WO2015172341A1 - 针对磷脂酰肌醇蛋白多糖-3和t细胞抗原的双特异性抗体 - Google Patents

针对磷脂酰肌醇蛋白多糖-3和t细胞抗原的双特异性抗体 Download PDF

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WO2015172341A1
WO2015172341A1 PCT/CN2014/077521 CN2014077521W WO2015172341A1 WO 2015172341 A1 WO2015172341 A1 WO 2015172341A1 CN 2014077521 W CN2014077521 W CN 2014077521W WO 2015172341 A1 WO2015172341 A1 WO 2015172341A1
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antibody
gpc3
cell
cells
amino acid
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WO2015172341A8 (zh
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李宗海
王华茂
蒋华
石必枝
王红阳
杨胜利
顾健人
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上海市肿瘤研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to tumor-associated antibodies, and more particularly to the field of genetically engineered antibodies. Background technique
  • Phosphatidylinositol-3 (Glypi can-3, GPC3, also known as DGSX, GTR2-2, MXR7, 0CI-5, SDYS, SGB, SGBS or SGBS 1) is a cell surface protein belonging to heparan sulfate Proteoglycan family.
  • the GPC3 gene encodes a precursor core protein of about 70-kDa, which can be cleaved by furin to produce a soluble amino terminal peptide capable of secreting into the blood around 40-kDa, and containing about 30-kDa.
  • GPC3 protein is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • GPC3 is highly expressed in the fetal liver and not in the liver tissue of normal adults. However, the expression is restored in hepatocellular carcinoma, which is closely related to the occurrence and development of liver cancer. Not only is the detection rate high in the early stage of liver cancer, but also the detection rate increases with the development of liver cancer. GPC3 expression was not detected in hepatic adenocarcinoma, cholangiocarcinoma, liver metastases, and 12 common solid tumors and 21 non-hepatoma cell lines. In addition, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, and neuroblastoma. Considering the high expression of GPC3 in hepatocellular carcinoma, melanoma and other tumors, GPC3 is considered to be a candidate for tumor immunotherapy.
  • anti-GPC3 antibodies for the detection of liver cancer and the use of anti-GPC3 antibodies for antibody-dependent (ADCC) or complement-dependent (CDC) cytotoxicity have been reported, for example, CN101 186650A (Xiwai Pharmaceutical Co., Ltd.), the invention name is "anti- An antibody against a secreted N-terminal peptide or a C-terminal peptide of GPC3 present in the blood"; CN102180969A (Microbial Epidemiology Institute of the Chinese Academy of Military Medical Sciences), the invention name is "anti-hepatocarcinoma active monoclonal antibody and Application ", which relates to an anti-GPC3 monoclonal antibody prepared by using amino acid residues 359-580 of amino terminus of GPC3 as an antigen; CN101633693A (Second Military Medical University of Chinese People's Liberation Army), which relates to a monoclonal antibody against an amino terminal antigen of GPC3; CN101052878A (Invit
  • Table 1 summarizes additional monoclonal antibodies (mAbs) against GP C3 reported since 2003.
  • mAbs monoclonal antibodies
  • a therapeutic mAb against GPC3 has recently been reported (Advances in Liver Cancer Antibody Therapies: A Focus on Glypi can-3 and Mesothel in, BioDrugs. 201 1 October 1 ; 25 (5): 275 - 284.), It is directed against an antigenic determinant of GPC3 located at amino acid residues 524-563 of the C-terminus.
  • monoclonal antibody GC33 (IgG2a, ⁇ ) induces antibody-dependent cytotoxicity (ADCC) and inhibits tumor growth of subcutaneously transplanted HepG2 and HuH-7 xenografts in mice, and its anti-tumor effect mainly originates from natural killing. cell.
  • Bispecific antibody (BsAb) technology is often used to improve the efficacy of therapeutic antibodies against tumors: a pair of antibodies specific for tumor-associated antigens and surface-trigger-specific antibodies that bind to effector cells that kill tumor cells
  • the specific antibody can efficiently enrich the immune effector cells in the body around the tumor cells, activate the immune effector cells to specifically kill the tumor cells, thereby treating the tumor.
  • bispecific antibodies are prepared by chemical coupling method, hybridoma method and genetic engineering method.
  • the chemical coupling method uses a reducing agent to melt different monoclonal antibodies or fragments thereof.
  • a monovalent antibody or antibody fragment, and a heterobifunctional cross-linker is used to crosslink two monovalent antibodies or fragments thereof having different antigen specificities.
  • This method can rapidly and quantitatively prepare BsAb, but the antibody is inactivated during the crosslinking process, and it is difficult to ensure the homogeneity of the product.
  • the hybridoma method is a method of cell fusion, in which a hybridoma cell secreting one of the monoclonal antibodies is fused with a spleen cell immunized with another antigen, or two hybridoma cells secreting different monoclonal antibodies are fused to each other.
  • the fused cells, the former are called trisomy, and the latter are called tetrasomy, which are collectively referred to as secondary hybridomas.
  • the BsAb prepared by the hybridoma method has high biological activity, but is cumbersome to prepare, time consuming, and difficult to separate from other non-active or unwanted antibodies produced at the same time. In addition, breakthroughs have not yet been made in human hybridoma technology.
  • the BsAb obtained by this method faces the problem of producing human anti-mouse antibody (HAMA) and excessive molecular weight in clinical application, and the cost of preparing clinical grade BsAb by this method is very high. High, difficult to use universally.
  • BsAb Genetic engineering preparation BsAb is developed on the basis of the development of genetically engineered antibody technology on the basis of small molecule antibodies. It has obvious advantages, such as stable method, mass production, and greatly reduced cost and easy operation. With the maturity of genetic engineering techniques, two different single-chain antibodies (ScFv) can be joined to form a small molecule BsAb by different methods. There are three forms of BsAb depending on the connection method.
  • Mini-antibody A molecule in which two ScFvs are assembled into one heterodimer using an oligomerized domain (for example, Fos or Jun leucine zipper).
  • Dibodies The VH and VL of two different antibodies are linked into two different single strands by a short linker peptide (eg, Gly 4 Ser): VH1-VL2 and VH2-VL1, Co-expression in the same cell, because the short linker peptide makes the VH and VL of the same chain difficult to pair, but only matches the V-region of the other chain but is actually the same source, and folds to form one with two A dimeric molecule of an antigen binding site.
  • a short linker peptide eg, Gly 4 Ser
  • Bispecific antibody-mediated biological immunotherapy has a good clinical application prospect in the biological treatment of tumors.
