)应用流式细胞仪数据分析软件Flowjo7.6分析数据。
流式细胞分析结果表明，四种抗体P1A6E和P3F2、以及对照抗体SS和C10，无论是单链抗体形式(图7)还是单克隆全抗形式(图8，其荧光峰在PANC-1-MSLN细胞上与空白对照(PBS)相比有显著的差异(图7B，图8B)，在PANC-1细胞上无明显差别(图7A，图8A)，表明其都可以特异识别人间皮素稳定表达的PANC-1-MSLN细胞，但是与人间皮素表达阴性的PANC-1细胞不结合，表明这四个抗体可以特异性识别人间皮素。抗体P1A6E和P3F2的荧光峰明显强于对照抗体SS和C10，表明P1A6E和P3F2对PANC-1-MSLN细胞的结合效率要高于SS和C10。
实施例7、含有人间皮素抗体的CAR T的制备
构建嵌合抗原受体，本发明示例的嵌合抗原受体各部分的连接顺序如表2。
表2
嵌合抗原受体 胞外结合区-跨膜区-胞内信号区1-胞内信号区2等 描述
P1A6E-δZ scFv(MSLN)-CD8-CD3δzeta 阴性对照
P1A6E-Z scFv(MSLN)-CD8-CD3zeta 第一代
P1A6E-BBZ scFv(MSLN)-CD8-CD137-CD3zeta 第二代
P1A6E-28Z scFv(MSLN)-CD28a-CD28b-CD3zeta 第二代
P1A6E-28BBZ scFv(MSLN)-CD28a-CD28b-CD137-CD3zeta 第三代
P3F2-δZ scFv(MSLN)-CD8-CD3δzeta 阴性对照
P3F2-Z scFv(MSLN)-CD8-CD3zeta 第一代
P3F2-BBZ scFv(MSLN)-CD8-CD137-CD3zeta 第二代
P3F2-28Z scFv(MSLN)-CD28a-CD28b-CD3zeta 第二代
P3F2-28BBZ scFv(MSLN)-CD28a-CD28b-CD137-CD3zeta 第三代
注：CD28a代表CD28分子的跨膜区，CD28b代表CD28分子的胞内信号区。
本实施例中使用的慢病毒质粒载体系统属于第三代慢病毒四质粒系统，该系统共有四个质粒即编码VSV-G蛋白的包膜质粒pCMV-VSV-G(购自addgene)、编码Rev蛋白的包装质粒pRSV-Rev(购自addgene)、编码Gal和Pol的pMDLg/pRRE(购自addgene)及基于空载体pRRLSIN.cPPT.PGK-GFP.WPRE(购自addgene)的编码目的基因CAR的重组表达载体。所有CAR的基因载体启动子均利用已经公开的专利201310164725.X中的载体上延长因子-1α(elongation factor-1α，EF-1α)。构建具体方法如下：
(1)获得启动子片段：利用专利201310164725.X中的载体pWPT-eGFP-F2A-CAR，引物pwpxlF(SEQ ID NO:25，5’-gcaggggaaagaatagtaga ca-3’)和pWPT-MluIR(SEQ ID NO:26，5’-aggccagcggcaggagcaaggcggtcactggta aggccatggtggcgaccggtagc-3’)，PCR扩增出带有启动子EF-1α的片段。
(2)获得目的CAR片段：分别利用前述获得V5-scFv-P1A6E-Fc和V5-scFv-P3F2-Fc为模板，引物P1A6E-F(SEQ ID NO:27，5’-ctcctgccgctggccttgctgctccacgccgccaggccgcaggtacagctggaaca-3’)和引物SEQ ID NO:28，P1A6E-R(5’-gcggcgctggcgtcgtggtacctaggacggtgacc-3’)；引物P3F2-F(SEQ ID NO:29，5’ctcctgccgctggccttgctgctccacgccgccaggccgcagatgcagctagt gca-3’)和P3F2-R(SEQ ID NO:30，5’gcggcgctggcgtcgtggtacgtttgatctccag-3’)，扩增出目的CAR片段P1A6E部分和P3F2部分
