WO2017012567A1 - 肿瘤特异性抗egfr抗体及其应用 - Google Patents

肿瘤特异性抗egfr抗体及其应用 Download PDF

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Publication number
WO2017012567A1
WO2017012567A1 PCT/CN2016/090892 CN2016090892W WO2017012567A1 WO 2017012567 A1 WO2017012567 A1 WO 2017012567A1 CN 2016090892 W CN2016090892 W CN 2016090892W WO 2017012567 A1 WO2017012567 A1 WO 2017012567A1
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Prior art keywords
seq
antibody
variable region
chain variable
set forth
Prior art date
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PCT/CN2016/090892
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English (en)
French (fr)
Inventor
王华茂
宋波
Original Assignee
上海益杰生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority to JP2018522842A priority Critical patent/JP2018528786A/ja
Application filed by 上海益杰生物技术有限公司 filed Critical 上海益杰生物技术有限公司
Priority to CA2993262A priority patent/CA2993262A1/en
Priority to RU2018104703A priority patent/RU2730605C2/ru
Priority to SG11201801003YA priority patent/SG11201801003YA/en
Priority to ES16827258T priority patent/ES2881807T3/es
Priority to KR1020187005181A priority patent/KR20180053648A/ko
Priority to US15/746,711 priority patent/US10927176B2/en
Priority to DK16827258.1T priority patent/DK3327036T3/da
Priority to EP16827258.1A priority patent/EP3327036B1/en
Priority to AU2016295601A priority patent/AU2016295601B2/en
Publication of WO2017012567A1 publication Critical patent/WO2017012567A1/zh
Priority to HK18110747.5A priority patent/HK1251240A1/zh
Priority to US17/182,956 priority patent/US20220017625A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention is in the field of immunity, and more particularly, the present invention relates to tumor-specific anti-EGFR antibodies and uses thereof.
  • EGFR is overexpressed or mutated in many tumors. How to selectively recognize these overexpressed or mutated EGFR is undoubtedly an important scientific issue. To date, antibodies against the EGFR 287-302 epitope have been identified for the purpose of recognizing the overexpression of EGFR, EGFRvIII, and de4 EGFR on the tumor surface, but do not recognize EGFR in normal cells. Unfortunately, antibodies against this epitope will still have side effects such as rash in clinical trials (http://meetinglibrary.asco.org/content/115945-132), indicating that it may still be only for this epitope. Identify EGFR in normal cells such as keratinocytes.
  • an antibody which specifically recognizes EGFRv ⁇ expressed by a tumor cell or an overexpressed EGFR, wherein the antibody has a light chain variable region and a heavy chain variable region, and
  • CDR1 of the light chain variable region thereof has an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55;
  • the CDR2 of the light chain variable region thereof has an amino acid sequence selected from the group consisting of SEQ ID NO: 42 and SEQ ID NO: 53;
  • the CDR3 of the light chain variable region thereof has an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57;
  • CDR1 of the heavy chain variable region thereof has an amino acid sequence: SEQ ID NO: 44;
  • the CDR2 of the heavy chain variable region thereof has an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52;
  • the CDR3 of its heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 50.
  • the antibody comprises:
  • Antibody having a light chain variable region having the CDR1 set forth in SEQ ID NO: 41, the CDR2 set forth in SEQ ID NO: 42, or the CDR3 set forth in SEQ ID NO: 43; or a heavy chain variable region thereof CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 45, and CDR3 shown in SEQ ID NO: 46;
  • Antibody (c) having a light chain variable region having the CDR1 set forth in SEQ ID NO: 41, the CDR2 set forth in SEQ ID NO: 42, or the CDR3 set forth in SEQ ID NO: 48; or a heavy chain variable region thereof CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 50;
  • Antibody (d) having a light chain variable region having the CDR1 set forth in SEQ ID NO: 41, the CDR2 set forth in SEQ ID NO: 42, and the CDR3 set forth in SEQ ID NO: 43; or a heavy chain variable region thereof CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 52, and CDR3 shown in SEQ ID NO: 50;
  • Antibody having a light chain variable region having the CDR1 set forth in SEQ ID NO: 41, the CDR2 set forth in SEQ ID NO: 42, and the CDR3 set forth in SEQ ID NO: 43; or a heavy chain variable region thereof CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 45, and CDR3 shown in SEQ ID NO: 50;
  • Antibody (f) having a light chain variable region having the CDR1 set forth in SEQ ID NO: 41, the CDR2 set forth in SEQ ID NO: 53, and the CDR3 set forth in SEQ ID NO: 54; or a heavy chain variable region thereof CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 50;
  • Antibody (g) having a light chain variable region having the CDR1 set forth in SEQ ID NO: 41, the CDR2 set forth in SEQ ID NO: 42, or the CDR3 set forth in SEQ ID NO: 54; or a heavy chain variable region thereof CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 50;
  • Antibody (h) having a light chain variable region having the CDR1 set forth in SEQ ID NO: 55, the CDR2 set forth in SEQ ID NO: 42, or the CDR3 set forth in SEQ ID NO: 56; or a heavy chain variable region thereof CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 45, and CDR3 shown in SEQ ID NO: 50;
  • Antibody having a light chain variable region having the CDR1 set forth in SEQ ID NO: 41, the CDR2 set forth in SEQ ID NO: 53, or the CDR3 set forth in SEQ ID NO: 56; or a heavy chain variable region thereof CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 52, and CDR3 shown in SEQ ID NO: 50;
  • Antibody (j) having a light chain variable region having the CDR1 set forth in SEQ ID NO: 41, the CDR2 set forth in SEQ ID NO: 42, or the CDR3 set forth in SEQ ID NO: 56; or a heavy chain variable region thereof CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 52, and CDR3 shown in SEQ ID NO: 50;
  • the antibody (k) having a light chain variable region having the CDR1 set forth in SEQ ID NO: 41, the CDR2 set forth in SEQ ID NO: 42, and the CDR3 set forth in SEQ ID NO: 57; or a heavy chain variable region thereof CDR1 represented by SEQ ID NO: 44, CDR2 represented by SEQ ID NO: 52, CDR3 represented by SEQ ID NO: 50;
  • the antibody (1) recognizes the same epitope as the antigenic epitope recognized by the antibody according to any one of (a) to (k).
  • the EGFRv ⁇ or the overexpressed EGFR specifically recognizing the expression of the tumor cell may be: a single chain antibody (scFV), a monoclonal antibody, a domain antibody, a Fab fragment, an Fd fragment, an Fv fragment, F(ab') 2 fragment and derivatives thereof, or other forms of antibodies; preferably single chain antibodies.
  • scFV single chain antibody
  • the antibody that specifically recognizes EGFRv ⁇ expressed by tumor cells or overexpressed EGFR is humanized, fully human, chimeric or murine.
  • amino acid sequence of the heavy chain variable region of the antibody is as shown at positions 124-239 of SEQ ID NO: 13; or the amino acid sequence of the light chain variable region of the antibody is as 1st to 10th in SEQ ID NO:13 Shown
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124-239 of SEQ ID NO: 59; or the amino acid sequence of the light chain variable region of the antibody is as described in SEQ ID NO: 59. 1-108 bits;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124-239 of SEQ ID NO: 61; or the amino acid sequence of the light chain variable region of the antibody is as described in SEQ ID NO: 61 1-108 bits;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124-239 of SEQ ID NO: 63; or the amino acid sequence of the light chain variable region of the antibody is as set forth in SEQ ID NO: 63 1-108 bits;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124-239 of SEQ ID NO: 65; or the amino acid sequence of the light chain variable region of the antibody is as set forth in SEQ ID NO: 65 1-108 bits;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124 to 239 of SEQ ID NO: 67; or the amino acid sequence of the light chain variable region of the antibody is as described in SEQ ID NO: 67. 1-108 bits;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124 to 239 of SEQ ID NO: 69; or the amino acid sequence of the light chain variable region of the antibody is as described in SEQ ID NO: 69. 1-108 bits;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124-239 of SEQ ID NO: 71; or the amino acid sequence of the light chain variable region of the antibody is as described in SEQ ID NO: 71 1-108 bits;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124-239 of SEQ ID NO: 73; or the amino acid sequence of the light chain variable region of the antibody is as set in SEQ ID NO: 73 1-108 bits;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124-239 of SEQ ID NO: 75; or the amino acid sequence of the light chain variable region of the antibody is as set forth in SEQ ID NO: 1-108; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in positions 124 to 239 of SEQ ID NO: 77; or the amino acid sequence of the light chain variable region of the antibody is as described in SEQ ID NO: 77. 1-108 bits are shown.
  • the antibody is antibody (a); more preferably, the amino acid sequence of the heavy chain variable region of the antibody is as shown at positions 124-239 of SEQ ID NO: 13; The amino acid sequence of the light chain variable region of the antibody is shown in positions 1-108 of SEQ ID NO: 13.
  • nucleic acid encoding an antibody as described above is provided.
  • an expression vector comprising the nucleic acid.
  • the expression vector is a PH/DHFR vector.
  • a host cell comprising the nucleic acid to which the described expression vector or genome is integrated is provided.
  • the host cell is a eukaryotic host cell or a prokaryotic host cell; preferably a eukaryotic host cell, more preferably a Chinese hamster ovary cell (CHO).
  • an antibody for the preparation of a targeted drug, antibody drug conjugate or multifunctional, which specifically targets tumor cells expressing EGFRv ⁇ or overexpressed EGFR
  • a multifunctional immunoconjugate comprising: An antibody according to any of the preceding claims; and a functional molecule linked thereto (including covalently linked, coupled, attached, adsorbed); said functional molecule being selected from the group consisting of a molecule targeting a tumor surface marker, inhibiting tumor Molecules, molecules or detectable markers that target surface markers of immune cells.
  • the molecule that targets the tumor surface marker is an antibody or ligand that binds to a tumor surface marker; or the tumor suppressing molecule is an antitumor cytokine or an antitumor toxin;
  • 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 molecule that targets a surface marker of an immune cell is an antibody or a ligand that binds to a surface marker of an immune cell; preferably, the Immunocellular surface markers include, but are not limited to, CD3, CD16, CD28.
  • the molecule that targets a surface marker of an immune cell is an antibody that binds to a T cell surface marker, and the antibody of any of the foregoing A bispecific T cell engager (BiTE) in which T cells are involved is formed.
  • BiTE bispecific T cell engager
  • the antibody that binds to an immunocyte 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.
  • the anti-CD3 antibody is humanized, fully human, chimeric or murine.
  • the multifunctional immunoconjugate is a fusion polypeptide, and a linker (linker) is further included between the antibody of any of the foregoing and a functional molecule linked thereto.
  • the multifunctional immunoconjugate is administered in the form of a polypeptide or by means of gene administration.
  • nucleic acid encoding the multifunctional immunoconjugate is provided.
  • a chimeric antigen receptor comprising an antibody according to any of the preceding embodiments, which is expressed on the surface of an immune cell, said chimeric antigen receptor comprising a sequence of any of the foregoing:
  • the transmembrane region comprises a transmembrane region of CD8 or CD28.
  • the immune cells comprise: T lymphocytes, NK cells or NKT cells.
  • the chimeric antigen receptor comprises the following sequentially linked antibodies, a transmembrane region and an intracellular letter. Area:
  • An antibody according to any of the preceding a transmembrane region of a CD28 molecule, an intracellular signal region of a CD28 molecule, and a CD3 ⁇ ; or
  • the antibody of any of the foregoing 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: 36 or the amino acid sequence set forth at positions 285-601;
  • SEQ ID NO: 37 or the amino acid sequence set forth at positions 285-702; or
  • SEQ ID NO: 38 or the amino acid sequence set forth at positions 285-744;
  • SEQ ID NO: 39 or the amino acid sequence set forth at positions 285-749; or
  • SEQ ID NO: 40 or the amino acid sequence set forth at positions 285-791.
  • nucleic acid encoding a chimeric antigen receptor according to any of the preceding claims is provided.
  • nucleic acid encoding the chimeric antigen receptor has:
  • SEQ ID NO: 31 or the nucleotide sequence set forth in 966-1916; or
  • SEQ ID NO: 32 or the nucleotide sequence set forth in 966-2219; or
  • SEQ ID NO: 34 or the nucleotide sequence set forth in 966-2360; or
  • SEQ ID NO: 35 or the nucleotide sequence set forth in 966-2486.
  • an expression vector comprising the nucleic acid described above is provided.
  • the expression vector is derived from the lentiviral plasmid pWPT (or pWPT-eGFP).
  • a virus comprising the vector described above.
  • 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 which does not contain CD3 purine but contains the intracellular signal domain of CD28, the intracellular signal domain of CD137 or this a combination of the two.
  • 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 use of the genetically modified immune cell for the preparation of a tumor suppressing drug the tumor being a tumor that expresses EGFRv ⁇ or overexpresses EGFR.
  • composition comprising:
  • a chimeric antigen receptor according to any of the preceding claims or a nucleic acid encoding the chimeric antigen receptor;
  • a genetically modified immune cell as described above is provided.
  • Antibodies 7B3 and Y022 specifically bind to the antigens EGFRvIII and N1N2-806 (phage ELISA assay).
  • Figure 4 Purified electropherogram of three scFv-Fc fusion antibodies.
  • FACS detects the ability of single-chain antibody scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc to bind to cell surface EGFR.
  • FIG. 1 Schematic diagram of the structure of the pH-Y022/CD3 expression vector.
  • FIG. 8 FACS detects the antigen binding specificity of the Y022/CD3 single chain bifunctional antibody.
  • Figure 10 Schematic diagram of the joining sequence of each part of the chimeric antigen receptor.
  • the present inventors After intensive research and screening, the present inventors obtained an antibody which specifically recognizes EGFRvIII or overexpressed EGFR of a tumor cell, and hardly recognizes EGFR of a normal cell.
  • the antibodies of the present invention can be applied to the preparation of various targeted antitumor drugs as well as drugs for diagnosing tumors.
  • the present inventors further screened and amino acid mutations based on the previously obtained humanized antibodies, and found an anti-EGFR antibody capable of more specifically targeting tumor cell EGFR, which selectively binds to tumors overexpressing EGFR or EGFRvIII. And does not bind to normal cell EGFR.
  • 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 Frame area (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
  • FR Frame area
  • 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 described herein. If the variant is capable of compete with the parent antibody for specific binding to SEQ ID NO: 1, and its ability to recognize tumor cell EGFRvIII or overexpressed EGFR is close to the specific antibody provided in the examples of the invention.
  • 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 on a region other than the CDR regions of the antibody.
  • 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 Column.