  • the method is characterized in that: the bispecific antibody-mediated killing effect on the tumor cells is achieved by stimulating the body's immune system, and the immune effector cells are highly tumor-specific for tumor cell killing, and Not subject to MHC restrictions.
  • bispecific antibodies are not harmful to normal tissues. Therefore, the use of bispecific antibodies to treat tumors is complementary to traditional methods such as surgery, radiotherapy, and chemotherapy. Its real role is to eliminate subclinical lesions, reduce or even eliminate tumor recurrence and metastasis. It can be used to cure tumors and stimulate the body to produce and maintain immune protection for a long time.
  • the preparation of clinical grade BsAbs for tumor therapy generally requires five characteristics: 1 high specificity and affinity for targeted tumor antigens; 2 monovalent binding effector cell surface The cytotoxic effect triggers the molecule and crosslinks only after encountering the tumor antigen, they do not contain the Fc segment; 3 can effectively initiate high levels of targeted cytotoxicity and the corresponding leukocyte population selectively in the tumor Locally produces an inflammatory response; 4 humanizes, reduces the HAMA response that occurs after repeated use; 5 BsAb should be small enough to penetrate into the tumor but should be large enough to remain in the circulation for a sufficient period of time.
  • immune effector cells include T lymphocytes, cells, and mononuclear macrophages.
  • Neutrophils and LAK cells lymphokine-activated killer cells
  • TIL cells tumor-infiltrating lymphocytes.
  • T lymphocytes are the main cells for specific cellular immune responses and are the most potent effector cells currently under investigation.
  • the CD3 molecule is expressed on the surface of all mature T cells and is a common surface marker on all T cell surfaces.
  • CD3 and TCR are non-covalently bound to form a complete TCR-CD3 complex, which is involved in the immune response to antigen stimuli. It is the most widely used and most successful triggering molecule on the surface of immune effector cells in bispecific antibodies. .
  • the anti-CD3 antibody in the BsAb When the anti-CD3 antibody in the BsAb binds to the CD3 molecule on the surface of the T cell, it can produce various effect functions to achieve killing of the tumor cells. These effector functions include (1) T cell proliferation and differentiation, (2) cytokine secretion, and (3) cytotoxicity.
  • the antibody used in the construction of BsAb is generally an Fv fragment, because the Fv fragment is the smallest unit containing the complete antigen binding site, the molecule is small (only 1/6 of the intact antibody), contains no Fc segment, and has low immunogenicity. Easy to pass through the vessel wall and into the solid tumor, but also can be expressed in E. coli, fermentation production, greatly reducing the production cost, but because Fv can not form covalent bonds between VH and VL, it is unstable in the body, it is easy Dissociation.
  • a polypeptide chain, a linker peptide is ligated between VH and VL to form a so-called single-chain antibody (ScFv).
  • the linker peptide is usually a flexible peptide of 15 amino acids in length, commonly used (Gly 4 Ser) 3 .
  • the GPC3 molecule is a liver cancer-specific marker, and despite the above-described description of the bispecific antibody involved in CD3, is it a bifunctional antibody (including bifunctional T cell mediated formation) of an antibody against any antigen combined with an anti-CD3 antibody; Object, ie bi spec i fic T-ce ll Engager , ⁇ ) It is unpredictable that the biological effects to be effectively killed and related tumor cells such as liver cancer. So far, there have been no reports on the bispecific antibodies against GPC3, and this view has been proved from one side. In a bispecific antibody, two functional domains are required to bind correctly to their binding sites, respectively.
  • a first aspect of the invention relates to a bispecific antibody comprising a first domain that specifically recognizes phosphatidylinositol-3, a second domain that specifically recognizes human T cell antigen CD3, and Connector for the functional domain.
  • the bispecific antibodies of the invention have unexpectedly improved specificity against the cytotoxic toxicity of liver cancer tumor cells.
  • a second aspect of the invention relates to a nucleotide sequence encoding the above antibody.
  • a second aspect of the invention relates to a vector comprising the above nucleotide sequence, including an expression vector.
  • a fourth aspect of the invention relates to a eukaryotic or prokaryotic expression system comprising the vector described above.
  • a fifth aspect of the invention relates to the use of the above antibody for the preparation of a medicament for treating or preventing a tumor.
  • the meanings of the terms used in the present invention are as follows:
  • “Functional domain” refers to a three-dimensional structure capable of specifically recognizing and/or binding to an epitope, such as an antibody or antibody fragment, including a natural intact antibody, a single chain antibody (scFV), an Fd fragment, a Fab fragment, F (ab) ') 2 fragments, single domain antibody fragments, isolated CDR fragments, and derivatives thereof.
  • the binding site of the functional domains of the invention may be derived not only from antibodies, but also from other proteins that bind to GPC3 and/or CD3, such as naturally occurring surface receptors or ligands.
  • the degree of "specific recognition” and specificity can be judged by classical immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like.
  • specific recognition is preferably determined by flow cytometry, and the criteria for specific recognition in specific cases can be judged by one of ordinary skill in the art based on the common knowledge in the art.
  • an “intact antibody” consists of two identical heavy and light chains, each of which contains a variable region (V region) and one or more constant regions (C region).
  • the variable region is responsible for binding to the antigen, while the constant region is primarily responsible for binding the effector molecule.
  • There are three highly diverse flexible loops in each variable region called complementarity determining regions (CDRs), which are primarily responsible for the recognition of antigens.
  • the rest of the variable region contains a rigid beta sheet and supports the so-called Frame area (FRs).
  • FRs Frame area
  • Single-chain antibody (scFV) fragment refers to an antibody fragment constructed by genetic engineering, which has a heavy chain variable region ( VH ) and a light chain variable region (; VL) linked by a linker. Recombinant proteins, linkers associate these two domains to form an antigen binding site.
  • the size of ScFV is typically 1/6 of that of an intact antibody.
  • the single chain antibody is preferably an amino acid chain sequence encoded by one nucleotide chain.
  • Fd fragment refers to a heavy chain V H and C H ⁇ fi into the antibody fragment.
  • Fab fragment refers to a heterodimer formed by an Fd fragment (consisting of heavy chains VH and CH1) and an entire light chain formed by interchain disulfide bonds.
  • the “Fab antibody” is 1/3 the size of an intact antibody and contains only one antigen binding site.
  • F(ab') 2 fragment refers to a bivalent fragment comprising two linked Fab fragments.
  • a "single domain antibody” consists of a heavy chain variable region or a light chain variable region. Since the antibody fragment consists of only one domain, it is named. The size of this fragment is 1/12 of an intact antibody.