(3)通过PCR分别获得CAR的第一代、第二代、第三代的共有序列和阴性对照序列：分别以室专利201310164725.X中的pWPT-eGFP-F2A-GPC3-δZ、pWPT-eGFP-F2A-GPC3-Z、pWPT-eGFP-F2A-GPC3-28Z、pWPT-eGFP-F2A-GPC3-28BBZ为模板，以引物HF(SEQ ID NO:63，5’accacgacgccagcgccgcgaccac)和引物pwpxlR(SEQ ID NO:64，5’-tagcgtaaaaggagcaacatag)，分别获得片段CD8-CD3δzeta(δZ)、CD8-CD3zeta(Z)、CD28a-CD28b-CD3zeta(28Z)和CD28a-CD28b-CD137-CD3zeta(28BBZ)序列。
(4)参考专利US 8,911,993B2(COMPOSITIONS FOR TREATMENT OF CANCER)中的BBZ序列，使用基于PCR搭桥的基因合成方法，合成共有序列CD8-CD137-CD3zeta(BBZ)片段部分。
(5)将上述获得的启动子片段、目的CAR片段分别与共有序列片段CD8-CD3δzeta(δZ)、CD8-CD3zeta(Z)、CD8-CD137-CD3zeta(BBZ)、CD28a-CD28b-CD3zeta(28Z)和CD28a-CD28b-CD137-CD3zeta(28BBZ)序列部分常规搭桥后，再用引物pwpxlF和pwpxlR扩增，获得含有EF-1α和目的基因CAT的片段，分别称为：
P1A6E-δZ(SEQ ID NO:31)；
P1A6E-Z(SEQ ID NO:32)；
P1A6E-BBZ(SEQ ID NO:33)；
P1A6E-28Z(SEQ ID NO:34)；
P1A6E-28BBZ(SEQ ID NO:35)。
P3F2-δZ(SEQ ID NO:36)；
P3F2-Z(SEQ ID NO:37)；
P3F2-BBZ(SEQ ID NO:38)；
P3F2-28Z(SEQ ID NO:39)；
P3F2-28BBZ(SEQ ID NO:40)。
(6)将上述步骤中获得的带有启动子和目的基因CAR的片段，通过ClaI和SalI双酶切后连接于同样酶切的载体pRRLSIN.cPPT.PGK-GFP.WPRE中，从而构建表达各嵌合抗原受体的慢病毒载体。构建成功的载体经Mlu和Sal酶切鉴定正确后测序核对，可以准备用于慢病毒包装。
得到的含有各目的CAR的载体如下：：
pRRLSIN-EF1α-P1A6E-δZ；
pRRLSIN-EF1α-P1A6E-Z；
pRRLSIN-EF1α-P1A6E-BBZ；
pRRLSIN-EF1α-P1A6E-28Z；
pRRLSIN-EF1α-P1A6E-28BBZ；
pRRLSIN-EF1α-P3F2-δZ；
pRRLSIN-EF1α-P3F2-Z；
pRRLSIN-EF1α-P3F2-BBZ；
pRRLSIN-EF1α-P3F2-28Z；
pRRLSIN-EF1α-P3F2-28BB。
通过以上构建，分别可获得十个CAR多肽序列，称为：
P1A6E-δZ(SEQ ID NO:41)；
P1A6E-Z(SEQ ID NO:42)；
P1A6E-BBZ(SEQ ID NO:43)；
P1A6E-28Z(SEQ ID NO:44)；
P1A6E-28BBZ(SEQ ID NO:45)。
P3F2-δZ(SEQ ID NO:46)；
P3F2-Z(SEQ ID NO:47)；
P3F2-BBZ(SEQ ID NO:48)；
P3F2-28Z(SEQ ID NO:49)；
P3F2-28BBZ(SEQ ID NO:50)。
质粒转染293T包装慢病毒