  • 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 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 an immune effector cell allows the immune effector cell to have a highly specific cytotoxic effect on tumor cells expressing EGFRvIII or overexpressing EGFR.
  • 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 ⁇ , CD28, CD137, the intracellular signal region of the CD134 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 the practice of the present invention.
  • 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 (EGFR)-CAR.
  • the present invention also provides a vector comprising the above nucleic acid encoding a chimeric antigen receptor protein expressed on the surface of an immune effector cell.
  • the vector used in the invention is 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.
  • 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 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 single-chain antibody 7B3 is a humanized antibody fragment that specifically recognizes a cryptic epitope formed by amino acid sequences 287-302 of EGFR exposed in tumor cells ( 287 CGADSYEMEEDGVRKC 302 (SEQ ID NO: 1) ).
  • the nucleotide sequences of the VL and VH genes are obtained from the sequences SEQ ID NO: 14 and SEQ ID NO: 13 shown in Patent 201210094008.x, and are ligated in the order of VL 7B3 - linker - VH 7B3 .
  • Nucleotide sequence of single-chain antibody 7B3 (717 base pairs, SEQ ID NO: 2):
  • the amino acid sequence of the single-chain antibody 7B3 (239 amino acids, SEQ ID NO: 3; the scribed region is 7B3 VL CDR1, CDR2, CDR3, 7B3VH CDR1, CDR2, CDR3):
  • dNTPs dATP, dCTP, dGTP, and dTTP, 2 mM each
  • 2 ⁇ l of 25 mM MgSO4 2 ⁇ l of 25 mM MgSO4
  • KOD Plus 1 U of KOD Plus (purchased from Takara) were supplemented with water and the PCR procedure was started in a thermocycler. The reaction was first heated to 94 ° C for pre-denaturation for 5 minutes and then held for 25 cycles, each cycle being 94 ° C for 30 seconds, 56 ° C for 30 seconds and 68 ° C for 30 seconds. Finally, it was kept at 68 ° C for 10 minutes.
  • the first fragment was amplified using primers pC7B3fw (SEQ ID NO: 4, ATAACAGGCCCAGCCGGCCATGGATATTCAGATGACCCAGAG) and LR3re (SEQ ID NO: 5, CACTTTGGTGCCCTGGCCAAATGTMNNTGGGNNMN NMNNMNNCTGMNNGCAATAATAGGTCGCAAAATC), and the second fragment utilized primers LR3f2fw (SEQ ID NO: 6, ACATTTGGCCAGGGCACCAAAG) and pC7B3re (SEQ ID NO: 7, ATAAATGCGGCC GCGCTGCTCACGGTCAC).
  • the PCR product was expected to be identified by analytical agarose gel electrophoresis and purified from the sample using the Wizard SV Gel and PCR Clean-up kit (purchased from Promega). The two fragments were added to the second round of duplex PCR as a template in an equimolar ratio.
  • the reaction system still used the KOD Plus system mentioned above. The reaction was first heated to 94 ° C for 5 minutes, then kept for 10 cycles, each The cycle reaction conditions were 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 30 seconds. Finally, it was kept at 68 ° C for 10 minutes.
  • primers pC7B3fw and pC7B3re at a final concentration of 0.2 ⁇ M were directly added to the reaction system, and a PCR procedure was started.
  • the reaction was first heated to 94 ° C for pre-denaturation for 5 minutes and then held for 25 cycles, each cycle being 94 ° C for 30 seconds, 56 ° C for 30 seconds and 68 ° C for 30 seconds. Finally, it was kept at 68 ° C for 10 minutes.
  • the PCR product was expected to be separated by preparative agarose gel electrophoresis and purified by the Wizard SV Gel and PCR Clean-up kit according to the manufacturer's instructions.
  • the complete library DNA fragment contains sfiI and NotI restriction endonuclease recognition sites, restriction endonuclease sfiI/NotI (purchased from New England Biolabs) for restriction digestion, and inserted into the same double digestion.
  • the ligation product was used to separate DNA from the sample using the Wizard SV Gel and PCR Clean-up kit and desalted for electroporation.
  • an electroporation flask and an electroporator Gene Pulser II purchasedd from Bio-Rad
  • E. coli ER2738 purchased from New England Biolabs
  • a DNA fragment encoding a 7B3 mutant library two DNA fragments were obtained by PCR using plasmid pCantab 5E-7B3 as a template, and then spliced by bridge PCR. Specifically, the following procedure was used: for the synthesis of the gene, PCR reaction was carried out in a volume of 50 ⁇ l, each using the plasmid pCantab 5E-7B3 as a template, the final concentration of each primer was 0.2 ⁇ M, and 5 ⁇ l of 10 ⁇ KOD Plus buffer, 4 ⁇ l.
  • the dNTPs (dATP, dCTP, dGTP, and dTTP, 2 mM each), 2 ⁇ l of 25 mM MgSO 4 and 1 U of KOD Plus were filled in water and the PCR procedure was started in a thermocycler. The reaction was first heated to 94 ° C for pre-denaturation for 5 minutes and then held for 25 cycles, each cycle being 94 ° C for 30 seconds, 56 ° C for 30 seconds and 68 ° C for 30 seconds. Finally, it was kept at 68 ° C for 10 minutes.
  • the amplification of the first fragment utilizes primers HR3f1fw (SEQ ID NO: 8, TCGCAATTCCTTTAGTTGTTCC) and HR3f1re (SEQ ID NO: 9, CAGGGTGCCCTGGCCCCAGTAANNMNNMNNMNNMNNMNNMNNMNNMNNMNNGGCGCGCAATAATACAC), and the second fragment utilizes primers HR3f2fw (SEQ ID NO: 10, TACTGGGGCCAGGGCACCCTG) and HR3f2re ( SEQ ID NO: 11, GGAATAGGTGTATCACCGTACTCAG).
  • the PCR product is expected to be identified by analytical agarose gel electrophoresis and purified from the sample using the Wizard SV Gel and PCR Clean-up kit.
  • the two fragments were added to the second round of duplex PCR as a template in an equimolar ratio.
  • the reaction system still uses the above-mentioned KOD Plus system. In the absence of primers, the reaction is heated to 94 ° C for 5 minutes. It was then kept for 10 cycles, and the reaction conditions for each cycle were 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 30 seconds. Finally, it was kept at 68 ° C for 10 minutes.
  • the primer HR3f1fw with a final concentration of 0.2 ⁇ M was directly added to the reaction system. And HR3f2re, and start the PCR program.
  • the reaction was first heated to 94 ° C for pre-denaturation for 5 minutes and then held for 25 cycles, each cycle being 94 ° C for 30 seconds, 56 ° C for 30 seconds and 68 ° C for 30 seconds. Finally, it was kept at 68 ° C for 10 minutes.
  • the PCR product was expected to be separated by preparative agarose gel electrophoresis and purified by the Wizard SV Gel and PCR Clean-up kit according to the manufacturer's instructions.
  • the complete library DNA fragment contains sfiI and NotI restriction endonuclease recognition sites, restriction endonuclease sfiI/NotI for restriction digestion, and inserted into the same double-digested phagemid vector pCANTAB 5E. .
  • the ligation product was used to separate DNA from the sample using the Wizard SV Gel and PCR Clean-up kit and desalted for electroporation. During electroporation, the electroporation flask and electroporator Gene Pulser II were used to transform into the self-made competent E. coli ER2738. It was finally confirmed that a library containing 6.0 ⁇ 10 9 mutants was obtained.
  • the screening scheme was as follows: The above library was infected with the helper phage M13KO7 to obtain the corresponding phage library.
  • the phage library was incubated with biotinylated antigen EGFRvIII (purchased from Ruijin Bio) at room temperature for 2 hours, and then with blocking solution 2% (w/v) BSA (bovine serum albumin, purchased from Shanghai Biotech)
  • BSA bovine serum albumin, purchased from Shanghai Biotech
  • the blocked streptavidin magnetic beads MyOne C1 purchased from Invitrogen 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.
  • 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, 25 ⁇ l, 10 ⁇ l and 10 ⁇ l, respectively, and the concentration of biotin-labeled antigen EGFRvIII was 10 nM, 1 nM, 0.5 nM and 0.1 nM, respectively, and the number of washings of PBST was 10 times and 10, respectively. Times, 15 times and 20 times.
  • 50, 500 and 1000 fold excess of the non-biotinylated antigen EGFRvIII was added to compete for elution to remove mutants with weaker binding ability.
  • the precipitate was separated by centrifugation (15 minutes, 1600 ⁇ g, 4 ° C), and resuspended in 400 ml of 2YT / ampicillin / kanamycin The medium was shake cultured at 37 ° C and 200 rpm for 16 hours. Finally, the cells were separated by centrifugation (20 min, 5000 ⁇ g, 4 ° C) and discarded. The supernatant was filtered through a 0.45 ⁇ m filter, and then 1/4 volume of 20% (w/v) PEG 8000, 2.5 M NaCl solution was added. 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.
  • each single colony was inoculated with 300 ⁇ l of 2YT/ampicillin medium (containing 2% glucose) in a 96-well deep well culture plate, and cultured with shaking at 37 ° C and 250 rpm for 16 hours.
  • 20 ⁇ l of the culture was inoculated to 500 ⁇ l of 2YT/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 2YT medium, and 50 ⁇ l/well was added to the culture plate. Incubate at 37 ° C and 150 rpm for 30 minutes, then add 50 ⁇ l/well of the prepared kanamycin solution (take 180 ⁇ l of 50 mg/ml kanamycin, add to 15 ml of 2YT medium), and shake at 37 ° C and 250 rpm. Incubate 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).
  • Clones with strong antigen binding signals in the ELISA but no binding signals to BSA were selected for subsequent evaluation and sequencing analysis.
  • the antibody obtained from the light chain affinity matured library is then further combined with the antibody obtained from Example 2 by the heavy chain affinity maturation library on the light chain variable region and the heavy chain variable region sequence, and the obtained antibody is also capable of specificity.
  • the binding antigens EGFRvIII and N1N2-806 do not bind to the control proteins BSA and N1N2.
  • Y022 contains 12 amino acid mutation sites (S31V, V89N, A92E, Q93N, F94I, Y96L, S182Q, L222M, R224K, G225N, F226W, R227D). As shown in Figure 1, Y022 was able to specifically bind to the antigens EGFRvIII and N1N2-806 in a single phage ELISA assay, without binding to the control proteins BSA and N1N2.
  • Nucleotide sequence of single chain antibody Y022 (717 bases; SEQ ID NO: 12):
  • amino acid sequence of single chain antibody Y022 (239 amino acids; SEQ ID NO: 13):
  • positions 1-108 are light chains, and the light chain CDR1 sequence: HASQDINVNIG (SEQ ID NO: 41), CDR2 sequence: HGKNLED (SEQ ID NO: 42), CDR3 sequence: NQYENIPLT (SEQ ID NO: 43).
  • positions 124-239 are heavy chains, and the heavy chain CDR1 sequence: GYSITSDYAWN (SEQ ID NO: 44), CDR2 sequence: YISYRGRTQYNPSLKS (SEQ ID NO: 45), CDR3 sequence: MGKNWDY (SEQ ID NO: 46).
  • the nucleotide sequence of Y022 was contained in pCantab5E, which was called pCantab 5E-Y022 plasmid.
  • the inventors Using the same method as the production of antibody Y022, the inventors also obtained another 10 antibody clones with significantly improved affinity and stability, namely M14, M15, M25, M26, S7, S8, S17, S22, S23 and S29. .
  • the amino acid mutation sites contained in all single-chain antibodies are shown in Table 1 as compared to the parent antibody 7B3.
  • Nucleotide sequence of single chain antibody M14 (717 bases; SEQ ID NO: 58):
  • amino acid sequence of the single-chain antibody M14 (239 amino acids; SEQ ID NO: 59):
  • amino acid sequences of M14 light chain CDR1 HASQDINSNIG
  • CDR2 HGKNLED
  • CDR3 NQYENNPIT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTNYNPSLKS
  • CDR3 LGRGFRY
  • amino acid sequences of M15 light chain CDR1 HASQDINVNIG
  • CDR2 HGKNLED
  • CDR3 NQYENNPIT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTSYNPSLKS
  • CDR3 LGRGFRY
  • Nucleotide sequence of single chain antibody M25 (717 bases; SEQ ID NO: 62):
  • amino acid sequence of the single-chain antibody M25 (239 amino acids; SEQ ID NO: 63):
  • amino acid sequences of M25 light chain CDR1 HASQDINVNIG
  • CDR2 HGKNLED
  • CDR3 NQYENIPLT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTRYNPSLKS
  • CDR3 LGRGFRY
  • amino acid sequence of single-chain antibody M26 (239 amino acids; SEQ ID NO: 65):
  • amino acid sequences of M26 light chain CDR1 HASQDINVNIG
  • CDR2 HGKNLED
  • CDR3 NQYENIPLT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTQYNPSLKS
  • CDR3 LGRGFRY
  • amino acid sequences of S7 light chain CDR1 HSSQDINVNIG
  • CDR2 HGTNLED
  • CDR3 NQYENNPIT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTSYNPSLKS
  • CDR3 LGRGFRY
  • Nucleotide sequence of single chain antibody S8 (717 bases; SEQ ID NO: 68):
  • amino acid sequences of S8 light chain CDR1 HASQDINVNIG
  • CDR2 HGKNLED
  • CDR3 NQYENNPIT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTSYNPSLKS
  • CDR3 LGRGFRY
  • amino acid sequences of S17 light chain CDR1 HASQDINTNIG
  • CDR2 HGKNLED
  • CDR3 NQYENNPLT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTQYNPSLKS
  • CDR3 LGRGFRY
  • amino acid sequences of S22 light chain CDR1 HASQDINVNIG
  • CDR2 HGTNLED
  • CDR3 NQYENNPLT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTRYNPSLKS
  • CDR3 LGRGFRY
  • Nucleotide sequence of single chain antibody S23 (717 bases; SEQ ID NO: 74):
  • amino acid sequence of the single-chain antibody S23 (239 amino acids; SEQ ID NO: 75):
  • amino acid sequences of S23 light chain CDR1 HASQDINVNIG
  • CDR2 HGKNLEDG
  • CDR3 NQYENNPLT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTRYNPSLKS
  • CDR3 LGRGFRY
  • Nucleotide sequence of single chain antibody S29 (717 bases; SEQ ID NO: 76):
  • amino acid sequences of S23 light chain CDR1 HASQDINVNIG
  • CDR2 HGKNLED
  • CDR3 NQYENFPLT
  • heavy chain CDR1 GYSITSDYAWN
  • CDR2 YISYRGRTRYNPSLKS
  • CDR3 LGRGFRY
  • each antibody was inserted into the NdeI/XhoI site of the expression vector pET22B(+), and the antibody protein was recombinantly produced in E. coli BL21 (DE3) and purified by a nickel column using a carboxy-terminally fused 6 ⁇ histidine polypeptide. .