  • Derivatives of antibodies includes, for example, when a derivative of the antibody is obtained by phage display technology, surface plasmon resonance techniques such as those used in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to GPC3 or CD3 epitopes ( Schier, Human Antibody Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • the antibodies or fragments thereof used in the present invention may be further modified, either singly or in combination, using conventional techniques known in the art, such as amino acid deletions, insertions, substitutions, additions, and/or recombinations, and/or other modification methods.
  • Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY Modifications are preferably made at the nucleic acid level.
  • the antibodies or antibody fragments used in the invention may be humanized, chimeric or murine.
  • CD3 refers to an antigen expressed in a T cell that is part of a T cell receptor complex, which is composed of three different chains, CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • the vast majority of anti-CD3 antibodies recognize the CD3 epsilon chain.
  • Figure 1 (a): Gel electrophoresis pattern of a nucleotide fragment encoding a CD3 scFv amplified by PCR.
  • the right column shows the target amplification product, and the left column shows the molecular weight marker molecular weight marker (DL2000, purchased from TAKARA Biotechnology Co., Ltd.).
  • the left column is the target amplification product, and the right column is the molecular weight marker molecular weight marker (DL2000, purchased from TAKARA Biotechnology Co., Ltd.).
  • the 55kD band indicated by the arrow is the target amplification product, and the molecular weight marker (DL2000, purchased from TAKARA Biotechnology Co., Ltd.) is on the right.
  • DL2000 molecular weight marker
  • FIG. 1 Schematic diagram of the construction of pH-GPC3/CD3 expression vector.
  • FIG 3 SDS-PAGE electropherogram of the purified GPC3/CD bispecific antibody polypeptide expressed by the PH-GPC3/CD3 expression vector.
  • the left band is the purified antibody molecule of interest, and the right side is the molecular weight marker (SDS-PAGE small molecular weight standard protein, purchased from Shanghai Shengzheng Biotechnology Co., Ltd.).
  • Figure 4A-E FACS (fluorescence activated cell sorter, or flow cytometry) analysis of the PH-GPC3/CD3 expression vector expressing purified GPC3/CD3 bispecific antibody binding to different cell lines.
  • FIG. 5 Cytotoxic targeting cleavage effects of different concentrations of GPC3/CD3 bispecific antibodies against three cell lines.
  • the vertical axis is the percentage of cytotoxicity (as defined below) and the horizontal axis is the concentration of the bispecific antibody (ng/mL).
  • the first domain that specifically recognizes phosphatidylinositol-3 can specifically bind to the C-terminal peptide of GPC3, or specifically bind to the N-terminal peptide of GPC3.
  • GPC3 CN101186650A; CN102180969A; CN101633693A; CN101052878A; CN1842540A; those GPC3 monoclonal antibodies and any currently known GPC3-specific antibodies such as GC33, or one or more heavy chain variable regions and one by monoclonal antibodies Or a single chain antibody formed by multiple light chain variable regions.
  • the first and/or second functional domains of the bispecific antibodies of the invention may be selected from the group consisting of intact antibodies, single chain antibodies (scFV;), Fab fragments, Fd fragments, Fv fragments, F(ab') 2 fragments and derivatives thereof. .
  • the first and/or second domain of the invention may be a single chain antibody (scFV) comprising a heavy chain variable region and a light chain variable region
  • the second functional domain of the present invention for specifically recognizing the T cell surface receptor CD3 is not specifically limited, as long as It is capable of specifically recognizing CD3.
  • the linker of the present invention linked to the first and/or second functional domains described above may be a short peptide chain such as, but not limited to, (Gly 4 Ser) n wherein n is from 1 to 5 and n is an integer. In a specific embodiment, n is preferably 3.
  • the amino acid sequence of the first domain is SEQ ID NO: 5, ie by VL (5 PC3 - (; G 4 S) 3 -VH CTC3 , or the similarity to the amino acid sequence is 95 Amino acid sequences of % and above.
  • the amino acid sequence of the second domain is SEQ ID NO: ⁇ , that is, VH CD3 - VE(GGS) 4 GG-VL CD3 , or 95% similarity to the amino acid sequence The above amino acid sequence.
  • the nucleotide sequence of the first domain is the nucleotide sequence of SEQ ID NO: 6, which encodes the amino acid sequence of SEQ ID NO: 5, or with the nucleoside
  • the nucleotide sequence similarity is 95% or more.
  • nucleotide sequence of the second domain is
  • nucleotide sequence of SEQ ID NO: 8 which encodes the amino acid sequence of SEQ ID NO: 7, or a nucleotide sequence having a similarity to the nucleotide sequence of 95% or more.
  • a third aspect of the invention is a genetically engineered vector comprising the nucleotide sequence encoding the bispecific antibody protein of the invention.
  • the vector may be a eukaryotic cell vector or a prokaryotic cell vector, as long as the vector satisfies: (a) its coding sequence comprises a sequence of initiation of replication such that the vector is capable of being replicated in a host cell, (b) it comprises A gene sequence encoding a marker that encodes a protein that is required for survival and growth of the host cell in a particular selection medium. In the case where the host cell is not transfected or transformed into a vector comprising the gene, the host cell cannot survive in a particular selection medium.
  • Typical screening marker genes encode proteins comprising proteins that are tolerant to antibiotics or toxins, including, for example, ampicillin, kanamycin, tetracycline, neomycin, hygromycin, methotrexate, and the like; make up A protein associated with auxotrophy that supplies key nutrients that are not present in the culture medium, such as the D-alanine racemase gene.
  • Examples of the use of the resistance screening include, by transfecting an exogenous vector containing a neomycin resistance gene, and allowing the host cell to obtain a growth medium in the case of a medium containing the drug neomycin or G418.
  • DHFR dihydrofolate reductase screening markers
  • mammalian cells such as Chinese hamster ovary cells (CHO).
  • Mammalian cell host cells refer to DHFR-deficient cells and do not contain the dihydrofolate reductase gene. , can not synthesize nucleic acids, must be grown in the medium containing HT.
  • a positive clone containing the exogenous vector containing the gene of interest and the DHFR gene can be selected by selection of the above culture conditions.
  • the expression vector may also include other constituent sequences, including a signal peptide sequence, a transcription termination sequence, an enhancer sequence, etc.
  • the vector of the present invention is a eukaryotic cell vector.
  • the vector of the present invention is a pH vector derived from eukaryotic expression of an antibody, which comprises a promoter of CMV, an internal ribosome entry site (IRES), a DHFR screening marker and the like.
  • IRS internal ribosome entry site
  • DHFR screening marker a DHFR screening marker and the like.
  • Methotrexate (MTX) is an inhibitor of DHFR that blocks its effects.