以6×106的密度接种培养至第6～10代的HEK-293T细胞(ATCC：CRL-11268)于10cm培养皿中，37℃，5％CO2培养过夜准备用于转染。培养基为含10％胎牛血清的DMEM。
转染的步骤如下：
A液配制：将10μg的目的基因质粒pRRLSIN-cPPT.EF-1α-CAR(选自pRRLSIN-EF1α-P1A6E-δZ、pRRLSIN-EF1α-P1A6E-Z、pRRLSIN-EF1α-P1A6E-BBZ、pRRLSIN-EF1α-P1A6E-28Z、pRRLSIN-EF1α-P1A6E-28BBZ；pRRLSIN-EF1α-P3F2-Z、pRRLSIN-EF1α-P3F2-BBZ、pRRLSIN-EF1α-P3F2-28Z、pRRLSIN-EF1α-P3F2-28BBZ)，分别与7.5μg包装质粒pMDLg RRE和pRSV-REV、以及3μg包膜质粒pCMV-VSV-G，溶 入800μL的无血清DMEM培养液中，混匀。
B液配制：将60μg PEI(聚乙烯亚胺，购自Polysciences公司)溶解于800μL的无血清DMEM培养液中，轻轻混匀，室温孵育5min。
转染复合物的形成：将A液加入B液中轻轻混合，加入后立即涡旋混合或轻轻混匀，室温下孵育20min。
将转染复合物1.6ml滴加入HEK-293T细胞中，4-5h小时后，用2％FBS的DMEM培基给转染的293T细胞换液。
在转染72h后，使用0.45μm滤器过滤收集病毒，然后采用Beckman Optima L-100XP超速离心机28000rpm，4℃离心2小时，弃离心上清，离心所得沉淀用1/10～1/50原液体积的AIM-V培养液(购自Invitrogen公司)进行重悬，以100μL/管分装冻存于-80℃，以待病毒滴定或感染T淋巴细胞。
实施例8、重组慢病毒感染CTL细胞
由健康人外周血通过密度梯度离心法获得人外周血单个核细胞(上海市血液中心提供)，外周血单个核细胞通过CTL细胞磁珠(购自Stem Cell Technologies)负性分选方法获得CTL，分选后的CTL细胞进行流式细胞检测CTL细胞的纯度，以CTL细胞的阳性率≥95％为宜进行下一步操作。以约1×106/mL密度加入Quantum 007淋巴细胞培养基液(购自PAA公司)培养并以细胞：磁珠比例为1:1加入同时包被有抗CD3和CD28抗体的磁珠(Invitrogen公司)和终浓度300U/mL的重组人IL-2(购自上海华新生物高技术有限公司)刺激培养24h。然后以MOI≈5用上述重组慢病毒感染CTL细胞。感染后的细胞每隔一天采用5×105/mL的密度进行传代，同时在淋巴细胞培养液中补加终浓度300U/mL的重组人IL-2。
感染的CTL细胞在培养第8天时通过流式细胞检测各不同嵌合抗原受体表达。首先，感染后的CAR T细胞与生物素标记的人间皮素重组蛋白37℃孵育1h，D-PBS洗2次，然后加入PE-标记的streptavidin在37℃孵育40min。D-PBS洗3次后流式细胞仪检测阳性细胞比率。以未感染的T淋巴细胞作为阴性对照，表达不同嵌合抗原受体的病毒感染CTL细胞其阳性率如表3所示。该阳性率结果表明，通过慢病毒感染的方法能够获得一定阳性率的CAR+CTL细胞。
表3
Figure PCTCN2016096292-appb-000009
CTL细胞在分别感染包装有不同嵌合抗原受体的病毒后，以细胞密度为5×105/ml隔天传代培养、计数、并对传代的细胞培养液补加IL-2(终浓度为300U/ml)，培养第11天约有20～40倍的扩增，表明表达不同嵌合抗原受体的CTL细胞在体外能够进行一定数量的扩增，为后续体外毒性试验及体内试验提供了保证。
实施例9、表达嵌合抗原受体的T淋巴细胞的体外毒性效果实验
体外毒性实验使用的材料如下：