  • E. coli BL21 DE3
  • carboxy-terminally fused 6 ⁇ histidine polypeptide 5 ml of 2 x YT/ampicillin medium was inoculated with each single colony, and cultured at 37 ° C and 220 rpm for 16 hours with shaking.
  • the sample was shaken at room temperature for 30 minutes to completely dissolve the cell debris.
  • the inclusion body pellet was then collected by centrifugation (15 minutes, 10,000 x g, 4 °C), and 20 ml of denaturing buffer (50 mM PB, 300 mM NaCl, 8 M urea, 10 mM imidazole, pH 8.0) was added and shaken at room temperature for one hour.
  • the precipitate was removed by centrifugation (15 min, 10,000 x g, 4 ° C), and the supernatant was collected, and the protein was purified using a 5 ml HisTrap HP purification column (purchased from GE Healthcare).
  • the purity of the purified antibody protein was analyzed by SDS polyacrylamide gel electrophoresis, and the protein concentration was determined by the BCA method.
  • the binding activity of the antibody to the antigen EGFRvIII was determined by a concentration gradient ELISA assay.
  • the antigen EGFRvIII was diluted with 0.1 M NaHCO 3 (pH 9.6) coating, coated with 200 ng per well, 50 ⁇ l/well, coated overnight at 4 ° C, and PBST containing 2% (w/v) BSA at room temperature. Closed for 2 hours. The plate was then rinsed three times with PBST and removed.
  • HRP-labeled goat anti-mouse antibody purchased from Santa Cruz
  • PBST PBST
  • 100 ⁇ l was added to each well and incubated for 1 hour at 37 °C.
  • the wells were rinsed three times with PBST, then rinsed three times with PBS, finally added to TMB for 15 minutes, and the color reaction was stopped with 50 ⁇ l of 2M H 2 SO 4 per well, using an enzyme-linked immunosorbent assay (Bio-Rad) at 450 nm.
  • Bio-Rad enzyme-linked immunosorbent assay
  • the extinction value is measured.
  • the obtained absorbance intensity values were evaluated using Sigma Plot software to calculate the binding strength of the antibody.
  • the extinction values measured in each case were plotted against the corresponding antibody concentrations and the resulting curves were fitted using the following non-linear regression.
  • the binding/dissociation balance between the identified immobilized antigen and the antibody protein is:
  • x concentration of antibody protein
  • y concentration of the antigen/antibody complex (indirectly measured by the absorbance after the color reaction);
  • the scFv-Y022 fragment was amplified from the resulting clone using primer pairs V5-Y022-F (SEQ ID NO: 14, ACAGTGCTAGCAGATATTCAGATGACCCAG) and V5-Y022-R (SEQ ID NO: 15, AAGATAGGCGGCCGCGCTGCTCACGGTCACCAG) according to standard protocols; ScFv-806 was amplified from V5-806-F (SEQ ID NO: 16, ACAGTGCTAGCAGACATCCTGATGACCCAAT) and V5-806-R (SEQ ID NO: 17, AAGAATGCGGCCGCTGCAGAGACAGTGACCAG) with pH-806/CD3 (see 201210094008.X) as template.
  • V5-C225-F SEQ ID NO: 18, ACAGTGCTAGCAGACATCTTGCTGACTCAG
  • V5-C225-R SEQ ID NO: 19, AAGAATGCGGCCGCTGCAGAGACAGTGACCAG
  • C225 VL-linker-VH DNA fragment
  • the scFv-C225 fragment was amplified as a template; the amplified product was digested with NheI/NotI (purchased from NEB), and the vector plasmid pCMV-V5 was digested with NheI/NotI by T4 DNA ligase (purchased from NEB).
  • -Fc this vector is fused downstream of the multiple cloning site to express the Fc fragment of human antibody IgG1, hereinafter referred to as V5-Fc, purchased from Shanghai Ruijin Biotechnology Co., Ltd.
  • V5-Fc this vector is fused downstream of the multiple cloning site to express the Fc fragment of human antibody IgG1, hereinafter referred to as V5-Fc, purchased from Shanghai Ruijin Biotechnology Co., Ltd.
  • V5-scFv-Y022-Fc, V5-scFv-806-Fc and V5-scFv-C225-Fc eukaryotic expression plasmids were obtained, respectively.
  • 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 processed samples were affinity-purified by protein A (purchased from GE) affinity column to finally obtain purified antibody-Fc fusion proteins scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc, and the results were as follows.
  • Figure 4 shows.
  • FACS detects the binding ability of single-chain antibody scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc to cell surface EGFR
  • 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
  • test antibodies scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc were separately added, and PBS was used as a negative control, and the final concentration of the antibody was 20 ⁇ g/ml, and 100 ul was added per tube. Ice bath, 45 minutes.
  • the single-chain antibody Y022 of the present invention and U87-EGFR exogenously overexpressing EGFR construction method according to: Wang H., et al., Identification of an Exex 4-Deletion Variant of Epidermal Growth Factor Receptor With Increased Metastasis-Promoting Capacity. Neoplasia, 2011, 13, 461-471
  • U87-EGFRvIII overexpressing EGFRvIII construction method according to: WO/2011/035465
  • A431, CAL 27 and MDA- endogenously overexpressing EGFR MB-468 cells have different degrees of binding, especially with U87-EGFRvIII and A431 cells, but the binding ability is not as high as single-chain antibody 806.
  • the single-chain antibody C225 has a very strong binding ability to the above cells. These single chain antibodies have little binding to U87 cells.
  • both single-chain antibodies, Y022 and 806, were hardly able to bind to the glioma cell line U87 cells.
  • the single-chain antibody Y022 does not bind to normal prostate epithelial cells RWPE-1 and human primary keratinocytes K2, whereas single-chain antibody 806 binds to different degrees with both normal cells.
  • the single-chain antibody Y022 can specifically bind to tumor cells overexpressing EGFR and EGFRvIII, and hardly binds to normal cells expressing EGFR.
  • Example 7 Construction of an expression vector comprising a nucleotide sequence encoding a Y022/CD3 single-chain bifunctional antibody
  • the pCantab 5E-Y022 plasmid obtained in Example 3 was used as a template, and the primers were obtained by PCR primer amplification using the primers pH7B3f2_fw (SEQ ID NO: 20, GATATTCAGATGACCCAGAGCCCGAGCAG) and the reverse primer pH7B3f2_re (SEQ ID NO: 21, AATAGGATCCACCACCTCCGCTGCTCACGGTCAC). DNA fragment of Y022scFv. Another DNA fragment containing the pH vector signal peptide sequence was obtained by PCR using the pH-7B3/CD3 plasmid (see 201210094008.X Example 3 and Figure 2) as a template, using the forward primer pH7B3f1_fw (SEQ ID NO: 22).
  • DNA polymerase and forward primer pH7B3f1_fw and reverse primer pH7B3f2_re were added, and amplification was carried out for 30 cycles.
  • the amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 60 ° C, 40 s; extension: At 68 ° C, 140 s, 30 cycles were performed, then a total extension of 68 ° C, 10 min.
  • the amplified sequence was simultaneously digested with restriction endonuclease NheI/BamHI and double digested according to the reaction conditions recommended by the enzyme supplier (New England Biolabs, NEB).
  • the expression vector pH (see 201210094008.X Example 3 and Figure 2) was also digested with the restriction endonuclease NheI/BamHI. Then follow the recommendations recommended by the enzyme supplier (NEB) Conditions, the double-cut Y022scFv fragment and the pH vector fragment were ligated with T4 DNA ligase.
  • nucleotide sequence encoding the Y022 single-chain antibody polypeptide is cloned into a vector, and together with the nucleotide sequence encoding the CD3 single-chain antibody polypeptide already contained on the vector, can be transcribed into one mRNA, and finally translated into Y022/ CD3 single chain bifunctional antibody polypeptide.
  • the new plasmid was named pH-Y022/CD3, and its detailed structure is shown in Fig. 6.
  • Example 8 Expression and purification of single-chain bifunctional antibody Y022/CD3, pH-806/CD3 and pH-C225/CD3
  • the expression vectors pH-Y022/CD3, pH-806/CD3 and pH-C225/CD3 were transfected into Chinese hamster ovary (CHO) cells according to the instructions of FreeStyle MAX Reagent Transfection Reagent (from Invitrogen). then expression kit according OptiCHO TM proteins (from Invitrogen) and stable clones were screened. Stable clones of CHO cells transfected with one of the above expression vectors, respectively, were cultured in shake flasks at 37 ° C, 130 rpm for 7 days, using CD OptiCHO (from Gibco). The culture supernatant was obtained by centrifugation and then stored at -20 °C.
  • Protein purification was performed using a histidine affinity chromatography column (His Trap HP column, available from GE Healthcare) according to the method steps provided by the manufacturer. Specifically, the column was equilibrated with buffer A (20 mM sodium phosphate pH 7.4, 0.4 M NaCl), and then PBS was dialyzed, and the cell culture supernatant (500 mL supernatant) was added to the column (1 mL) at a flow rate of 3 ml/min. The column was then washed with 5 volumes of buffer A and 10 volumes of buffer A containing 50 mM imidazole to remove the heteroprotein. The bound protein of interest was eluted with the same buffer A supplemented with 250 mM imidazole. All purification steps were performed at 4 °C.
  • the purified single-chain bifunctional antibody was detected by reducing SDS-PAGE. As shown in Fig. 7, the molecular weight of these antibody molecules was about 60 kD, which was in accordance with the molecular weight of the single-chain bifunctional antibody calculated from the amino acid sequence.
  • the binding ability of the single-chain bifunctional antibody Y022/CD3 to EGFR was analyzed by a fluorescence activated cell sorter (FACS) (BD, FACSCalibur).
  • FACS fluorescence activated cell sorter
  • the tumor cells listed in Table 2 in the logarithmic growth phase were inoculated into a 6 cm dish, and the inoculated cell density was about 90%, and cultured overnight at 37 ° C in an incubator.
  • the cells were digested with 10 mM EDTA, and the cells were collected by centrifugation at 200 g x 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 bifunctional antibody Y022/CD3 of the present invention is capable of binding to U87-EGFR, U87-EGFRvIII and A431 cells, but hardly binds to U87 and human keratinocytes.
  • Y022/CD3 can also bind to human peripheral blood mononuclear cells (PBMC) or Jurkat cells (human peripheral blood leukemia T cells, positive for CD3 expression) as shown, suggesting that the bifunctional antibodies of the present invention can specifically CD3 antigen binding on the surface of T cells.
  • PBMC peripheral blood mononuclear cells
  • Jurkat cells human peripheral blood leukemia T cells, positive for CD3 expression
  • Expression plasmids were constructed according to the methods mentioned in Examples 7 and 8 (substituting Y022 in Examples 7 and 8 with other mutant forms) and expressing M14/CD3, M15/CD3, M25/CD3, M26 /CD3, S7/CD3, S8/CD3, S17/CD3, S22/CD3, S23/CD3, S29/CD3. According to the method of the present example, the binding ability of these antibodies to U87-EGFRvIII overexpressing EGFRvIII and CAL27 cells endogenously overexpressing EGFR was determined, respectively. The above antibodies were able to bind to both cells, and the mean fluorescence intensity (MFI) values are shown in Table 13.
  • MFI mean fluorescence intensity
  • Example 10 Biological Activity Analysis of Single-Chain Bifunctional Antibodies Such as Y022/CD3 - Cytotoxicity to Various Tumor Cells
  • PBMC Peripheral blood mononuclear cells
  • PBS phosphate buffered saline
  • RPMI 1640 complete medium Gibco
  • the mixed cell suspension was added to a 96-well plate at a volume of 75 ⁇ L/well. Then, each well was added with 25 ⁇ L of a ten-fold serial dilution of the following reagents from 1000 ng/mL to 0.1 ng/mL:
  • NGR/CD3 single-chain bifunctional antibody (negative control, NGR is a neovascular targeting peptide which does not have a cross-binding site with EGFR. It is prepared according to a conventional method).
  • CytoTox The non-radioactive cytotoxicity test is based on the colorimetric detection method and can replace the 51Cr 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 51Cr 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.
  • LDH lactate dehydrogenase
  • INT tetrazolium salt
  • the five EGFR-associated tumor cells listed in Table 3 below were used to separately analyze the bifunctional antibody Y022/CD3 of the present invention and the NGR/CD3 single-chain bifunctional antibody-mediated T cell tumor which is not associated with EGFR as a control. Killing ability.
  • the kill rate of tumor cells (ie, % cytotoxicity) is based on Non-radioactive cytotoxicity assays are calculated using the following formula provided in the G1780 product instruction manual:
  • Effective cell spontaneous refers to the spontaneous release of LDH by effector cells
  • Target cell spontaneous refers to the release of LDH produced by cells when they are not treated by other factors.
  • Maximum target cells is the release of LDH from complete lysis of target cells after treatment with 0.8% Triton X-100,
  • Target cell max-target cell spontaneous represents the release of LDH produced by complete lysis of cells after external treatment.
  • tumor cells expressing mutant EGFR and/or overexpressing EGFR are as U87-EGFRvIII, U87-EGFR and A431 are all specifically killed by T-specificity-specific antibody Y022/CD3-directed T cells.
  • the minimum specific cytotoxicity was 32.1%, and the maximum was 66.2%.
  • the cytotoxicity of Y022/CD3 to cells expressing low levels of EGFR and human primary keratinocytes was very low, 3.4% and 4.5%, respectively, which was significantly lower than that of the above-mentioned tumor cells expressing mutant EGFR and/or overexpressing EGFR. Cytotoxicity.
  • cytotoxic % results for Y022/CD3 and control antibody NGR/CD3 at various concentrations for each tumor are shown in Tables 4-8 below.
  • tumor cells expressing mutant EGFR and/or overexpressing EGFR are all bifunctionally specific antibodies M14/CD3, M15/CD3, M25/CD3, M26/CD3, S7/CD3, S8/CD3, S17/CD3, S22/CD3, S23/CD3, S29/CD3 antibody-directed T cells with different degrees of killing. There is almost no killing effect on U87 cells expressing low levels of EGFR.
  • Example 11 Construction of a lentiviral plasmid expressing a chimeric antigen receptor protein encoded by a nucleic acid of the present invention and viral packaging
  • the chimeric antigen receptor is constructed, and the joining sequence of each part of the chimeric antigen receptor exemplified in the present invention is shown in Table 9 and FIG.