  • DHFR is inhibited, and the gene is self-amplified by feedback regulation, and the upstream and downstream genes are amplified, and the target gene is also amplified, thereby increasing the expression level of the target protein.
  • a fourth aspect of the invention relates to an expression system comprising the above genetically engineered vector, i.e., to a host cell comprising the vector for expression of a desired multifunctional antibody polypeptide.
  • the host cell of the present invention may be any prokaryotic host cell or eukaryotic host cell, in accordance with the vector used.
  • Eukaryotic host cells including yeast, insect cells, plant cells, mammalian cells, etc., may be preferred, since eukaryotic cells have complex post-translational modifications (eg, glycosylation) of the protein of interest, and are increasingly used. For large-scale cultivation.
  • Common host cell lines include monkey kidney cells (COS-7 ATCC CRL 1651), human embryonic kidney cells 293 and their subcloned cell lines, baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells (CHO), and the like.
  • the eukaryotic host cell of the invention is a Chinese hamster ovary cell.
  • a fifth aspect of the invention relates to the use of the above-described GPC3/CD3 bispecific antibody for the preparation of a medicament for treating or preventing a tumor.
  • tumors include, but are not limited to, liver cancer, melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma.
  • VL GPC 3 and VH Grc3 nucleotide fragments are respectively corresponding to the corresponding VL portion of the GC33 antibody ('086 patent SEQ ID NO: 91) and the VH portion ('086 patent SEQ ID NO: 91) published in the patent document US7919086B2 (hereinafter referred to as '086 patent). : 81 )
  • the gene sequence was obtained.
  • a sequence encoding a nucleotide of a GPC3 single-chain antibody fragment by introducing a nucleotide encoding a (Gly 4 Ser) 3 (simplified as (G 4 S) 3 ) amino acid sequence linker between the VL GPC3 and VH GPC3 nucleotide fragments Composition.
  • VL CD3 and VH CD3 gene fragments were obtained according to nucleotide sequence 857-1585 of SEQ ID NO: 9 published in the patent document US7112324B1, respectively, wherein the nucleotide sequence encoding the CD3 single-chain antibody fragment passed in VL eD3 and VH A nucleotide sequence encoding the amino acid sequence VE(GGS) 4 GG linker is introduced between the eD3 nucleotide fragments.
  • the nucleotide fragment of the GPC3 single-chain antibody was used as a template PCR for 15 cycles, and the 5'CD3-l/3 'CD3-2 primer was used for CD3.
  • the nucleotide fragment of the single-chain antibody was subjected to 15 cycles of template PCR, and the PCR products were collected and mixed, and an appropriate mixture was used as a template to carry out the second round of PCR bridge with 5'GPC3-1 and 3'CD3-2 primers.
  • SEQ ID NO: 4 The amplified sequence SEQ ID NO: 9 was simultaneously digested with restriction endonuclease Nhel/Notl-HF according to the reaction conditions recommended by the enzyme supplier (New England Biolabs, NEB) in buffer 2. Perform double digestion.
  • the expression pH vector (see WO/2011/035465 Example 7 and Figure 15) was also subjected to the same digestion with restriction endonuclease Nhel/Notl-HF to obtain a pH/DHFR vector fragment.
  • the fragment of SEQ ID NO: 9 and the pH/DHFR vector fragment were then ligated with T4 DNA ligase according to the reaction conditions recommended by the enzyme supplier (NEB) to form a nucleotide containing the GPC3/CD3 bispecific antibody. Expression of the fragment
  • the body pH-GPC3/CD, its detailed connection structure is shown in Figure 2.
  • the expression vector PH-GPC3/CD3 obtained in the above procedure was transfected into Chinese hamster ovary (CHO-S) cells according to the instructions of FreeStyle MAX Reagent transfection reagent (from Invitrogen), and then according to the OptiCHOTM protein expression kit (from Invitrogen). ) Screening for stable clones. Stable clones of CHO-S cells transfected with one of the above expression vectors, respectively, were cultured in shake flasks at 37 ° C, 130 rpm for 7 days, and the medium used was CD OptiCHO (from Gibco ). The culture supernatant was obtained by centrifugation and then stored at -20 ° (.
  • the GPC3/CD3 protein in the culture supernatant was enriched by immobilized metal affinity chromatography (IMAC), using a histidine affinity column (His Trap HP column, purchased from GE Healthcare). ) Perform protein purification. According to the method steps provided by the column manufacturer, equilibrate with buffer A (20 mM phosphate pH 7.4, 0.4 M NaCl), and then add the PBS dialyzed cell culture supernatant (500 mL supernatant) to the column ( 10 mL;), flow rate 3 mL/min. The column was then washed with 5 volumes of buffer A and 10 volumes of buffer A containing 20 mM imidazole to remove the heteroprotein.
  • IMAC immobilized metal affinity chromatography
  • 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 protein was dialyzed against PBS to measure the spectral absorption at a wavelength of 280 nm, and the protein concentration was calculated as a coefficient of 0.53 mg/OD.
  • the purified GPC3/CD3 bispecific antibody protein was analyzed by SDS-PAGE electrophoresis, as shown in Fig. 3, and its molecular weight was about 55 kD.
  • Example 2 Analysis of binding specificity of GPC3/CD3 bispecific antibody to target cells
  • T cells are contained in PBMC.
  • Fluorescence activated cell sorter also commonly known as flow cytometry FACScalibur, by BD provides analysis of the binding ability of the bispecific antibody GPC3/CD3.
  • the tumor cells in the logarithmic growth phase as listed in Table 1 were inoculated into a 6 cm dish, and the inoculated cell density was about 90%, and cultured overnight at 37 °C.
  • Cells were digested with 10 mM EDTA and harvested by centrifugation at 200 g x 5 min. Resuspended in 1% calf serum-containing phosphate buffer (NBS PBS) at a concentration of 1 ⁇ 10 6 to lx 10 7 /mL, and added to a flow-type dedicated tube in an amount of 100 ⁇ l/tube.
  • NBS PBS calf serum-containing phosphate buffer
  • the two experimental groups were added with the test antibodies 7B3/CD3 and 806/CD3, and one control group was added with the antibody NGR/CD3 as the negative control, and the other control group was the PBS blank without antibody.
  • the final concentration of each antibody was 2 ( ⁇ g/ml, 100 ul per tube. Ice bath, 45 minutes.
  • Each sample was analyzed for at least 10,000 cells using a flow cytometer.
  • the GPC3/CD3 bispecific antibody of the present invention can bind to the hepatoma cell lines Huh7 and HepG2 expressing GPC3, but not to the CHO-K1 cell line which does not express GPC3.