如表4所示的间皮素阴性胰腺癌细胞系(PANC-1)和转染了间皮素基因的PANC-1(PANC-1-MSLN)细胞系作为靶细胞，效应细胞为如实施例4所验证的体外培养12天的FACS检测嵌合抗原受体表达的阳性细胞记为嵌合抗原受体阳性(CAR+)的CTL，效靶比视情况分别为3:1，1:1和1:3，靶细胞数量为10000个/孔，根据不同效靶比对应效应细胞。各组均设5个复孔，取5个复孔的平均值。检测时间为第18h。
其中各实验组和各对照组如下：
各实验组：各靶细胞+表达不同嵌合抗原受体的CTL，
对照组1：靶细胞最大释放LDH，
对照组2：靶细胞自发释放LDH，
对照组3：效应细胞自发释放LDH。
检测方法：采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。该方法是基于比色法的检测方法，可替代51Cr释放法。CytoTox 
Figure PCTCN2016096292-appb-000010
检测定量地测量乳酸脱氢酶(LDH)。LDH是一种稳定的胞质酶，在细胞裂解时会释放出来，其释放方式与51Cr在放射性分析中的释放方式基本相同。释放出的LDH培养基上清中，可通过30分钟偶联的酶反应来检测，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。具体参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。
细胞毒性计算公式为：
Figure PCTCN2016096292-appb-000011
具体如表4所示，本发明的抗间皮素的单链抗体(P1A6E，P3F2)的CAR均有明显的杀伤间皮素阳性胰腺癌细胞的活性，其中第二代和第三代的抗mesothelin的CAR T细胞比第一代抗肿瘤活性略强。mock组没有明显杀伤。此外，所有CAR T细胞对间皮素阴性的PANC-1胰腺癌细胞均没有杀伤活性。这些结果表明本发明的抗间皮素的CAR-T细胞(包括一、二、三代CAR T)可以选择性地针对间皮素阳性的胰腺癌细胞，并进行有效的杀伤。此外，本发明的抗间皮素的第一、二、三代CAR T对间皮素阳性肿瘤细胞呈现效靶比梯度依赖性即效靶比越高细胞毒性作用越强。
表4、融合表达单链抗体的CAR T细胞的体外抗肿瘤细胞毒性
Figure PCTCN2016096292-appb-000012
Figure PCTCN2016096292-appb-000013
Figure PCTCN2016096292-appb-000014
实施例10、人间皮素抗体的表位分析
通过PCR从SEQ ID NO:1中扩增人间皮素基因片段，通过NheI/BamHI双酶切，将人间皮素基因片段连接至含有鼠Fc段的真核表达载体pCMV-V5-muFc中。根据实施例4瞬时转染HEK-293F细胞，细胞培养上清经处理后通过protein G(购自GE)亲和柱进行亲和纯化，最终获得纯化的人间皮素片段-muFc融合蛋白，然后通过ELISA鉴定抗体scFv-P1A6E-Fc和scFv-P3F2-Fc的结合。根据Genbank登陆号NP_001170826.1(SEQ ID NO：65)，把成熟的人间皮素分成三个区域，区域R1(E296-T390，SEQ ID NO：66)，区域R2(S391-Q486,SEQ ID NO：67)，区域R3(N487-G581,SEQ ID NO：68)，ELISA结果显示抗体scFv-P1A6E-Fc和scFv-P3F2-Fc都只和区域1(E296-T390)结合。进一步把区域1分割成5个小的片断，分别和muFc融合表达。区域R1A(296E-337D,SEQ ID NO：69)，区域R1B(328D-369I,SEQ ID NO：70)，区域R1C(360Y-405T,SEQ ID NO：71)，区域R1AB(296E-359L,SEQ ID NO：72)，R1BC(328D-405T,SEQ ID NO：73)。ELISA结果如图9和图10所示，其中抗体scFv-P1A6E-Fc和scFv-P3F2-Fc和区域R1AB有明显的结合，和区域R1A有很弱的结合，和区域R1B不结合。