  • CD28a represents the transmembrane region of the CD28 molecule and CD28b represents the intracellular signal region of the CD28 molecule.
  • a forward primer (SEQ ID NO: 24, a sequence comprising a partial CD8 signal peptide) and a reverse primer (SEQ ID NO: 25, a sequence comprising a partial CD8 hinge) were used as primer pairs.
  • Y022scFv was obtained by PCR amplification.
  • the nucleic acid sequences of the anti-EGFRvIII chimeric antigen receptor protein other than Y022scFv were obtained by PCR using the sequences SEQ ID NO: 26, 27, 28, 29 and 30 disclosed in Patent Application No. 201310164725.X, respectively.
  • the eGFP-F2A-CD8sp sequence was obtained by PCR amplification of the primer pair (SEQ ID NO: 26, 27) using the plasmid of SEQ ID NO: 27 contained in Patent Application No. 201310164725.X as a template.
  • CD8-CD3 ⁇ zeta ( ⁇ Z) was obtained by PCR amplification using the primer pair (SEQ ID NO: 28, 29) using the plasmid of SEQ ID NO: 26 in the patent 201310164725.X as a template.
  • CD8-CD3zeta (Z), CD8-CD137-CD3zeta (BBZ), CD28a-CD28b-CD3zeta (28Z) and CD28a-CD28b-CD137-CD3zeta (28BBZ) sequences are respectively claimed in patent 201310164725.X
  • the plasmid corresponding to SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30 was used as a template, which was obtained by PCR amplification using a primer pair (SEQ ID NO: 28, 30).
  • eGFP-F2A-CD8sp nucleic acid fragment obtained as described above, and the equimolar Y022scFv nucleic acid fragment and equimolar CD8-CD3 ⁇ zeta ( ⁇ Z) or CD8-CD3zeta (Z) or CD8-CD137-CD3zeta (BBZ) or CD28a-, respectively CD28b-CD3zeta (28Z) or CD28a-CD28b-CD137-CD3zeta (28BBZ) nucleic acid fragment, three-fragment splicing and PCR as shown in Figure 9, splicing conditions: pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s ; annealing: 60 ° C, 40 s; extension: 68 ° C, 140 s, 5 cycles, then total extension 68 ° C, 10 min, supplement DNA polymerase and forward primer (SEQ ID NO: 24) and reverse primer (CD8-
  • the vector system used for the lentiviral plasmid vector constructed below belongs to the third generation auto-inactivated lentiviral vector system, which has three plasmids, the coding protein Gag/Pol, and the packaging protein psPAX2 encoding Rev protein (purchased from addgene).
  • the envelope plasmid PMD2.G (purchased from addgene) encoding the VSV-G protein and the recombinant expression vector encoding the gene of interest CAR based on the empty vector pWPT-eGFP (purchased from addgene).
  • the promoter of elongation factor-1 ⁇ can regulate the expression of enhanced green fluorescent protein (eGFP) in empty vector.
  • eGFP enhanced green fluorescent protein
  • F2A food and mouth disease virus
  • F2A is a core sequence of 2A (or "self-cleaving polypeptide 2A") from foot-and-mouth disease virus. It has a "self-shearing" function of 2A and can achieve co-expression of upstream and downstream genes.
  • 2A has an effective and feasible strategy for constructing gene therapy polycistronic vectors due to its high shear efficiency, high balance of upstream and downstream gene expression and short self-sequence.
  • the sequence is used to achieve co-expression of the target gene with GFP or eGFP, and GFP is detected.
  • eGFP can detect the expression of CAR indirectly.
  • This example constructs a lentiviral expression vector in which eGFP linked to F2A is co-expressed with a specific CAR, collectively referred to as pWPT-eGFP-F2A-CAR.
  • the target gene eGFP-F2A-CAR obtained in the above step 2 (see 2 in Example 7, the component after F2A is abbreviated as CAR) is digested by MluI and SalI restriction enzymes, and ligated into the same double digestion.
  • a lentiviral vector expressing each chimeric antigen receptor is constructed.
  • the successfully constructed vector can be prepared for lentiviral packaging after MluI and SalI digestion and sequencing.
  • eGFP-F2A-CAR is transcribed into one mRNA, but is finally translated into two peptide chains, eGFP and anti-EGFRvIII chimeric antigen receptor, in which the anti-EGFRvIII chimeric antigen receptor will be localized under the guidance of CD8 ⁇ signal peptide.
  • CD8 ⁇ signal peptide On the cell membrane.
  • the obtained carrier containing each of the following CARs is as follows (the component following F2A can be simply referred to as CAR):
  • 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 (purchased from PAA) (purchased from PAA)
  • PEI polyethyleneimine, purchased from Polysciences
  • transfection complex 4.4 1.6 ml of the transfection complex was added dropwise to HEK-293T cells. After 4-5 h, the transfected 293T cells were exchanged with 2% FBS DMEM.
  • the transfection efficiency (ie, the proportion of cells showing green fluorescence) was observed on the next day of transfection, and the positive transfection efficiency of ⁇ 80% was successful in the transfection experiment.
  • the virus was collected by filtration using a 0.45 ⁇ m filter (purchased from Millipore), and then used.
  • 293T cells were seeded at 1 ⁇ 10 5 /mL in a 96-well culture plate, 100 ⁇ L/well, and cultured at 37 ° C, 5% CO 2 , and the culture solution was DMEM containing 10% fetal bovine serum.
  • 50 ⁇ L/well of the culture supernatant was discarded, 50 ⁇ L/well of the above fresh culture solution was added, and a polybrene having a final concentration of 6 ⁇ g/mL was incubated at 37 ° C, 5% CO 2 for 30 min.
  • the titers of the above-mentioned virus containing the mock empty vector control and each eGFP-F2A-CAR packaged by the PEI transfection method were all at a level of about 0.5 to 1 ⁇ 10 7 U/mL, and the virus titer measured after concentration was about It is 0.5 to 1 x 10 8 U/mL.
  • Example 12 recombinant lentivirus-infected T cells
  • Peripheral blood mononuclear cells were obtained from peripheral blood of healthy humans by density gradient centrifugation, and cultured in AIM-V lymphocyte medium (purchased from Invitrogen) at a density of about 2 ⁇ 10 6 /mL.
  • Magnetic beads coated with anti-CD3 and CD28 antibodies were added at a ratio of cells: magnetic beads to 1:1, and recombinant human IL-2 was added at a final concentration of 300 U/mL (purchased from Shanghai Huaxin Biotech Co., Ltd.).
  • the company was stimulated for 48 hours and then infected with T cells with the above recombinant lentivirus (MOI ⁇ 15).
  • Infected T cells were tested for expression of different chimeric antigen receptors by flow cytometry on day 8 of culture. Since eGFP was co-expressed with CAR, positive cells detecting eGFP were positive cells expressing chimeric antigen receptors. The positive rate of virus-infected T cells expressing different chimeric antigen receptors using uninfected T lymphocytes as a negative control is shown in Table 10. The positive rate results indicate that a certain positive rate of CAR T cells can be obtained by a method of lentivirus infection.
  • T cells After infecting the virus packed with different chimeric antigen receptors, T cells were subcultured, counted, and supplemented with IL-2 at a cell density of 5 ⁇ 10 5 /ml every other day (final concentration was 300U/ml), about 100-1000 times amplification on the 11th day of culture, indicating that T cells expressing different chimeric antigen receptors can be expanded in vitro, providing for subsequent in vitro toxicity tests and in vivo experiments. Guarantee.
  • the target cells were U87, U87-EGFR, U87-EGFRvIII, A431, CAL 27, MDA-MB-468, RWPE-1 cells and human primary keratinocytes K2 as shown in Table 5, respectively.
  • the effector cells were subjected to FACS for 12 days in vitro to detect chimeric antigen receptor-positive T lymphocytes (CAR T cells).
  • the effective target ratios were 3:1, 1:1 and 1:3, the target cell number was 10000/well, and each group had 5 replicate wells.
  • the detection time is 18h.
  • Each experimental group each target cell + CAR T lymphocytes expressing different chimeric antigen receptors;
  • Control group 1 maximum release of LDH from target cells
  • Control group 2 spontaneous release of LDH from target cells
  • 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 51Cr 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 Y022-28Z CAR T and Y022-28BBZ CAR T pairs expressing the chimeric antigen receptor of the present invention are higher than the 806-CAR T at different effect ratios.
  • Cells expressing EGFR and EGFRvIII have significant killing effects, and exhibit a potent target ratio that is more cytotoxic than a gradient-dependent immediate target ratio.
  • the target-to-dependent data further shows that the CAR T cells expressing the chimeric antigen receptor of the present invention have a specific cytotoxic effect on the high expression of EGFR and its variant cells.
  • Y022-CAR T has almost no killing effect on RWPE-1 cells that normally express EGFR and human primary keratinocytes K2.
  • chimeric antigen receptor Y022-28BBZ CAR T lymph The cytotoxicity of cells to RWPE-1 cells and human primary keratinocytes K2 was 12% and 2%, respectively.
  • the cytotoxicity of Y022-28Z CAR T lymphocytes to RWPE-1 cells and human primary keratinocytes K2 was 8%. And 3%.
  • 806-CAR T has different degrees of killing on both cells, and cytotoxicity of 806-28BBZ CAR T lymphocytes to RWPE-1 cells and human primary keratinocytes K2 is 25% and 22%, respectively.
  • the cytotoxicity of 806-28Z CAR T lymphocytes to RWPE-1 cells and human primary keratinocytes K2 was 15% and 13%, respectively.
  • CAR T which was transfected with a virus containing a mock plasmid (carrying scFv-Y022- ⁇ Z) as a negative control, showed very low cytotoxic effects on the above cell lines.
  • the chimeric antigen receptor Y022-CAR T constructed by a single-chain antibody against EGFR and its variants, It can selectively kill tumor cells that highly express EGFR and its variant (EGFRvIII), but has almost no killing effect on cells that normally express EGFR.