  • the GPC3/CD3 antibody efficiently binds to Jurkat and PBMC cells, showing the binding specificity of the GPC3/CD3 bispecific antibody to T cells expressing CD3 on the surface.
  • the GPC3/CD3 bispecific antibody of the present invention has the ability to specifically recognize GPC3 and CD3 at the same time.
  • Example 3 GPC3/CD3 bispecific antibody mediated cytotoxic killing effect on tumor cells
  • PBMC Peripheral blood mononuclear cells
  • the mixed cell suspension was added to a 96-well plate at a volume of 75 ⁇ L/well. Then, 25 L of a ten-fold serial dilution of GPC3/CD3 single-chain bifunctional antibody from 1000 ng/mL to 0.1 ng/mL was added to each well. After incubating for 40 hours in a 37 ° C, 5% CO 2 incubator, the cells of the antibody were detected using the CytoTox96® Non-Radioactive Cytotoxicity Assay Kit (Promega) according to the manufacturer's instructions. Toxic effect.
  • the CytoTox 96® non-radioactive cytotoxicity assay is based on a colorimetric assay that replaces the 51 Cr release method.
  • the CytoTox 96® assay quantitatively measures lactate dehydrogenase (LDH).
  • LDH lactate dehydrogenase
  • 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 foraiazan.
  • INT tetrazolium salt
  • the killing rate of tumor cells (ie, % cytotoxicity) is calculated according to the following formula provided by the CytoT OX 96® Non-Radioactive Cytotoxicity Test G1780 Product Instructions:
  • Experiment refers to the release of LDH from an experimental well added to an antibody/effector/target cell
  • effector cell spontaneous refers to the spontaneous release of LDH from a control well containing only effector cells. In the present invention, it refers to a control well of an effector cell.
  • Target cell spontaneous refers to the release of LDH produced by a control well containing only target cells when the target cells are not treated by other factors.
  • Maximum target cells is the release of LDH from the control wells after treatment with 0.8% Triton X-100 to completely lyse the target cells.
  • Target cell max-target cell spontaneous represents the release of LDH from the complete lysis of target cells by external treatment.
  • the EC of the bifunctional antibody against tumor cell killing was calculated using the GraphPad Prism 5 software (GraphPad Software inc., San Diego, USA) analysis program. 5Q value.
  • the EC 5Q value of the GPC3/CD3 bispecific antibody to HepG2 cell line was 14.22 ng/ml, respectively, and to the Huh-7 cell line was 656.7 ng/ml.

Abstract

公开了一种针对磷脂酰肌醇蛋白多糖-3和T细胞抗原的双特异性抗体及其制备方法和用途，所述抗体包含特异性识别磷脂酰肌醇蛋白多糖-3的第一功能域、特异性识别人T细胞抗原CD3的第二功能域和连接上述功能域的接头，可用于制备治疗或预防肿瘤的药物。

Description

针对磷脂酰肌醇蛋白多糖 -3和 T细胞抗原的双特异性抗体 技术领域
本发明涉及肿瘤相关抗体， 更具体的， 基因工程抗体领域。 背景技术
磷脂酰肌醇蛋白多糖 -3 (Glypi can-3 , GPC3， 又称 DGSX， GTR2-2, MXR7, 0CI-5, SDYS, SGB, SGBS或 SGBS 1)是一种细胞表面蛋白， 属于硫酸乙酰肝素蛋白 多糖家族。 GPC3基因编码产生 70-kDa左右的前体核心蛋白， 该前体蛋白能够被弗 林蛋白酶 （fur in ) 剪切产生 40-kDa左右能够分泌进入血液的可溶性氨基端肽， 和 30-kDa左右含有 2个硫酸乙酰肝素（HS)糖链的膜结合的羧基端肽。 GPC3蛋白通 过糖基磷脂酰肌醇 （GPI ) 锚依附在细胞膜上。