因此，抗体scFv-P1A6E-Fc和scFv-P3F2-Fc的结合位点应该位于R1A和R1B重叠的位置附近。这个区域包含有10个氨基酸“DAALLATQMD”，基于该序列向两端各延长10个氨基酸，或者5个氨基酸，形成两个肽段R1J10:“YKKWELEACVDAALLATQMDRVNAIPFTYE(SEQ ID NO:74)”和R1J5:“LEACVDAALLATQMDRVNAI(SEQ ID NO:75)”分别和muFc融合表达纯化。ELISA结果显示抗体scFv-P1A6E-Fc和scFv-P3F2-Fc和R1J10以及R1J5都不结合。根据以上结果，抗体scFv-P1A6E-Fc和scFv-P3F2-Fc的表位应该是位于区域R1AB(SEQ ID NO：72)的构象表位。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (37)

  1. 可特异结合间皮素的全人抗体，其特征在于，该全人抗体选自：
    (a)抗体，其包含重链可变区，所述重链可变区具有包含SEQ ID NO:54所示的氨基酸序列的CDR1、包含SEQ ID NO:55所示的氨基酸序列的CDR2、包含SEQ ID NO:56所示的氨基酸序列的CDR3；
    (b)抗体，其包含轻链可变区，所述轻链可变区具有包含SEQ ID NO:51所示的氨基酸序列的CDR1、包含SEQ ID NO:52所示的氨基酸序列的CDR2、包含SEQ ID NO:53示氨基酸序列的CDR3；
    (c)抗体，包含(a)所述抗体的重链可变区及(b)所述抗体的轻链可变区；
    (d)抗体，包含重链可变区，所述重链可变区具有包含SEQ ID NO:60所示的氨基酸序列的CDR1、包含SEQ ID NO:61所示的氨基酸序列的CDR2、包含SEQ ID NO:62所示的氨基酸序列的CDR3；
    (e)抗体，包含轻链可变区，所述轻链可变区具有包含SEQ ID NO:57所示的氨基酸序列的CDR1、包含SEQ ID NO:58所示的氨基酸序列的CDR2、包含SEQ ID NO:59所示的氨基酸序列的CDR3；
    (f)包含(d)所述抗体的重链可变区及(e)所述抗体的轻链可变区；
    (g)抗体，识别与(a)～(f)中任一项所述的抗体所识别的抗原决定部位相同的抗原决定部位。
  2. 如权利要求1所述的全人抗体，其特征在于，该全人抗体包括重链可变区和轻链可变区，其重链可变区的氨基酸序列如SEQ ID NO:6中第1-123位所示；其轻链可变区的氨基酸序列如SEQ ID NO:6中第139-254位所示；或
    该全人抗体包括重链可变区和轻链可变区，其重链可变区的氨基酸序列如SEQ ID NO:8中第1-124位所示；其轻链可变区的氨基酸序列如SEQ ID NO:8中第140-247位所示。
  3. 编码权利要求1-2任一所述的抗体的核酸。
  4. 一种表达载体，其包含权利要求3所述的核酸。
  5. 一种宿主细胞，其包含权利要求4所述的表达载体或基因组中整合有权利要求3所述的核酸。
  6. 权利要求1-2任一所述的抗体的用途，用于制备特异性靶向表达间皮素的肿瘤细胞的靶向性药物，抗体药物偶联物或多功能抗体；或
    用于制备诊断肿瘤的试剂，该肿瘤表达间皮素；或
    用于制备嵌合抗原受体修饰的免疫细胞。
  7. 一种嵌合抗原受体，所述嵌合抗原受体包含顺序连接的：权利要求1-2任一所述的抗体、跨膜区和胞内信号区。
  8. 如权利要求7所述的嵌合抗原受体，其特征在于，所述的胞内信号区选自：CD3ζ，FcεRIγ，CD27，CD28，CD137，CD134，MyD88，CD40的胞内信号区序列，或其组合。
  9. 如权利要求7所述的嵌合抗原受体，其特征在于，所述的跨膜区包含CD8或CD28的跨膜区。