  • the third generation (Y022-28BBZ) CAR T is more toxic to target cells than the second generation (Y022-28Z) CART.

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Abstract

本发明提供了一种肿瘤特异性抗EGFR抗体及其应用。该抗体可用于制备靶向性抗肿瘤药物以及诊断肿瘤的药物。

Description

肿瘤特异性抗EGFR抗体及其应用 技术领域
本发明属于免疫性领域，更具体地，本发明涉及肿瘤特异性抗EGFR抗体及其应用。
背景技术
EGFR在很多肿瘤中过量表达或变异，如何选择性地识别这些过量表达或变异的EGFR无疑是一个很重要的科学问题。迄今为止，针对EGFR287-302表位的抗体被认为可以达到识别肿瘤表面过量表达EGFR、EGFRvIII、de4EGFR的目的，但却不识别正常细胞的EGFR。很遗憾的是针对该表位的抗体在临床实验中，还是会有病人出现皮疹等副作用(http://meetinglibrary.asco.org/content/115945-132)，表明仅仅针对该表位可能还是会识别正常细胞(如角质上皮细胞)的EGFR。
因此，如何筛选更高肿瘤特异性的抗EGFR抗体显得非常迫切。高度肿瘤特异的抗体，不管是用于肿瘤影像学诊断、个体化诊断或者是肿瘤靶向治疗等都具有非常大的潜在应用价值。
发明内容
本发明的目的在于提供肿瘤特异性抗EGFR抗体及其应用。
在本发明的第一方面，提供一种特异性识别肿瘤细胞表达的EGFRvШ或者过量表达的EGFR的抗体，该抗体具有轻链可变区和重链可变区，且，
其轻链可变区的CDR1具有选自下组的氨基酸序列：SEQ ID NO:41，SEQ ID NO:47，SEQ ID NO:55；
其轻链可变区的CDR2具有选自下组的氨基酸序列：SEQ ID NO:42，SEQ ID NO:53；
其轻链可变区的CDR3具有选自下组的氨基酸序列：SEQ ID NO:43，SEQ ID NO:48，SEQ ID NO:54，SEQ ID NO:56，SEQ ID NO:57；
其重链可变区的CDR1具有氨基酸序列：SEQ ID NO:44；
其重链可变区的CDR2具有选自下组的氨基酸序列：SEQ ID NO:45，SEQ ID NO:49，SEQ ID NO:51，SEQ ID NO:52；
其重链可变区的CDR3具有选自下组的氨基酸序列：SEQ ID NO:46，SEQ ID NO:50。
在一个优选例中，所述的抗体包括：
抗体(a)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:43所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:45所示的CDR2、SEQ ID NO:46所示的CDR3；
抗体(b)，其轻链可变区具有SEQ ID NO:47所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:48所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:49所示的CDR2、SEQ ID NO:50所示的CDR3；
抗体(c)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:48所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:51所示的CDR2、SEQ ID NO:50所示的CDR3；
抗体(d)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:43所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:52所示的CDR2、SEQ ID NO:50所示的CDR3；
抗体(e)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:43所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:45所示的CDR2、SEQ ID NO:50所示的CDR3；
抗体(f)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:53所示的CDR2、SEQ ID NO:54所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:51所示的CDR2、SEQ ID NO:50所示的CDR3；
抗体(g)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:54所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:51所示的CDR2、SEQ ID NO:50所示的CDR3；
抗体(h)，其轻链可变区具有SEQ ID NO:55所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:56所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:45所示的CDR2、SEQ ID NO:50所示的CDR3；
抗体(i)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:53所示的CDR2、SEQ ID NO:56所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:52所示的CDR2、SEQ ID NO:50所示的CDR3；
抗体(j)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:56所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:52所示的CDR2、SEQ ID NO:50所示的CDR3；
抗体(k)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:57所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:52所示的CDR2、SEQ ID NO:50所示的CDR3；或
抗体(l)，识别与(a)～(k)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。
在另一优选例中，所述的特异性识别肿瘤细胞表达的EGFRvШ或者过量表达的EGFR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。
在另一优选例中，所述的特异性识别肿瘤细胞表达的EGFRvШ或者过量表达的EGFR的抗体是人源化的，全人源的，嵌合的或鼠源的。
在另一优选例中，所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:13中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:13中第1-108位 所示；
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:59中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:59中第1-108位所示；
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:61中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:61中第1-108位所示；
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:63中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:63中第1-108位所示；
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:65中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:65中第1-108位所示；
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:67中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:67中第1-108位所示；
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:69中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:69中第1-108位所示；
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:71中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:71中第1-108位所示；
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:73中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:73中第1-108位所示；
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:75中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:75中第1-108位所示；或
所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:77中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:77中第1-108位所示。
在另一优选例中，所述的抗体是抗体(a)；更佳的，所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:13中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:13中第1-108位所示。
在本发明的另一方面，提供编码前面所述的抗体的核酸。
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。在另一优选例中，所述的表达载体是PH/DHFR载体。
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。在另一优选例中，所述的宿主细胞是真核宿主细胞或原核宿主细胞；较佳地位真核宿主细胞，更佳地为中国仓鼠卵巢细胞(CHO)。
在本发明的另一方面，提供前面任一所述的抗体的用途，用于制备特异性靶向表达EGFRvШ或过量表达的EGFR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EGFRvШ或过量表达的EGFR；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，提供一种多功能免疫辍合物，所述的多功能免疫辍合物包括： 前面任一所述的抗体；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，所述的靶向免疫细胞的表面标志物的分子是结合免疫细胞表面标志物的抗体或配体；较佳地，所述的免疫细胞表面标志物包括(但不限于)：CD3，CD16，CD28。
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，其与前面任一所述的抗体形成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，所述的结合免疫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，全人源的，嵌合的或鼠源的。
在另一优选例中，所述的多功能免疫辍合物是融合多肽，且，前面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(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的形式给药或采用基因给药的方式。
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，提供前面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EGFRvШ或过量表达的EGFR；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，提供包含前面任一所述的抗体的嵌合抗原受体，其表达于免疫细胞表面，所述的嵌合抗原受体包含顺序连接的：前面任一所述的抗体，跨膜区和胞内信号区；所述的胞内信号区选自：CD3ζ，FcεRIγ，CD27，CD28，CD137，CD134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，所述的免疫细胞包括：T淋巴细胞，NK细胞或NKT细胞。
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：
前面任一所述的抗体、CD8和CD3ζ；
前面任一所述的抗体、CD8、CD137和CD3ζ；
前面任一所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
前面任一所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
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:36或其中第285-601位所示的氨基酸序列；或
SEQ ID NO:37或其中第285-702位所示的氨基酸序列；或
SEQ ID NO:38或其中第285-744位所示的氨基酸序列；或
SEQ ID NO:39或其中第285-749位所示的氨基酸序列；或
SEQ ID NO:40或其中第285-791位所示的氨基酸序列。
在本发明的另一方面，提供编码前面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966-1916所述的核苷酸序列；或
SEQ ID NO:32或其中第966-2219所述的核苷酸序列；或
SEQ ID NO:33或其中第966-2345所述的核苷酸序列；或
SEQ ID NO:34或其中第966-2360所述的核苷酸序列；或
SEQ ID NO:35或其中第966-2486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，其包含前面所述的核酸。
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，所述的病毒包含前面所述载体。
前面任一所述的嵌合抗原受体或编码其的核酸、或含有该核酸的表达载体或病毒的用途，用于制备靶向表达EGFRvШ或者过量表达EGFR的肿瘤细胞的基因修饰的免疫细胞。
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。
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EGFRvШ或者过量表达EGFR的肿瘤。
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：
前面任一所述的抗体或编码该抗体的核酸；或
前面任一所述的免疫辍合物或编码该辍合物的核酸；或
前面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
前面任一所述的基因修饰的免疫细胞。
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、抗体7B3和Y022能特异性结合抗原EGFRvIII和N1N2-806(噬菌体ELISA实验)。
图2、抗体7B3对抗原EGFRvIII的结合曲线。
图3、抗体Y022对抗原EGFRvIII的结合曲线。
图4、三种scFv-Fc融合抗体的纯化电泳图。
图5、FACS检测单链抗体scFv-Y022-Fc，scFv-806-Fc以及scFv-C225-Fc与细胞表面EGFR的结合能力。
图6、pH-Y022/CD3表达载体结构示意图。
图7、单链双功能抗体Y022/CD3、806/CD3和C225/CD3的SDS-PAGE检测。
图8、FACS检测Y022/CD3单链双功能抗体的抗原结合特异性。
图9、单链双功能抗体的细胞毒性图。
图10、嵌合抗原受体各部分的连接顺序示意图。
具体实施方式
本发明人经过深入的研究筛选，获得了一种特异性识别肿瘤细胞EGFRvIII或过量表达的EGFR、而几乎不识别正常细胞EGFR的抗体。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。
抗EGFR抗体
本发明人在前期已经获得的人源化抗体基础上进一步进行筛选以及氨基酸突变，找到了一种能够更特异地针对肿瘤细胞EGFR的抗EGFR抗体，其选择性地结合过量表达EGFR或者EGFRvIII的肿瘤，而与正常细胞EGFR不结合。
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SEQ ID NO:1，且其识别肿瘤细胞EGFRvIII或过量表达的EGFR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。
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。
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达EGFRvIII或过量表达EGFR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，CD28，CD137，CD134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，在本发明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(EGFR)-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。
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。
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 PCTCN2016090892-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、单链抗体7B3亲和力成熟库的构建
单链抗体7B3是一个经过人源化改造的抗体片段，能特异性识别在肿瘤细胞中暴露的EGFR的第287-302位氨基酸序列形成的隐蔽表位(287CGADSYEMEEDGVRKC302(SEQ ID  NO:1))。其VL和VH基因的核苷酸序列得自专利201210094008.x中所示的序列SEQ ID NO:14和SEQ ID NO:13，并按照VL7B3-接头-VH7B3的顺序连接而成。
单链抗体7B3的核苷酸序列(717个碱基对，SEQ ID NO:2)：
Figure PCTCN2016090892-appb-000002
单链抗体7B3的氨基酸序列(239个氨基酸，SEQ ID NO:3；划线区依次为7B3VL CDR1、CDR2、CDR3，7B3VH CDR1、CDR2、CDR3)：
Figure PCTCN2016090892-appb-000003
为提高7B3单链抗体对EGFR结合能力，分别对其轻链CDR3和重链CDR3区域的部分氨基酸进行随机突变，并构建对应的亲和力成熟文库。
1、7B3轻链CDR3亲和力成熟库的构建
通过对7B3单链抗体进行序列比对和分析，7B3轻链第三个CDR区域的部分氨基酸被选定，通过引物引入随机化突变，用于构建轻链亲和力成熟文库。
为制备编码7B3突变体文库的DNA片段，以质粒pCantab 5E-7B3(将7B3插入到pCantab5E-7B3的sfiI/NotI位点中)为模板，通过PCR方法分别得到两段DNA片段，随后通过搭桥PCR的方式拼接而成。具体使用以下操作步骤：为合成基因，在50μl体积重进行PCR反应，每个反应使用质粒pCantab 5E-7B3作为模板，每条引物的终浓度为0.2μM，以及5μl 10×KOD Plus缓冲液、4μl dNTPs(dATP、dCTP、dGTP和dTTP，每种2mM)、2μl 25mM MgSO4和1U KOD Plus(购自Takara)，用水补足体积后，在热循环仪中开始PCR程序。反应先加热样品到94℃预变性5分钟，然后保温25个循环，每个循环是94℃30秒、56℃30秒和68℃30秒。最后在68℃保温10分钟。第一个片段的扩增利用引物pC7B3fw(SEQ ID NO:4，ATAACAGGCCCAGCCGGCCATGGATATTCAGATGACCCAGAG)和LR3re(SEQ ID NO:5，CACTTTGGTGCCCTGGCCAAATGTMNNTGGGNNMN NMNNMNNCTGMNNGCAATAATAGGTCGCAAAATC)，第二个片段利用引物LR3f2fw(SEQ ID NO:6，ACATTTGGCCAGGGCACCAAAG)和pC7B3re(SEQ ID NO:7，ATAAATGCGGCC GCGCTGCTCACGGTCAC)。