GPC3高度表达于胎儿肝脏， 而不表达于正常成年人的肝组织。 但在肝细胞肝 癌中恢复表达， 与肝癌的发生发展有十分密切的关系， 不仅在肝癌发生的早期检 出率较高， 而且随着肝癌的发展， 其检出率也随之增高。 而在肝腺癌， 胆管细胞 癌， 肝转移癌和 12种常见实体瘤和 21种非肝癌细胞系中均未检测出 GPC3的表达。 此外， GPC3也在例如黑色素瘤， 卵巢透明细胞癌、 卵黄囊瘤、 神经母细胞瘤等肿 瘤中表达。 考虑到 GPC3在肝细胞肝癌， 黑色素瘤等肿瘤中的高表达， GPC3被认为 是肿瘤免疫治疗的一个候选靶标。
利用抗 GPC3抗体进行肝癌检测和利用抗 GPC3抗体的抗体依赖的 （ADCC ) 或补 体依赖的（CDC)细胞毒性的研宄己有报道， 例如 CN101 186650A (中外制药株式会 社）， 发明名称为 "抗存在于血液中的 GPC3的分泌性 N-端肽或 C-端肽的抗体" ； CN102180969A (中国人民解放军军事医学科学院微生物流行病研宄所） ，发明名 称为 "抗肝癌活性单克隆抗体及其应用" ， 其涉及用 GPC3氨基端 359-580位氨基 酸残基为抗原制备的抗 GPC3单克隆抗体； CN101633693A (中国人民解放军第二军 医大学） ，其涉及针对 GPC3的氨基端抗原的单克隆抗体； CN101052878A (株式会社 英仙蛋白质科学）， 其涉及以使用识别 GPC3的 N端部位的抗体测定 GPC3为特征的监 控肝炎重症化的方法； CN1842540A (中外制药株式会社），其涉及一种据称与传统 抗体相比具有较高 ADCC活性和 CDC活性的抗 GPC3单克隆抗体。 此外， 表 1总结了另外一些自 2003年来报道的针对 GP C 3的单克隆抗体 ( mAbs ) 。 其中， 一种针对 GPC3的治疗性 mAb最近被报道（Advances in Liver Cancer Antibody Therapies： A Focus on Glypi can-3 and Mesothel in ， BioDrugs. 201 1 October 1 ; 25 (5) ： 275 - 284. )，其针对 GPC3位于 C端 524- 563位 氨基酸残基形成的抗原决定簇。 根据该报道单克隆抗体 GC33 ( IgG2a， κ ) 诱导 抗体依赖细胞毒作用 （ADCC ) 并表现出抑制小鼠皮下移植 HepG2和 HuH-7异种移植 物的肿瘤生长， 其抗肿瘤作用主要源于自然杀伤细胞。
表 1.己知的一些抗 GPC3单克隆抗体
Figure imgf000004_0001
然而， 仍然有强烈的需要继续探索特异性针对 GPC3的治疗性抗体以用于肝 癌， 黑色素瘤， 卵巢透明细胞癌、 卵黄囊瘤、 神经母细胞瘤等肿瘤的治疗。 令人 期待的是， 这些治疗性抗体需要具有改善的特异性， 肿瘤细胞杀伤毒性， HAMA问 题， 肿瘤杀伤所需的最小剂量等优点。
双特异性抗体（BsAb ) 技术常被用于改善针对肿瘤的治疗性抗体的功效： 将 肿瘤相关抗原特异的抗体与具杀伤肿瘤细胞功能的效应细胞的表面触发分子特异 的抗体偶联构成的双特异性抗体， 可高效地将体内的免疫效应细胞富集于肿瘤细 胞周围， 激活免疫效应细胞特异性地杀伤肿瘤细胞， 从而起到***的目的。 目前双特异性抗体的制备有化学偶联法， 杂交瘤法和基因工程法三种。
化学偶联法是先采用还原剂， 使不同的两种单克隆抗体或其片段解链， 获得 单价抗体或抗体片段， 再采用异双功能交联剂把两种具有不同抗原特异性的单价 抗体或其片段交联在一起。 该方法可快速， 大量制备 BsAb， 但是在交联过程中抗 体时有失活， 而且难于保证产物的均质性。
杂交瘤法是通过细胞融合的方法， 将分泌其中一种单克隆抗体的杂交瘤细胞 与经另一种抗原免疫的脾细胞融合， 或两种分泌不同单克隆抗体的杂交瘤细胞彼 此融合。 融合而成的细胞， 前者称三体瘤， 后者称四体瘤， 两者统称二次杂交 瘤。 杂交瘤方法制备的 BsAb 生物活性高， 但制备繁琐， 耗时长， 且不易与同时 产生的其他没有活性或不需要的抗体分离。 此外， 人源杂交瘤技术尚未取得突破 性进展， 该法获得的 BsAb在临床应用上面临产生人抗鼠抗体（HAMA)问题和分子量 过大的问题， 且用该方法制备临床级的 BsAb成本很高， 难以普及使用。
基因工程法制备 BsAb是随着基因工程抗体技术的发展， 在小分子抗体的基础 上发展起来的， 有其明显的优越性， 如方法稳定， 可大量生产， 且成本大大降 低， 操作简便等。 随着基因工程技术的成熟， 用不同的方法可将两种不同的单链 抗体（ScFv)连接构成小分子的 BsAb。 根据连接方法不同有三种 BsAb的构成形式。
(1)微型抗体 （Mini-antibody ) ： 利用一个寡聚化的结构域（例如， Fos或 Jun亮 氨酸拉链） 将两个 ScFv组装成一个异二聚体的分子。 （2)二聚体抗体 ( Diabodies ) ： 将两个不同抗体的 VH和 VL用一个短的连接肽（例如， Gly4Ser)连 接成两条不同的单链： VH1-VL2和 VH2-VL1 , 在同一个细胞中共表达， 由于短的连 接肽使得同一条链的 VH和 VL难以配对， 而仅与另外一条链的、 但实际上却是来源 相同的 V区相匹配， 折叠形成一个具有两个抗原结合位点的二聚体分子。 （3)单链 双功能抗体 ( s ingle chain bi spec if ic ant ibody, ScBsAb ) ： 在同一个载体上 将两种特异性的 scFv用一个域间连接肽 （interl inker)相连构成， 其中构成两个 scFv的链内连接肽（intral inker)常用的是（Gly4Ser) 3， 而连接两个 scFv的域间连 接肽有两种设计， 一种为短的肽链， 一般不超过 10个氨基酸残基， 常用的是 Gly4Ser， 这种设计主要目的是防止异源可变区之间的错配。 另一种设计方案为长 的肽链。 域间连接肽设计总的原则是保证抗体可变区结构域发生正确的配对和折 叠并保持其生物学活性及稳定性， 其次还可根据需要赋予产物一些新的特性， 如 便于纯化、 增强其在体内的半衰期等。
双特异抗体介导的生物免疫导向治疗在肿瘤的生物治疗中有良好的临床应用 前景。 其特征在于： 其一， 双特异抗体介导的对肿瘤细胞的杀伤作用是靠激发机 体的免疫***完成的， 免疫效应细胞对肿瘤细胞的杀伤是高度肿瘤特异性的， 且 不受 MHC的限制。 其二， 双特异抗体对正常组织是无害的。 因此， 应用双特异抗 体***是手术， 放疗， 化疗等传统方法的补充， 其真正作用在于消除亚临床 病灶， 减少乃至消灭肿瘤的复发和转移。 表现为既可以根治肿瘤， 又可以激发机 体产生和维持较长时间的免疫保护作用。
综合小鼠及临床研宄结果， 制备用于肿瘤治疗的临床级 BsAb—般优选需要具 备五种特性： ①对靶向的肿瘤抗原具有很高的特异性和亲和力； ②可单价结合效 应细胞表面的细胞毒效应触发分子， 并只在遭遇了肿瘤抗原后发生交联， 它们不 含有 Fc段； ③可有效地启动高水平的靶向性细胞毒作用以及相应的白细胞群有选 择性地在肿瘤局部产生炎症反应； ④人源化， 降低重复使用后发生的 HAMA反应； ⑤ BsAb应小到足以渗透入肿瘤但又应大到足以在循环中维持足够的时间。