  10. 如权利要求7所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体包括如下的顺序连接的抗体，跨膜区和胞内信号区：
    权利要求1-2任一所述的抗体、CD8和CD3ζ；
    权利要求1-2任一所述的抗体、CD8、CD137和CD3ζ；
    权利要求1-2任一所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
    权利要求1-2任一所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
  11. 如权利要求7所述的嵌合抗原受体，其特征在于，所述的抗体是单链抗体或结构域抗体。
  12. 如权利要求7所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体具有：
    SEQ ID NO:41或其中第22-353位所示的氨基酸序列；
    SEQ ID NO:42或其中第22-454位所示的氨基酸序列；
    SEQ ID NO:43或其中第22-498位所示的氨基酸序列；
    SEQ ID NO:44或其中第22-501位所示的氨基酸序列；
    SEQ ID NO:45或其中第22-543位所示的氨基酸序列；
    SEQ ID NO:46或其中第22-346位所示的氨基酸序列；
    SEQ ID NO:47或其中第22-447位所示的氨基酸序列；
    SEQ ID NO:48或其中第22-491位所示的氨基酸序列；
    SEQ ID NO:49或其中第22-494位所示的氨基酸序列；或
    SEQ ID NO:50或其中第22-536位所示的氨基酸序列。
  13. 编码权利要求7-12任一所述的嵌合抗原受体的核酸。
    在另一优选例中，编码所述的嵌合抗原受体的核酸具有：
    SEQ ID NO:31或其中第473-1468位所述的核苷酸序列；
    SEQ ID NO:32或其中第473-1771位所述的核苷酸序列；
    SEQ ID NO:33或其中第473-1903位所述的核苷酸序列；
    SEQ ID NO:34或其中第473-1912位所述的核苷酸序列；
    SEQ ID NO:35或其中第473-2038位所述的核苷酸序列；
    SEQ ID NO:36或其中第473-1447位所述的核苷酸序列；
    SEQ ID NO:37或其中第473-1750位所述的核苷酸序列；
    SEQ ID NO:38或其中第473-1882位所述的核苷酸序列；
    SEQ ID NO:39或其中第473-1891位所述的核苷酸序列；
    SEQ ID NO:40或其中第473-2017位所述的核苷酸序列。
  14. 一种表达载体，其特征在于，其包含权利要求13所述的核酸。
  15. 一种病毒，其特征在于，所述的病毒包含权利要求14所述载体。
  16. 权利要求7-12任一所述的嵌合抗原受体、或权利要求13所述的核酸、或权利要求14所述的表达载体、或权利要求15所述的病毒的用途，用于制备靶向表达间皮素的肿瘤细胞的基因修饰的免疫细胞。
  17. 如权利要求16所述的嵌合抗原受体，其特征在于，所述的表达间皮素的肿瘤包括：胰腺癌、卵巢癌，胸腺间皮瘤。
  18. 一种基因修饰的免疫细胞，其特征在于，其转导有权利要求13所述的核酸，或权利要求14所述的表达载体或权利要求15所述的病毒；或
    其表面表达权利要求7-12任一所述的嵌合抗原受体。
  19. 如权利要求18所述的免疫细胞，其特征在于，其还携带外源的细胞因子的编码序列；较佳地，所述的细胞因子包括：IL-12，IL-15或IL-21。
  20. 如利要求18所述的免疫细胞，其特征在于，其还表达另一种嵌合抗原受体，该受体不含有CD3ζ，但含有CD28的胞内信号结构域、CD137的胞内信号结构域或者这两者的组合。