预期PCR产物通过分析琼脂糖凝胶电泳鉴定，并用Wizard SV Gel and PCR Clean-up试剂盒(购自Promega)从样品中纯化回收。两个片段以等摩尔比加入到第二轮搭桥PCR中作为模板，反应体系仍然使用上述提到的KOD Plus体系，反应先加热样品到94℃预变性5分钟，然后保温10个循环，每个循环反应条件是94℃30秒、60℃30秒和68℃30秒。最后在68℃保温10分钟。随后在反应体系中直接加入终浓度为0.2μM的引物pC7B3fw和pC7B3re，并开始PCR程序。反应先加热样品到94℃预变性5分钟，然后保温25个循环，每个循环是94℃30秒、56℃30秒和68℃30秒。最后在68℃保温10分钟。预期PCR产物通过制备性琼脂糖凝胶电泳分离，并用Wizard SV Gel and PCR Clean-up试剂盒根据制造商的说明纯化回收。
完整的文库DNA片段两端分别含有sfiI和NotI限制性内切酶识别位点，经限制性内切酶sfiI/NotI(购自New England Biolabs)进行限制性消化，插入到经过同样双酶切的噬菌粒载体pCANTAB 5E中。连接产物使用Wizard SV Gel and PCR Clean-up试剂盒分离样品中的DNA并脱盐，用于电转化。在电转化时，使用电转化杯和电穿孔仪Gene Pulser II(购自Bio-Rad)，转化到自制的感受态大肠杆菌ER2738(购自New England Biolabs)。最终确认得到一个含有1.9x109个突变体的文库。
2、7B3重链CDR3亲和力成熟库的构建
通过对7B3单链抗体进行序列比对和分析，7B3重链第三个CDR区域的部分氨基酸被选定，通过引物引入随机化突变，用于构建重链亲和力成熟突变体文库。
为制备编码7B3突变体文库的DNA片段，以质粒pCantab 5E-7B3为模板，通过PCR方法分别得到两段DNA片段，随后通过搭桥PCR的方式拼接而成。具体使用以下操作步骤：为合成基因，在50μl体积中进行PCR反应，每个反应使用质粒pCantab 5E-7B3作为模板，每条引物的终浓度为0.2μM，以及5μl 10×KOD Plus缓冲液、4μl dNTPs(dATP、dCTP、dGTP和dTTP，每种2mM)、2μl 25mM MgSO4和1U KOD Plus，用水补足体积后，在热循环仪中开始PCR程序。反应先加热样品到94℃预变性5分钟，然后保温25个循环，每个循环是94℃30秒、56℃30秒和68℃30秒。最后在68℃保温10分钟。第一个片段的扩增利用引物HR3f1fw(SEQ ID NO:8，TCGCAATTCCTTTAGTTGTTCC)和HR3f1re(SEQ ID NO:9，CAGGGTGCCCTGGCCCCAGTAANNMNNMNNMNNMNNMNNGCGCGCGCAATAATACAC)，第二个片段利用引物HR3f2fw(SEQ ID NO:10，TACTGGGGCCAGGGCACCCTG)和HR3f2re(SEQ ID NO:11，GGAATAGGTGTATCACCGTACTCAG)。
预期PCR产物通过分析琼脂糖凝胶电泳鉴定，并用Wizard SV Gel and PCR Clean-up试剂盒从样品中纯化回收。两个片段以等摩尔比加入到第二轮搭桥PCR中作为模板，反应体系仍然使用上述提到的KOD Plus体系，在没有引物存在的情况下，反应先加热样品到94℃预变性5分钟，然后保温10个循环，每个循环反应条件是94℃30秒、60℃30秒和68℃30秒。最后在68℃保温10分钟。随后在反应体系中直接加入终浓度为0.2μM的引物HR3f1fw 和HR3f2re，并开始PCR程序。反应先加热样品到94℃预变性5分钟，然后保温25个循环，每个循环是94℃30秒、56℃30秒和68℃30秒。最后在68℃保温10分钟。预期PCR产物通过制备性琼脂糖凝胶电泳分离，并用Wizard SV Gel and PCR Clean-up试剂盒根据制造商的说明纯化回收。
完整的文库DNA片段两端分别含有sfiI和NotI限制性内切酶识别位点，经限制性内切酶sfiI/NotI进行限制性消化，插入到经过同样双酶切的噬菌粒载体pCANTAB 5E中。连接产物使用Wizard SV Gel and PCR Clean-up试剂盒分离样品中的DNA并脱盐，用于电转化。在电转化时，使用电转化杯和电穿孔仪Gene Pulser II，转化到自制的感受态大肠杆菌ER2738。最终确认得到一个含有6.0×109个突变体的文库。
实施例2、利用7B3亲和力成熟库针对EGFRvIII进行筛选
为得到更高亲和力的7B3突变体，分别利用轻链和重链突变体文库，各自实施了四轮筛选，筛选方案如下：上述文库经辅助噬菌体M13KO7感染，得到相应的噬菌体库。该噬菌体库与生物素标记的抗原EGFRvIII(购自锐劲生物)，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，购自上海生工)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。
在上述四轮筛选中，磁珠的用量分别为50μl、25μl、10μl和10μl，生物素标记的抗原EGFRvIII浓度分别为10nM、1nM、0.5nM和0.1nM，PBST的洗涤次数分别为10次、10次、15次和20次。从第二轮筛选开始，在洗脱前分别加入50倍、500倍和1000倍过量的未经生物素标记的抗原EGFRvIII用作竞争，以除去结合能力较弱的突变体。
为生产在表面展示了7B3单链抗体突变体的噬菌体，在400ml 2YT/氨苄青霉素培养基接种由实施例1中获得的甘油菌，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 2YT/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。
实施例3、特异性结合EGFRvIII的7B3突变体的鉴定
经过四轮针对抗原EGFRvIII的筛选，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)分析其和抗原EGFRvIII和N1N2-806(购自锐劲生物)的结合，其中N1N2-806是M13噬菌体PIII蛋白N1N2结构域和EGFR的第287-302位氨基酸的融合蛋白。为此目的，每个单菌落接种300μl 2YT/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 2YT/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 2YT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 2YT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EGFRvIII、N1N2-806以及阴性对照蛋白BSA和N1N2(购自锐劲生物)，50μl/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。
选取ELISA中对抗原结合信号较强，但是对BSA没有结合信号的克隆，用于后续评估和测序分析。随后将从轻链亲和力成熟文库中获得的抗体与从重链亲和力成熟文库中通过实施例2获得的抗体在轻链可变区和重链可变区序列上进一步组合，得到的抗体同样能够特异性的结合抗原EGFRvIII和N1N2-806，不与对照蛋白BSA和N1N2结合。
从抗体及抗原决定簇的晶体结构考虑，对7B3轻重链突变后的组合抗体与EGFR287-302的结合进行结构分析，最后选定一些氨基酸位点进行进一步突变，以获得更高亲和力和稳定性。所有发生改变的氨基酸位点包括位于轻链CDR1区域的S31，轻链CDR3区域的V89，A92，Q93，F94和Y96，重链CDR2区域的S182，重链CDR3区域的L222，R224，G225，F226和R227。在轻重链突变后的组合抗体序列基础上，进一步引入突变位点，得到了抗体Y022。同亲本抗体7B3相比，Y022包含12个氨基酸突变位点(S31V，V89N，A92E，Q93N，F94I，Y96L，S182Q，L222M，R224K，G225N，F226W，R227D)。如图1所示，在单噬菌体ELISA实验中，Y022能够特异性的结合抗原EGFRvIII和N1N2-806，不与对照蛋白BSA和N1N2结合。
单链抗体Y022的核苷酸序列(717个碱基；SEQ ID NO:12)：
Figure PCTCN2016090892-appb-000004
单链抗体Y022的氨基酸序列(239个氨基酸；SEQ ID NO:13)：
Figure PCTCN2016090892-appb-000005
其中第1-108位是轻链，且轻链CDR1序列：HASQDINVNIG(SEQ ID NO:41)，CDR2序列：HGKNLED(SEQ ID NO:42)，CDR3序列：NQYENIPLT(SEQ ID NO:43)。
其中第124-239位是重链，且重链CDR1序列：GYSITSDYAWN(SEQ ID NO:44)，CDR2序列：YISYRGRTQYNPSLKS(SEQ ID NO:45)，CDR3序列：MGKNWDY(SEQ ID NO:46)。
由于筛选自前面构建的突变体文库并进行定点突变，Y022的核苷酸序列包含于pCantab5E中，该质粒称为pCantab 5E-Y022质粒。
采用与产生抗体Y022相同的方法，本发明人还得到了另外10个亲和力和稳定性有显著改善的抗体克隆，分别是M14、M15、M25、M26、S7、S8、S17、S22、S23和S29。与亲本抗体7B3相比，所有单链抗体包含的氨基酸突变位点如表1所示。
表1
抗体 氨基酸突变位点
Y022 S31V，V89N，A92E，Q93N，F94I，Y96L，S182Q，L222M，R224K，G225N，F226W，R227D
M14 V89N，A92E，Q93N，F94N，Y96I，S182N
M15 S31V，V89N，A92E，Q93N，F94N，Y96I
M25 S31V，V89N，A92E，Q93N，F94I，Y96L，S182R
M26 S31V，V89N，A92E，Q93N，F94I，Y96L，S182Q
S7 S31V，K53T，V89N，A92E，Q93N，F94N，Y96I
S8 S31V，A44S，V89N，A92E，Q93N，F94N，Y96I
S17 S31T，V89N，A92E，Q93N，F94N，Y96L，S182Q
S22 S31V，K53T，V89N，A92E，Q93N，F94N，Y96L，S182R
S23 S31V，A44S，V89N，A92E，Q93N，F94N，Y96L，S182R
S29 S31V，V89N，A92E，Q93N，Y96L，S182R
单链抗体M14的核苷酸序列(717个碱基；SEQ ID NO:58)：
Figure PCTCN2016090892-appb-000006
单链抗体M14的氨基酸序列(239个氨基酸；SEQ ID NO:59)：
Figure PCTCN2016090892-appb-000007
M14轻链CDR1(HASQDINSNIG)、CDR2(HGKNLED)、CDR3(NQYENNPIT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTNYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:47，42，48，44，49，50。
单链抗体M15的核苷酸序列(717个碱基；SEQ ID NO:60)：
Figure PCTCN2016090892-appb-000008
单链抗体M15的氨基酸序列(239个氨基酸；SEQ ID NO:61)：
Figure PCTCN2016090892-appb-000009
Figure PCTCN2016090892-appb-000010
M15轻链CDR1(HASQDINVNIG)、CDR2(HGKNLED)、CDR3(NQYENNPIT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTSYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:41，42，48，44，51，50。
单链抗体M25的核苷酸序列(717个碱基；SEQ ID NO:62)：
Figure PCTCN2016090892-appb-000011
单链抗体M25的氨基酸序列(239个氨基酸；SEQ ID NO:63)：
Figure PCTCN2016090892-appb-000012
M25轻链CDR1(HASQDINVNIG)、CDR2(HGKNLED)、CDR3(NQYENIPLT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTRYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:41，42，43，44，52，50。
单链抗体M26的核苷酸序列(717个碱基；SEQ ID NO:64)：
Figure PCTCN2016090892-appb-000013
单链抗体M26的氨基酸序列(239个氨基酸；SEQ ID NO:65)：
Figure PCTCN2016090892-appb-000014
M26轻链CDR1(HASQDINVNIG)、CDR2(HGKNLED)、CDR3(NQYENIPLT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTQYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:41，42，43，44，45，50。
单链抗体S7的核苷酸序列(717个碱基；SEQ ID NO:66)：
Figure PCTCN2016090892-appb-000015
单链抗体S7的氨基酸序列(239个氨基酸；SEQ ID NO:67)：
Figure PCTCN2016090892-appb-000016
S7轻链CDR1(HASQDINVNIG)、CDR2(HGTNLED)、CDR3(NQYENNPIT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTSYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:41，53，54，44，51，50。
单链抗体S8的核苷酸序列(717个碱基；SEQ ID NO:68)：
Figure PCTCN2016090892-appb-000017
单链抗体S8的氨基酸序列(239个氨基酸；SEQ ID NO:69)：
Figure PCTCN2016090892-appb-000018
S8轻链CDR1(HASQDINVNIG)、CDR2(HGKNLED)、CDR3(NQYENNPIT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTSYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:41，42，54，44，51，50。
单链抗体S17的核苷酸序列(717个碱基；SEQ ID NO:70)：
Figure PCTCN2016090892-appb-000019
Figure PCTCN2016090892-appb-000020
单链抗体S17的氨基酸序列(239个氨基酸；SEQ ID NO:71)：
Figure PCTCN2016090892-appb-000021
S17轻链CDR1(HASQDINTNIG)、CDR2(HGKNLED)、CDR3(NQYENNPLT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTQYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:55，42，56，44，45，50。
单链抗体S22的核苷酸序列(717个碱基；SEQ ID NO:72)：
Figure PCTCN2016090892-appb-000022
单链抗体S22的氨基酸序列(239个氨基酸；SEQ ID NO:73)：
Figure PCTCN2016090892-appb-000023
S22轻链CDR1(HASQDINVNIG)、CDR2(HGTNLED)、CDR3(NQYENNPLT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTRYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:41，53，56，44，52，50。
单链抗体S23的核苷酸序列(717个碱基；SEQ ID NO:74)：
Figure PCTCN2016090892-appb-000024
Figure PCTCN2016090892-appb-000025
单链抗体S23的氨基酸序列(239个氨基酸；SEQ ID NO:75)：
Figure PCTCN2016090892-appb-000026
S23轻链CDR1(HASQDINVNIG)、CDR2(HGKNLEDG)、CDR3(NQYENNPLT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTRYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:41，42，56，44，52，50。
单链抗体S29的核苷酸序列(717个碱基；SEQ ID NO:76)：
Figure PCTCN2016090892-appb-000027
单链抗体S29的氨基酸序列(239个氨基酸；SEQ ID NO:77)：
Figure PCTCN2016090892-appb-000028
S23轻链CDR1(HASQDINVNIG)、CDR2(HGKNLED)、CDR3(NQYENFPLT)和重链CDR1(GYSITSDYAWN)、CDR2(YISYRGRTRYNPSLKS)、CDR3(LGRGFRY)的氨基酸序列分别为SEQ ID NO:41，42，57，44，52，50。
实施例4、抗体的表达和纯化
将各抗体的基因插入到表达载体pET22B(+)的NdeI/XhoI位点中，在E.coli BL21(DE3)重组制备抗体蛋白，并用羧基端融合的6×组氨酸多肽通过镍柱进行纯化。具体来说，为制备抗体蛋白，用各单菌落接种5ml 2×YT/氨苄青霉素培养基，并在37℃和220rpm下振荡培养16小时。用这种预培养物1ml接种100ml 2×YT/氨苄青霉素培养基，并在37℃和220rpm下振荡培养，直到细胞密度达到OD600＝0.5。加入1mMα-D-异丙基硫代半乳糖苷(IPTG)诱导外来基因表达后，继续在30℃和220rpm下振荡培养6小时。然后离心沉淀细胞(15分钟，3500×g，4℃)并重悬于35ml破碎缓冲液(50mM PB，300mM NaCl，2M尿素，0.5％Triton X-100，pH 8.0)。超声破碎后，样品放置于室温摇晃30分钟，彻底溶解细胞碎片。然后离心(15分钟，10,000×g，4℃)收集包涵体沉淀，加入20ml变性缓冲液(50mM PB，300mM NaCl，8M尿素，10mM咪唑，pH 8.0)，室温摇晃一小时。离心(15分钟，10,000×g，4℃)去除沉 淀，收集溶解上清，用5ml HisTrap HP纯化柱(购自GE Healthcare)纯化蛋白。通过SDS聚丙烯酰胺凝胶电泳分析纯化后抗体蛋白的纯度，并用BCA法测定蛋白浓度。
实施例5、抗体的结合活性测定
通过浓度梯度ELISA实验，测定抗体对抗原EGFRvIII的结合活性。为此目的，用0.1M NaHCO3(pH 9.6)包被液稀释抗原EGFRvIII，每孔包被200ng，50μl/孔，4℃包被过夜，并用含2％(w/v)BSA的PBST于室温下封闭2小时。然后用PBST漂洗平板三次并去除干净。随后，向每个孔板加入100μl含一系列浓度(起始浓度50ng/孔，18nM，直至稀释到1:81)的各抗体蛋白的PBST溶液，每个样品的测定使用平行三孔分析。37℃保温2小时后，用PBST漂洗三次，随后加入1:2000稀释的鼠抗His-tag抗体(购自Santa cruz)100μl/孔，37℃反应1小时。为检测结合的抗体，HRP标记的羊抗鼠抗体(购自Santa cruz)以1:15000稀释度稀释于PBST中，并向每个孔加入100μl，37℃孵育1小时。为了检测，用PBST漂洗孔三次，然后用PBS漂洗三次，最后加入TMB显示15分钟，用每孔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。用Sigma Plot软件评估所得到的吸光强度值，计算抗体的结合强度。为此目的，每种情况下测量的消光值对相应的抗体浓度作图，并用下面的非线性回归对所得曲线进行拟合。
Figure PCTCN2016090892-appb-000029
其中鉴定固定的抗原和抗体蛋白之间的结合/解离平衡是：
x＝抗体蛋白的浓度；
y＝抗原/抗体复合体的浓度(通过显色反应后的吸光值间接测量)；
a＝固定化抗原的总浓度；
b＝解离常数(KD)。
抗体7B3在浓度梯度ELISA实验中获得的结合曲线示例性表示于图2，其KD值约为22.4nM；抗体Y022对EGFRvIII的结合曲线如图3所示，其表观KD值约为2.7nM。
实施例6、Y022与细胞表面EGFR结合的活性分析
1、scFv-Y022-Fc，scFv-806-Fc及scFv-C225-Fc融合抗体的表达和纯化
根据标准方案，使用引物对V5-Y022-F(SEQ ID NO：14，ACAGTGCTAGCAGATATTCAGATGACCCAG)和V5-Y022-R(SEQ ID NO：15，AAGAATGCGGCCGCGCTGCTCACGGTCACCAG)从所得克隆中扩增出scFv-Y022片段；使用引物对V5-806-F(SEQ ID NO：16，ACAGTGCTAGCAGACATCCTGATGACCCAAT)和V5-806-R(SEQ ID NO：17，AAGAATGCGGCCGCTGCAGAGACAGTGACCAG)以pH-806/CD3(参见201210094008.X)为模板扩增出scFv-806片段；使用引物对V5-C225-F(SEQ ID NO：18，ACAGTGCTAGCAGACATCTTGCTGACTCAG)和V5-C225-R(SEQ ID NO：19，AAGAATGCGGCCGCTGCAGAGACAGTGACCAG)以C225(VL-linker-VH)DNA片段(序列 根据专利US20090099339A1中SEQ ID NO:10和SEQ ID NO:12确定，由上海锐劲生物技术有限公司通过全基因合成获得。)为模板扩增出scFv-C225片段；扩增产物通过NheI/NotI(购自NEB)双酶切，以T4DNA连接酶(购自NEB)于同样以NheI/NotI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-Y022-Fc、V5-scFv-806-Fc和V5-scFv-C225-Fc真核表达质粒。