以上述几点为依据， 目前， 人们开发研宄了多种激发不同免疫效应细胞， 针 对不同肿瘤细胞的双特异性抗体， 其中的免疫效应细胞包括 T淋巴细胞、 置细 胞、 单核巨噬细胞、 中性粒细胞以及 LAK细胞（淋巴因子 激活的杀伤细胞）和 TIL 细胞（激活的肿瘤侵润性淋巴细胞）等。 T淋巴细胞是特异性细胞免疫应答的主要 细胞， 是目前研宄的最多的效应细胞。 CD3分子表达于所有成熟 T细胞表面， 是所 有 T细胞表面共同的表面标志。 CD3与 TCR呈非共价结合， 形成完整的 TCR-CD3复 合物， 共同参于对抗原剌激的免疫应答， 是目前在双特异性抗体中应用最多且最 成功的免疫效应细胞表面的触发分子。
BsAb中的抗 CD3抗体与 T细胞表面的 CD3分子结合后， 可产生多种效应功能， 实现对肿瘤细胞的杀伤。 这些效应功能包括（1) T细胞增殖分化， （2)细胞因子分 泌， （3)细胞毒作用。
目前， 构建 BsAb时所用的抗体一般为 Fv片段， 这是因为 Fv片段是含有完整抗 原结合位点的最小单位， 分子小（只有完整抗体的 1/6)， 不含 Fc段， 免疫原性 低， 易于穿过血管壁和进入实体瘤， 而且还可以用大肠杆菌表达， 发酵生产， 大 大地降低了生产成本， 但由于 Fv中 VH和 VL间不能形成共价键， 在体内不稳定， 很 容易解离。 为了提高 Fv片段的稳定性， 在 VH和 VL间用一条多肽链即连接肽把它们 连接起来形成所谓的单链抗体 （ScFv)。 连接肽通常为一条长 15个氨基酸的具有 柔韧性的短肽， 常用的是（Gly4Ser) 3。
尽管 GPC3分子是肝癌特异的标记物， 以及尽管有上述关于涉及 CD3的双特异 性抗体的记载， 但是是否针对任何抗原的抗体与抗 CD3抗体组合形成的双功能抗 体 (包括双功能 T细胞介导物， 即 b i spec i fic T-ce l l Engager , ΒΪΤΕ ) 都具有期 待的生物学效果从而有效地杀伤肝癌等相关肿瘤细胞是无法预料的。 而迄今为止 还没有任何关于针对 GPC3的双特异抗体的报道， 也从一个侧面证明了上述看法。 双特异性抗体中， 需要两个功能域分别与其结合位点正确结合。 而即使每个抗体 单独能与其抗原正确结合， 也不能保证其在形成双特异性抗体后能保留原来的结 合能力， 并且由于空间位阻的关系， 不能保证形成双特异性抗体后， 能顺利地形 成协同作用。 空间位阻问题可以部分通过柔性接头的设计来解决， 但在双特异性 抗体的构建的设计不能事先保证解决抗原结合位点的正确折叠和抗原结合能力。
发明内容
本发明的第一方面涉及一种双特异性抗体， 其包含特异性识别磷脂酰肌醇 蛋白多糖 -3的第一功能域， 特异性识别人 T细胞抗原 CD3的第二功能域， 和连接上 述功能域的接头。 本发明的双特异性抗体具有意想不到的改善的特异性针对肝癌 肿瘤细胞的细胞杀伤毒性。
本发明的第二方面涉及编码上述抗体的核苷酸序列。
本发明的第二方面涉及包含上述核苷酸序列的载体， 包括表达性载体。
本发明的第四方面涉及包含上述载体的真核或原核表达***。
本发明的第五方面涉及上述抗体在制备治疗或预防肿瘤的药物中的应用。 本发明中所使用的术语的含义如下：
"功能域" 指的是能够特异性识别和 /或结合到表位上的三维结构， 例如抗体 或抗体片段， 包括天然完整抗体， 单链抗体 (scFV)， Fd片段， Fab片段， F(ab')2片 段， 单结构域抗体片段， 分离的 CDR片段， 及其衍生物。 值得注意的是， 本发明 的功能域的结合位点可以不仅来自于抗体， 也可以来自其他与 GPC3和 /或 CD3结 合的蛋白质， 例如天然存在的表面受体或配体。
"特异性识别" 和特异性的程度可以通过经典的免疫学技术来判断， 包括但 不限于免疫印迹， 免疫亲和层析， 流式细胞分析等。 在本发明中， 特异性识别优 选通过流式细胞技术来确定， 而具体情况下特异性识别的标准可由本领域一般技 术人员根据其掌握的本领域常识来判断。
"完整抗体" 由两个同样的重链和轻链组成， 各条链分别包含一个可变区 (V 区)和一个或多个恒定区 (C区）。 可变区负责与抗原结合， 而恒定区主要负责结合 效应分子。 在各可变区有三个具有高度多样性的柔性的环， 称作互补决定区 ( CDR) ，其主要负责识别抗原。 可变区的其他部分包含刚性的 β片层并支持所谓 的框架区 （FRs) 。 CDR和 FR间隔排列形成三明治结构。
"单链抗体 (scFV)片段" 指的是通过基因工程构建的抗体片段， 其是有通过 接头 （linker) 连接的重链可变区 (VH)和轻链可变区 (； VL)的重组蛋白， 接头使得这 两个结构域相关联以形成抗原结合位点。 ScFV的大小一般是一个完整抗体的 1/6。 单链抗体优选是由一个核苷酸链编码的一条氨基酸链序列。
" Fd片段" 指的是由重链 VH和 CH^fi成抗体片段。
" Fab片段" 指的是由 Fd片段 （由重链 VH和 CH1组成） 和整条轻链通过链间 二硫键形成的异二聚物。 " Fab抗体" 的大小是完整抗体的 1/3， 其只包含一个抗 原结合位点。
" F(ab')2片段 "指的是包含两个相连的 Fab片段的二价片段。
"单结构域抗体" 由重链可变区或轻链可变区组成。 由于该抗体片段只由一 个结构域组成， 所以得名。 该片段的大小是一个完整抗体的 1/12。
"抗体的衍生物" 包括例如当通过噬菌体展示技术获得所述抗体的衍生物 时， 可使用如 BIAcore***中使用的表面等离子共振技术来增加与 GPC3或 CD3抗 原表位结合的噬菌体抗体的效率 （Schier, 人抗体杂交瘤 7 ( 1996 ) ， 97-105； Malmborg,免疫学方法杂志 183(1995)， 7-13 ) 。 还包括， 例如 WO 89/09622中描述 的嵌合抗体的产生的方法， EP- A 10239400和 WO90/07861中描述的人源化抗体产 生的方法， WO91/ 10741 , WO94/02602和 WO96/33735中有描述的产生异种抗体例 如小鼠中的人抗体的方法。
本发明使用的抗体或其片段可单独或联合使用本领域己知的常规技术， 例如 氨基酸缺失、 ***、 取代、 增加、 和 /或重组以及 /或其他修饰方法作进一步修 饰。 根据一种抗体的氨基酸序列在其 DNA序列中引入这种修饰的方法对本领域技 术人员来说是众所周知的； 见例如， Sambrook , 分子克隆： 实验手册， Cold Spring Harbor Laboratory(1989)N.Y 所指的修饰优选在核酸水平上进行。
本发明使用的抗体或抗体片段可以是人源化的， 嵌合的或鼠源的。
" CD3 " 指的是作为 T细胞受体复合物的一部分的表达于 T细胞的抗原， 其由 三个不同的链 CD3 ε ， CD3 δ和 CD3 γ组成。 CD3在 Τ细胞上通过例如抗 CD3抗体 对其的固定作用而产生的集中 （clustering) ， 导致 T细胞的活化， 与 T细胞受体介 导的活化类似， 但是不依赖于 TCR克隆的特异性。 绝大多数抗 CD3抗体识别 CD3 ε链。 附图说明