  21. 如权利要求18所述的免疫细胞，其特征在于，其还表达趋化因子受体；较佳地，所述的趋化因子受体包括：CCR2。
  22. 如权利要求18所述的免疫细胞，其特征在于，其还表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白。
  23. 如权利要求18所述的免疫细胞，其特征在于，其还表达安全开关；较佳地，所述的安全开关包括：iCaspase-9，Truancated EGFR或RQR8。
  24. 如权利要求18所述的免疫细胞，其特征在于，所述的免疫细胞包括：T淋巴细胞、NK细胞或者NKT淋巴细胞。
  25. 权利要求18-24任一所述的基因修饰的免疫细胞的用途，其特征在于，用于制备抑制肿瘤的药物，所述的肿瘤是表达间皮素的肿瘤。
  26. 一种多功能免疫辍合物，其特征在于，所述的多功能免疫辍合物包括：
    权利要求1-2任一所述的抗体；以及
    与之连接的功能性分子；所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。
  27. 如权利要求26所述的多功能免疫辍合物，其特征在于，所述的靶向肿瘤表面标志物的分子是结合肿瘤表面标志物的抗体或配体；或
    所述的抑制肿瘤的分子是抗肿瘤的细胞因子或抗肿瘤的毒素；较佳地，所述的细胞因子包括：IL-12、IL-15、IFN-beta、TNF-alpha。
  28. 如权利要求26所述的多功能免疫辍合物，其特征在于，所述的可检测标记物包括：荧光标记物、显色标记物。
  29. 如权利要求27所述的多功能免疫辍合物，其特征在于，所述的结合肿瘤表面标志物的抗体是指：识别间皮素以外的其它抗原的抗体，所述的其它抗原包括：EGFR，EGFRvIII， mesothelin，HER2，EphA2，Her3，EpCAM，MUC1，MUC16，CEA，Claudin 18.2，叶酸受体，Claudin 6，CD3，WT1，NY-ESO-1，MAGE 3，ASGPR1或CDH16。
  30. 如权利要求26所述的多功能免疫辍合物，其特征在于，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体，其与权利要求1-2任一所述的抗体形成T细胞参与的双功能抗体。
  31. 如权利要求30所述的多功能免疫辍合物，其特征在于，所述的结合T细胞表面标志物的抗体是抗CD3抗体。
  32. 如权利要求31所述的多功能免疫辍合物，其特征在于，其是融合多肽，权利要求1-2任一所述的抗体以及与之连接的功能性分子之间，还包括连接肽。
  33. 编码权利要求26-32任一所述的多功能免疫辍合物的核酸。
  34. 权利要求26-32任一所述的多功能免疫辍合物的用途，用于制备抗肿瘤药物，或
    用于制备诊断肿瘤的试剂，该肿瘤表达间皮素；或
    用于制备嵌合抗原受体修饰的免疫细胞；较佳地，所述免疫细胞包括：T淋巴细胞、NK细胞或者NKT淋巴细胞。
  35. 药物组合物，其特征在于，其包括：
    权利要求1-2任一所述的抗体或编码该抗体的核酸；或
    权利要求26-32任一所述的免疫辍合物或编码该辍合物的核酸；或
    权利要求7-12任一所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或
    权利要求18-24任一所述的基因修饰的免疫细胞。
  36. 能够与权利要求1-2任一所述的抗体竞争结合间皮素的抗体。
  37. 能够结合如SEQ ID NO:66所示的间皮素表位的抗体；优选地，所述抗体能结合SEQ ID NO：72所示的间皮素表位。
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