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-Y022-Fc、scFv-806-Fc和scFv-C225-Fc，鉴定结果如图4所示。
2、FACS检测单链抗体scFv-Y022-Fc，scFv-806-Fc以及scFv-C225-Fc与细胞表面EGFR的结合能力
通过荧光激活细胞分选仪(FACS)(BD公司，FACS Calibur)分析单链抗体scFv-Y022-Fc，scFv-806-Fc以及scFv-C225-Fc各自与下列细胞系的结合能力。
具体方法如下：
1)取对数生长期的如表2所列各肿瘤细胞接种到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)分别加入待测抗体scFv-Y022-Fc，scFv-806-Fc和scFv-C225-Fc，同时以PBS作为阴性对照，抗体终浓度为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1:50稀释的FITC荧光标记的羊抗人抗体(来自上海康成生物工程有限公司)，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WinMDI 2.9分析数据。
表2
Figure PCTCN2016090892-appb-000030
Figure PCTCN2016090892-appb-000031
结果如图5所示，本发明的单链抗体Y022与外源过表达EGFR的U87-EGFR(构建方法根据：Wang H.，et al.，Identification of an Exon 4-Deletion Variant of Epidermal Growth Factor Receptor with Increased Metastasis-Promoting Capacity.Neoplasia，2011，13，461-471)及过表达EGFRvIII的U87-EGFRvIII(构建方法根据：WO/2011/035465)，内源过表达EGFR的A431，CAL 27以及MDA-MB-468细胞均有不同程度的结合，尤其与U87-EGFRvIII以及A431细胞结合较高，但结合能力均不如单链抗体806高。而单链抗体C225与上述细胞的结合能力均非常强。这些单链抗体与U87细胞几乎没有结合。
此外，Y022和806这两个单链抗体都几乎不能与脑胶质瘤细胞系U87细胞结合。尤为值得关注的是：单链抗体Y022与正常前列腺上皮细胞RWPE-1以及人原代角质细胞K2亦不结合，而单链抗体806与这两种正常细胞均有不同程度的结合。
这些结果表明，单链抗体Y022可以特异性地和过表达EGFR以及EGFRvIII的肿瘤细胞结合，而几乎不与表达EGFR的正常细胞结合。
实施例7、包含编码Y022/CD3单链双功能抗体的核苷酸序列的表达载体的构建
以实施例3获得的pCantab 5E-Y022质粒为模板，采用正向引物pH7B3f2_fw(SEQ ID NO:20，GATATTCAGATGACCCAGAGCCCGAGCAG)和反向引物pH7B3f2_re(SEQ ID NO:21，AATAGGATCCACCACCTCCGCTGCTCACGGTCAC)为引物对，PCR扩增获得Y022scFv的DNA片段。另外一个包含pH载体信号肽序列的DNA片段通过PCR的方式获得，以pH-7B3/CD3质粒(参见201210094008.X实施例3和图2)为模板，采用正向引物pH7B3f1_fw(SEQ ID NO:22，CCATTGACGCAAATGGGCGGTAGG)和反向引物pH7B3f1_re(SEQ ID NO:23，CTGCTCGGGCTCTGGGTCATCTGAATATC)。两个片段以等摩尔比混合，进行片段拼接并PCR，拼接条件为：预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行5个循环，然后总延伸68℃，10min。随后补充DNA聚合酶及正向引物pH7B3f1_fw和反向引物pH7B3f2_re，扩增30个循环，扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行30个循环，然后总延伸68℃，10min。
扩增得到的序列用限制性内切酶NheI/BamHI同时酶切，按照酶供应商(New England Biolabs，NEB)建议的反应条件进行双酶切。表达载体pH(参见201210094008.X实施例3和图2)也用限制性内切酶NheI/BamHI进行同样的酶切。然后按照酶供应商(NEB)建议的反应 条件，用T4DNA连接酶连接双酶切后的Y022scFv片段和pH载体片段。由此得到编码Y022单链抗体多肽的核苷酸序列被克隆到载体中，与载体上已经含有的编码CD3单链抗体多肽的核苷酸序列一起，可以转录为一条mRNA，最终翻译为Y022/CD3单链双功能抗体多肽。新质粒命名为pH-Y022/CD3，其详细结构如图6所示。
实施例8、单链双功能抗体Y022/CD3、pH-806/CD3和pH-C225/CD3的表达和纯化
表达载体pH-Y022/CD3、pH-806/CD3和pH-C225/CD3(参见201210094008.X)分别根据FreeStyle MAX Reagent转染试剂(来自Invitrogen)说明书操作步骤转染到中国仓鼠卵巢(CHO)细胞中，然后根据OptiCHOTM蛋白表达试剂盒(来自Invitrogen)筛选稳定的克隆。分别转染有上述表达载体之一的CHO细胞的稳定克隆在摇瓶中37℃，130rpm培养7天，所用培养基为CD OptiCHO(来自Gibco)。通过离心获得培养上清，然后储存于-20℃。
按照生产商提供的方法步骤，采用组氨酸亲和层析柱(His Trap HP column，购自GE Healthcare)进行蛋白纯化。具体而言，层析柱用缓冲液A(20mM sodium phosphate pH 7.4，0.4M NaCl)平衡，然后PBS透析后将细胞培养上清(500mL上清)加入到层析柱上(1mL)，流速为3ml/min。然后用5倍体积的缓冲液A和10倍体积的含有50mM咪唑的缓冲液A清洗柱子，以去除杂蛋白。结合的目的蛋白用添加250mM咪唑的同样缓冲液A进行洗脱。所有的纯化步骤都在4℃下操作。
纯化的单链双功能抗体通过还原性SDS-PAGE进行检测，如图7所示，这些抗体分子的分子量均在60kD左右，符合根据氨基酸序列计算得到的单链双功能抗体的分子量。
实施例9、Y022/CD3等单链双功能抗体的抗原结合特异性分析
通过荧光激活细胞分选仪(FACS)(BD公司，FACSCalibur)分析单链双功能抗体Y022/CD3与EGFR的结合能力。
具体方法如下：
1、取对数生长期的如上表2所列各肿瘤细胞接种到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、分别加入待测抗体Y022/CD3，同时以无关抗体NGR/CD3作为阴性对照，抗体终浓度为5μg/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1:50稀释的小鼠抗组氨酸标签抗体(来自上海睿星基因技术有限公司)，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，加入1:50稀释的FITC荧光标记的羊抗小鼠抗体(来自上海康成生物工程 有限公司)，每管加入100ul。冰浴，45分钟。
9、每管加入2ml 1％NBS PBS，以200g×5min离心，共二遍。
10、弃上清，重悬于300ul 1％NBS PBS中，流式细胞仪检测。
11、应用流式细胞仪数据分析软件WinMDI 2.9分析数据。
结果如图8所示，本发明的双功能抗体Y022/CD3能够与U87-EGFR，U87-EGFRvIII以及A431细胞结合，但几乎不能与U87以及人角质上皮细胞结合。这些结果表明，Y022/CD3可以特异性地和突变的人EGFR及过表达的EGFR的肿瘤细胞结合，而不与正常表达EGFR的组织结合。
此外，如图所示Y022/CD3也能和人外周血单核细胞(PBMC)或Jurkat细胞(人外周血白血病T细胞，CD3表达阳性)结合，提示本发明的双功能抗体能够特异性地与T细胞表面的CD3抗原结合。
根据实施例7和8中提到的方法分别构建表达质粒(将实施例7和8中的Y022替换为其它突变形式的抗体)并表达纯化了M14/CD3，M15/CD3，M25/CD3，M26/CD3，S7/CD3，S8/CD3，S17/CD3，S22/CD3，S23/CD3，S29/CD3。根据本实施例的方法，分别测定了这些抗体对过表达EGFRvIII的U87-EGFRvIII和内源过表达EGFR的CAL 27细胞的结合能力。以上抗体均能够结合这两种细胞，其平均荧光强度(MFI)值如表13所示。
表13
抗体 U87MG-EGFRvIII CAL 27
PBS 1 3.11
M14 36.52 28.39
M15 37.86 29.43
M25 36.52 24.14
M26 41.42 24.58
S7 42.17 27.88
S8 38.54 29.96
S17 31.62 25.03
S22 31.34 24.58
S23 32.2 29.69
S29 34.6 25.71
实施例10、Y022/CD3等单链双功能抗体的生物学活性分析-对各种肿瘤细胞的细胞毒性
外周血单核细胞(PBMC)用Ficoll(来自Biochrom)密度梯度离心方法，按照标准步骤从健康人供主的血液中分离。离心后，用浓度为0.1M的磷酸盐缓冲液(PBS)洗涤细胞然后重悬于RPMI 1640完全培养基(Gibco)，将细胞浓度调整到5×105/mL。PBMC用作细胞毒性实验中的效应细胞。不同的肿瘤细胞作为靶细胞(target cells)。用RPMI 1640完全培养基将靶细胞浓度调整到5×104/mL。同样体积的靶细胞和效应细胞混合，使效应细胞：靶细胞(E:T)比值 为10:1。
将混合后的细胞悬液以75μL/孔的体积加到96孔板中。然后各孔分别添加25μL从1000ng/mL到0.1ng/mL的十倍系列梯度稀释的下列试剂：
(1)Y022/CD3单链双功能抗体(BiTe)；
(2)RPMI 1640完全培养基(背景对照)；
(3)NGR/CD3单链双功能抗体(阴性对照，NGR为新生血管靶向肽，其与EGFR没有交叉结合位点。其根据常规方法制备)。
在37℃，5％CO2的培养箱中孵育40小时后，根据生产商的操作说明，用
Figure PCTCN2016090892-appb-000032
非放射性细胞毒性检测试剂盒(Non-Radioactive Cytotoxicity Assay kit，来自Promega)检测抗体的细胞毒作用。
CytoTox
Figure PCTCN2016090892-appb-000033
非放射性细胞毒性检测是基于比色法的检测方法，可替代51Cr释放法。CytoTox
Figure PCTCN2016090892-appb-000034
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。
如下表3所列举的5种与EGFR有关的肿瘤细胞被用来分别分析本发明双功能抗体Y022/CD3以及作为对照的与EGFR不相关的NGR/CD3单链双功能抗体介导的T细胞肿瘤杀伤能力。
肿瘤细胞的杀伤率(即，细胞毒性％)是根据
Figure PCTCN2016090892-appb-000035
非放射性细胞毒性检测G1780产品使用说明书提供的下列公式计算的：
Figure PCTCN2016090892-appb-000036
其中：
“实验”指的是加入抗体/效应细胞/靶细胞的实验孔所产生的LDH释放值，
“效应细胞自发”指的是效应细胞自发产生的LDH释放，
“靶细胞自发”是指细胞不受其他因素处理时产生的LDH释放，
“靶细胞最大”是用0.8％Triton X-100处理后靶细胞完全裂解所产生的LDH释放，
“靶细胞最大-靶细胞自发”代表着细胞受外界处理后完全裂解所产生的LDH释放。
表3
Figure PCTCN2016090892-appb-000037
上述表3的结果表明，表达突变的EGFR和/或过表达EGFR的肿瘤细胞如 U87-EGFRvIII，U87-EGFR以及A431，都会被双功能特异性抗体Y022/CD3导向的T细胞特异性杀伤。
具体而言，用Y022/CD3处理的上述肿瘤细胞组中，最小的特异性细胞毒性为32.1％，最大可达66.2％。而Y022/CD3对表达低水平EGFR的细胞U87以及人原代角质细胞的细胞毒性非常低，分别为3.4％和4.5％，显著低于对上述表达突变EGFR和/或过表达EGFR的肿瘤细胞的细胞毒性。
更具体的，Y022/CD3和对照抗体NGR/CD3在不同浓度下对各肿瘤的细胞毒性％结果如下列表4-8所示。
表4
Figure PCTCN2016090892-appb-000038
表5
Figure PCTCN2016090892-appb-000039
表6
Figure PCTCN2016090892-appb-000040
表7
Figure PCTCN2016090892-appb-000041
表8
Figure PCTCN2016090892-appb-000042
Figure PCTCN2016090892-appb-000043
另外，采用相同的方法分别对表达纯化的如下BiTe：M14/CD3，M15/CD3，M25/CD3，M26/CD3，S7/CD3，S8/CD3，S17/CD3，S22/CD3，S23/CD3，S29/CD3进行了体外毒性实验分析，结果如图9所示。
由图9可以看到，表达突变的EGFR和/或过表达EGFR的肿瘤细胞如U87-EGFRvIII，U87-EGFR以及CAL27，都会被双功能特异性抗体M14/CD3，M15/CD3，M25/CD3，M26/CD3，S7/CD3，S8/CD3，S17/CD3，S22/CD3，S23/CD3，S29/CD3抗体导向的T细胞不同程度的杀伤。而对表达低水平EGFR的细胞U87几乎没有产生杀伤作用。
实施例11、表达本发明核酸编码的嵌合抗原受体蛋白的慢病毒质粒的构建及病毒包装
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9和图10。
表9
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
Y022-δZ scFv(EGFR)-CD8-CD3δzeta 阴性对照
Y022-Z scFv(EGFR)-CD8-CD3zeta 第一代
Y022-BBZ scFv(EGFR)-CD8-CD137-CD3zeta 第二代
Y022-28Z scFv(EGFR)-CD28a-CD28b-CD3zeta 第二代
Y022-28BBZ scFv(EGFR)-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。
1、核酸片段的扩增
(1)scFv序列的扩增
以pCantab 5E-Y022质粒为模板，采用正向引物(SEQ ID NO:24，包含部分CD8信号肽的序列)和反向引物(SEQ ID NO:25，包含部分CD8绞链的序列)为引物对，PCR扩增获得Y022scFv。
Figure PCTCN2016090892-appb-000044
(2)嵌合抗原受体其他部分的核酸序列
抗EGFRvIII嵌合抗原受体蛋白的除Y022scFv外其它部分的核酸序列分别以专利申请号为201310164725.X中公开的序列SEQ ID NO:26，27，28，29和30为模板通过PCR方式获得。具体地，其中eGFP-F2A-CD8sp序列以专利申请号201310164725.X中所载的SEQ ID NO:27质粒为模板，以引物对(SEQ ID NO:26，27)进行PCR扩增获得。CD8-CD3δzeta(δZ)以申请专利201310164725.X中SEQ ID NO:26质粒为模板，采用引物对(SEQ ID NO:28，29)通过PCR扩增获得。CD8-CD3zeta(Z)、CD8-CD137-CD3zeta(BBZ)、CD28a-CD28b-CD3zeta(28Z)和CD28a-CD28b-CD137-CD3zeta(28BBZ)序列分别以申请专利201310164725.X中 SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30对应的质粒为模板，采用引物对(SEQ ID NO:28，30)通过PCR扩增获得。
Figure PCTCN2016090892-appb-000045
2、核酸片段的拼接
分别将如前述获得的eGFP-F2A-CD8sp核酸片段，与等摩尔的Y022scFv核酸片段以及等摩尔的CD8-CD3δzeta(δZ)或CD8-CD3zeta(Z)或CD8-CD137-CD3zeta(BBZ)或CD28a-CD28b-CD3zeta(28Z)或CD28a-CD28b-CD137-CD3zeta(28BBZ)核酸片段，按图9所示进行三片段拼接并PCR，拼接条件为：预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行5个循环，然后总延伸68℃，10min，补充DNA聚合酶及正向引物(SEQ ID NO:24)和反向引物(CD8-CD3δzeta对应的反向引物为SEQ ID NO:29，其余的为SEQ ID NO:30)后PCR扩增30个循环，扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行30个循环，然后总延伸68℃，10min。扩增获得的片段分别称为(表2)：
Figure PCTCN2016090892-appb-000046
3、慢病毒质粒载体的构建
作为示例，以下构建的慢病毒质粒载体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(购自addgene)；编码VSV-G蛋白的包膜质粒PMD2.G(购自addgene)及基于空载体pWPT-eGFP(购自addgene)的编码目的基因CAR的重组表达载体。
在空载体pWPT-eGFP中，自带的延长因子-1α(elongation factor-1α，EF-1α)的启动子可调控增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的表达，在空载体中插入本实施例前述构建的构建体后，形成编码目的基因CAR的重组表达载体，其中通过来自口蹄疫病毒的核糖体跳跃序列(food and mouth disease virus，FMDV，ribosomal skipping sequence，F2A)实现eGFP与目的基因CAR的共表达。F2A是来自口蹄疫病毒的2A(或称为“自剪切多肽2A”)的一段核心序列，具备2A的“自剪切”功能，可以实现上游和下游基因共表达。2A由于其剪切效率高、上下游基因表达平衡性高及自身序列短小的优点为构建基因治疗多顺反子载体提供了一种有效的可行策略。尤其在基于嵌合抗原受体基因修饰T淋巴细胞的免疫治疗中多应用该序列实现目的基因与GFP或者eGFP的共表达，通过检测GFP 或者eGFP即可间接检测CAR的表达。
本实施例构建了由F2A相连的eGFP与特异性CAR共表达的慢病毒表达载体，统称为pWPT-eGFP-F2A-CAR。上述步骤2中获得的目的基因eGFP-F2A-CAR(参见实施例7中的2，F2A后面的组件简称为CAR)通过MluI和SalI限制性内切酶双酶切，连入同样双酶切的pWPT载体中，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MluI和SalI酶切鉴定及序列测定正确后，可以准备用于慢病毒包装。如前所述，eGFP-F2A-CAR转录为一条mRNA，但最终翻译为eGFP和抗EGFRvIII嵌合抗原受体两条肽链，其中在CD8α信号肽的引导下抗EGFRvIII嵌合抗原受体将定位在细胞膜上。
得到的含有各目的CAR的载体如下(F2A后面的组件可简称为CAR)：
Figure PCTCN2016090892-appb-000047
通过以上构建，分别可获得五个eGFP-F2A-CAR多肽序列，称为：
Figure PCTCN2016090892-appb-000048
4、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(购自PAA公司)的DMEM(购自PAA公司)
转染的步骤如下：
4.1A液配制：将10μg mock对照或10μg的各目的基因质粒pWPT-eGFP-F2A-CAR，分别与7.5μg包装质粒PAX2：和3μg包膜质粒pMD2.G，溶入800μL的无血清DMEM培养液中，混匀。
4.2B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。
4.3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。
4.4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。
在转染次日观察转染效率(即呈绿色荧光的细胞比例)，～80％的阳性转染效率即为转染实验成功。在转染72h后，使用0.45μm滤器(购自Millipore公司)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。
5、测定包装有mock或者eGFP-F2A-CAR的慢病毒滴度
第一天，以1×105/mL接种293T细胞于96孔培养板，100μL/孔，37℃，5％CO2培养，培养液为含10％胎牛血清的DMEM。第二天，弃50μL/孔培养上清，补加50μL/孔新鲜上述培养液，并含终浓度为6μg/mL的polybrene，37℃，5％CO2孵育30min。加10μL/孔的病毒原液或1μL/孔的病毒浓缩液，3倍稀释，6个梯度，两个复孔，37℃，5％CO2培养。感染48h后，流式细胞仪检测eGFP，以阳性率为5～20％的细胞数为宜，计算滴度(U/mL)＝阳性率×稀释倍数×100×104。PEI转染法包装的上述包含mock即空载体对照和各eGFP-F2A-CAR的病毒的滴度均为约0.5～1×107U/mL的水平，经浓缩后所测的病毒滴度约为0.5～1×108U/mL。
实施例12、重组慢病毒感染T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)，以约2×106/mL密度加入AIM-V淋巴细胞培养基(购自Invitrogen公司)培养，并以细胞：磁珠比例为1:1加入包被有抗CD3和CD28抗体的磁珠(Invitrogen公司)，同时加入终浓度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48h，然后以用上述重组慢病毒(MOI≈15)感染T细胞。感染的T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，由于eGFP与CAR共表达，检测eGFP的阳性细胞即为表达嵌合抗原受体的阳性细胞。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T细胞其阳性率如表10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 T细胞。
表10
转染有下列CAR的T细胞 CAR T细胞eGFP阳性率
Y022-δZ(Mock) 66％
Y022-Z 58％
Y022-BBZ 53％
Y022-28Z 54％
Y022-28BBZ 52％
T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100～1000倍的扩增，表明表达不同嵌合抗原受体的T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13、CAR-Y022的体外抗肿瘤活性
体外毒性实验使用的材料如下：
靶细胞分别为如表5所示的U87，U87-EGFR，U87-EGFRvIII，A431，CAL 27，MDA-MB-468，RWPE-1细胞以及人原代角质细胞K2。效应细胞为体外培养12天的FACS检测嵌合抗原受体表达阳性的T淋巴细胞(CAR T细胞)。
效靶比分别为3:1，1:1和1:3，靶细胞数量为10000/孔，每组均设5个复孔。检测时间为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CAR T淋巴细胞；
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 PCTCN2016090892-appb-000049
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 PCTCN2016090892-appb-000050
具体如表11和表12所示，在不同效靶比的情况下，与806-CAR T相比，本发明的表达嵌合抗原受体的Y022-28Z CAR T和Y022-28BBZ CAR T对高表达EGFR以及EGFRvIII的细胞具有明显的杀伤作用，并呈现效靶比梯度依赖性即效靶比越高细胞毒性作用越强。效靶比依赖性的数据进一步显示表达本发明的嵌合抗原受体的CAR T细胞对高表达EGFR及其变异体细胞的特异性细胞毒作用。
值得关注的是，Y022-CAR T对正常表达EGFR的RWPE-1细胞以及人原代角质细胞K2几乎没有杀伤作用，在效靶比3：1时，嵌合抗原受体Y022-28BBZ CAR T淋巴细胞对RWPE-1细胞以及人原代角质细胞K2的细胞毒性分别为12％和2％，Y022-28Z CAR T淋巴细胞对RWPE-1细胞以及人原代角质细胞K2的细胞毒性分别为8％和3％。相比较而言，806-CAR T对这两种细胞均有不同程度的杀伤，806-28BBZ CAR T淋巴细胞对RWPE-1细胞以及人原代角质细胞K2的细胞毒性分别为25％和22％，806-28Z CAR T淋巴细胞对RWPE-1细胞以及人原代角质细胞K2的细胞毒性分别为15％和13％。
另外，被含有mock质粒(携带scFv-Y022-δZ)的病毒转染的作为阴性对照的CAR T显示对上述细胞系的细胞毒性作用均非常低。
以上结果表明，针对EGFR及其变异体的单链抗体所构建的嵌合抗原受体Y022-CAR T， 能够选择性地杀伤高表达EGFR及其变异体(EGFRvIII)的肿瘤细胞，而对正常表达EGFR的细胞几乎没有杀伤作用。此外，从细胞毒性数据来看，第三代(Y022-28BBZ)的CAR T比第二代(Y022-28Z)的CART对靶细胞的杀伤毒性更强。
表11
Figure PCTCN2016090892-appb-000051
表12
Figure PCTCN2016090892-appb-000052
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 (36)

  1. 一种特异性识别肿瘤细胞表达的EGFRvШ或者过量表达的EGFR的抗体，其特征在于，该抗体具有轻链可变区和重链可变区，且，
    其轻链可变区的CDR1具有选自下组的氨基酸序列：SEQ ID NO:41，SEQ ID NO:47，SEQ ID NO:55；
    其轻链可变区的CDR2具有选自下组的氨基酸序列：SEQ ID NO:42，SEQ ID NO:53；
    其轻链可变区的CDR3具有选自下组的氨基酸序列：SEQ ID NO:43，SEQ ID NO:48，SEQ ID NO:54，SEQ ID NO:56，SEQ ID NO:57；
    其重链可变区的CDR1具有氨基酸序列：SEQ ID NO:44；
    其重链可变区的CDR2具有选自下组的氨基酸序列：SEQ ID NO:45，SEQ ID NO:49，SEQ ID NO:51，SEQ ID NO:52；
    其重链可变区的CDR3具有选自下组的氨基酸序列：SEQ ID NO:46，SEQ ID NO:50。
  2. 如权利要求1所述的抗体，其特征在于，所述的抗体包括：
    抗体(a)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:43所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:45所示的CDR2、SEQ ID NO:46所示的CDR3；
    抗体(b)，其轻链可变区具有SEQ ID NO:47所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:48所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:49所示的CDR2、SEQ ID NO:50所示的CDR3；
    抗体(c)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:48所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:51所示的CDR2、SEQ ID NO:50所示的CDR3；
    抗体(d)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:43所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:52所示的CDR2、SEQ ID NO:50所示的CDR3；
    抗体(e)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:43所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:45所示的CDR2、SEQ ID NO:50所示的CDR3；
    抗体(f)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:53所示的CDR2、SEQ ID NO:54所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:51所示的CDR2、SEQ ID NO:50所示的CDR3；
    抗体(g)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:54所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:51所示的CDR2、SEQ ID NO:50所示的CDR3；
    抗体(h)，其轻链可变区具有SEQ ID NO:55所示的CDR1、SEQ ID NO:42所示的CDR2、 SEQ ID NO:56所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:45所示的CDR2、SEQ ID NO:50所示的CDR3；
    抗体(i)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:53所示的CDR2、SEQ ID NO:56所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:52所示的CDR2、SEQ ID NO:50所示的CDR3；
    抗体(j)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:56所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:52所示的CDR2、SEQ ID NO:50所示的CDR3；
    抗体(k)，其轻链可变区具有SEQ ID NO:41所示的CDR1、SEQ ID NO:42所示的CDR2、SEQ ID NO:57所示的CDR3；或其重链可变区具有SEQ ID NO:44所示的CDR1、SEQ ID NO:52所示的CDR2、SEQ ID NO:50所示的CDR3；或
    抗体(l)，识别与(a)～(k)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。
  3. 如权利要求2所述的抗体，其特征在于，所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:13中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:13中第1-108位所示；
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:59中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:59中第1-108位所示；
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:61中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:61中第1-108位所示；
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:63中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:63中第1-108位所示；
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:65中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:65中第1-108位所示；
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:67中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:67中第1-108位所示；
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:69中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:69中第1-108位所示；
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:71中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:71中第1-108位所示；
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:73中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:73中第1-108位所示；
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:75中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:75中第1-108位所示；或
    所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:77中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:77中第1-108位所示。
  4. 如权利要求2所述的抗体，其特征在于，所述的抗体是抗体(a)；更佳的，所述的抗体的重链可变区的氨基酸序列如SEQ ID NO:13中第124-239位所示；或所述的抗体的轻链可变区的氨基酸序列如SEQ ID NO:13中第1-108位所示。
  5. 编码权利要求1-4任一所述的抗体的核酸。
  6. 一种表达载体，其包含权利要求5所述的核酸。
  7. 一种宿主细胞，其包含权利要求6所述的表达载体或基因组中整合有权利要求5所述的核酸。
  8. 权利要求1-4任一所述的抗体的用途，用于制备特异性靶向表达EGFRvШ或过量表达的EGFR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EGFRvШ或过量表达的EGFR；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。
  9. 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1-4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。
  10. 如权利要求9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。
  11. 如权利要求9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。
  12. 如权利要求9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免疫细胞表面标志物的抗体或配体；较佳地，所述的免疫细胞表面标志物包括：CD3，CD16，CD28。
  13. 如权利要求12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1-4任一所述的抗体形成T细胞参与的双功能抗体。
  14. 如权利要求13所述的多功能免疫辍合物，其特征在于，所述的结合免疫细胞表面标志物的抗体是抗CD3抗体。
  15. 如权利要求9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1-4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。
  16. 编码权利要求9-15任一所述的多功能免疫辍合物的核酸。
  17. 权利要求9-15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EGFRvШ或过量表达的EGFR；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。
  18. 包含权利要求1-4任一所述的抗体的嵌合抗原受体，其表达于免疫细胞表面，其特征在于，所述的嵌合抗原受体包含顺序连接的：权利要求1-4任一所述的抗体，跨膜区和胞内信号区。
  19. 如权利要求18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的胞内信号区序列，或其组合。
  20. 如权利要求19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。
  21. 如权利要求所述19的嵌合抗原受体，其特征在于，所述的免疫细胞包括：T淋巴细胞，NK细胞或NKT细胞。
  22. 如权利要求19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1-4任一所述的抗体、CD8和CD3ζ；
    权利要求1-4任一所述的抗体、CD8、CD137和CD3ζ；
    权利要求1-4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1-4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。
  23. 如权利要求19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。
  24. 如权利要求19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:36或其中第285-601位所示的氨基酸序列；或
    SEQ ID NO:37或其中第285-702位所示的氨基酸序列；或
    SEQ ID NO:38或其中第285-744位所示的氨基酸序列；或
    SEQ ID NO:39或其中第285-749位所示的氨基酸序列；或
    SEQ ID NO:40或其中第285-791位所示的氨基酸序列。
  25. 编码权利要求18-24任一所述的嵌合抗原受体的核酸。
  26. 一种表达载体，其特征在于，其包含权利要求25所述的核酸。
  27. 一种病毒，其特征在于，所述的病毒包含权利要求26所述载体。
  28. 权利要求18-24任一所述的嵌合抗原受体、或权利要求25所述的核酸、或权利要求26所述的表达载体、或权利要求27所述的病毒的用途，用于制备靶向表达EGFRvШ或者过量表达EGFR的肿瘤细胞的基因修饰的免疫细胞。
  29. 一种基因修饰的免疫细胞，其特征在于，其转导有权利要求25所述的核酸，或权利要求26所述的表达载体或权利要求27所述的病毒；或
    其表面表达权利要求18-24任一所述的嵌合抗原受体。
  30. 如权利要求29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。
  31. 如利要求29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。
  32. 如权利要求29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。
  33. 如权利要求29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。
  34. 如权利要求29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。
  35. 权利要求29-34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EGFRvШ或者过量表达EGFR的肿瘤。
  36. 药物组合物，其特征在于，其包括：
    权利要求1-4任一所述的抗体或编码该抗体的核酸；或
    权利要求9-15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18-24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29-34任一所述的基因修饰的免疫细胞。
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CA2993262A CA2993262A1 (en) 2015-07-21 2016-07-21 Tumor-specific anti-egfr antibody and application thereof
RU2018104703A RU2730605C2 (ru) 2015-07-21 2016-07-21 Опухолеспецифичное антитело против egfr и его применение
SG11201801003YA SG11201801003YA (en) 2015-07-21 2016-07-21 Tumor-specific anti-egfr antibody and application thereof
ES16827258T ES2881807T3 (es) 2015-07-21 2016-07-21 Anticuerpo anti-EGFR específico de tumor y aplicación del mismo
JP2018522842A JP2018528786A (ja) 2015-07-21 2016-07-21 腫瘍特異的抗ｅｇｆｒ抗体およびその使用
US15/746,711 US10927176B2 (en) 2015-07-21 2016-07-21 Tumor-specific anti-EGFR antibody and application thereof
AU2016295601A AU2016295601B2 (en) 2015-07-21 2016-07-21 Tumor-specific anti-EGFR antibody and application thereof
EP16827258.1A EP3327036B1 (en) 2015-07-21 2016-07-21 Tumor-specific anti-egfr antibody and application thereof
DK16827258.1T DK3327036T3 (da) 2015-07-21 2016-07-21 Tumor-specifikt anti-EGFR-antistof samt anvendelse deraf
HK18110747.5A HK1251240A1 (zh) 2015-07-21 2018-08-21 腫瘤特異性抗egfr抗體及其應用
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