图 1 (a) : PCR扩增的编码 CD3 scFv的核苷酸片段凝胶电泳图。 右栏为目标扩 增产物， 左栏为分子量标记分子量标记物 （DL2000， 购自 TAKARA生物技术有限公 司） 。
图 1 (b) : PCR扩增的编码 GPC3 scFv的核苷酸片段凝胶电泳图。 左栏为目标扩 增产物， 右栏为分子量标记分子量标记物 （DL2000， 购自 TAKARA生物技术有限公 司） 。
图 1 (c )： PCR扩增的编码 GPC3/CD3双特异性抗体的核苷酸片段凝胶电泳图。 箭头所指 55kD条带是目的扩增产物， 右侧为分子量标记物 （DL2000， 购自 TAKARA 生物技术有限公司） 。
图 2 : pH-GPC3/CD3表达载体构建示意图。
图 3 : PH-GPC3/CD3表达载体表达纯化的 GPC3/CD双特异性抗体多肽的 SDS- PAGE电泳图。 左侧条带为纯化的目的抗体多肽， 右侧为分子量标记物 （SDS-PAGE 小分子量标准蛋白质， 购自上海升正生物技术有限公司） 。
图 4A-E : PH-GPC3/CD3表达载体表达纯化的 GPC3/CD3双特异抗体与不同细胞 系表面结合情况的 FACS (荧光激活细胞分类器， 或称流式细胞） 分析。
图 5 : 不同浓度的 GPC3/CD3双特异性抗体针对三种细胞系的细胞毒性靶向裂 解效果。 纵轴是细胞毒性百分数 （根据下文所定义） ， 横轴为双特异性抗体的浓 度 ( ng/mL ) 。 具体实施方式
在本发明第一方面的一个具体实施方案中， 特异性识别磷脂酰肌醇蛋白多糖- 3的第一功能域可以特异性结合 GPC3的 C末端肽， 或者特异性结合于 GPC3的 N末 端肽。 例如 CN101186650A； CN102180969A； CN101633693A； CN101052878A； CN1842540A中所公开的那些 GPC3单克隆抗体和任何目前公开己知的 GPC3特异性抗 体如 GC33 , 或由单克隆抗体的一个或多个重链可变区和一个或多个轻链可变区形 成的单链抗体。 本发明双特异性抗体的第一和 /或第二功能域可以选自完整抗体， 单链抗体 (scFV；)， Fab片段， Fd片段， Fv片段， F(ab')2片段和其衍生物。 本发明的 第一和 /或第二功能域可以是包含一个重链可变区和一个轻链可变区的单链抗体 (scFV
本发明的特异性识别 T细胞表面受体 CD3的第二功能域不受具体的限制， 只要 其能够特异性地识别 CD3。 例如但不限于在下列专利中提到的 CD3抗体： US7,994,289, US6,750,325 ； US6,706,265 ； US5,968,509 ； US8,076,459 ； US7,728,114; US20100183615。
本发明的连接上述第一和 /或第二功能域的接头可以是短的肽链， 例如但不限 于 （Gly4Ser) n， 其中 n从 1到 5， n为整数。 在一个具体实施方案中， n优选为 3。
在本发明的一个具体实施方案中， 第一功能域的氨基酸序列 SEQ ID NO: 5,即 通过 VL(5PC3- (； G4S)3-VHCTC3， 或与该氨基酸序列相似性为 95%及以上的氨基酸序 列。
SEQ ID NO: 5
1 DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV HSNANTYLHW YLQKPGQSPQ LLIYKVSNRF 61 SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQNTHVP PTFGQGTKLE IKRGGGGSGG 121 GGSGGGGSQV QLVQSGAEVK KPGASVKVSC KASGYTFTDY EMHWVRQAPG QGLEWMGALD 181 PKTGDTAYSQ KFKGRVTLTA DESTSTAYME LSSLRSEDTA VYYCTRFYSY TYWGQGTLVT 241 VSS
在本发明的另一个具体实施方案中， 第二功能域的氨基酸序列为 SEQ ID NO: Ί, 即 VHCD3-VE(GGS)4GG-VLCD3， 或与该氨基酸序列相似性为 95%及以上的氨基 酸序列。
SEQ ID NO:7
1 DIKLQQSGAE LARPGASVKM SCKTSGYTFT RYTMHWVKQR PGQGLEWIGY
51 INPSRGYTNY NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY
101 DDHYCLDYWG QGTTLTVSSV EGGSGGSGGS GGSGGVDDIQ LTQSPAIMSA 151 SPGEKVTMTC RASSSVSYMN WYQQKSGTSP KRWIYDTSKV ASGVPYRFSG 201 SGSGTSYSLT ISSMEAEDAA TYYCQQWSSN PLTFGAGTKL ELK
在本发明第二方面的一个具体实施方案中， 第一功能域的核苷酸序列为 SEQ ID NO: 6的核苷酸序列， 其编码 SEQ ID NO: 5的氨基酸序列， 或与该核苷酸序列 相似性为 95 %及以上的核苷酸序列。
1 gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga
51 gccggcctcc atctcctgca gatctagtca gagccttgta cacagtaatg
101 ccaacaccta tttacattgg tacctgcaga agccagggca gtctccacag
151 ctcctgatct ataaagtttc caaccgattt tctggggtcc ctgacaggtt
201 cagtggcagt ggatcaggca cagattttac actgaaaatc agcagagtgg
251 aggctgagga tgttggggtt tattactgct ctcaaaatac acatgttcct 301 cctacgtttg gccaggggac caagctggag atcaaacgtg gtggaggcgg
351 ttcaggcgga ggtggctctg gcggtggcgg atcgcaggtg cagctggtgc
401 agtctggagc tgaggtgaag aagcctgggg cctcagtgaa ggtctcctgc
451 aaggcttctg gatacacctt caccgactat gaaatgcact gggtgcgaca
501 ggcccctgga caagggcttg agtggatggg agctcttgat cctaaaactg
551 gtgatactgc ctacagtcag aagttcaagg gcagagtcac gctgaccgcg
601 gacgaatcca cgagcacagc ctacatggag ctgagcagcc tgagatctga 在本发明第二方面的另一个具体实施方案中， 第二功能域的核苷酸序列为
SEQ ID NO: 8 的核苷酸序列， 其编码 SEQ IDNO: 7 的氨基酸序列， 或与该核苷 酸序列相似性为 95 %及以上的核苷酸序列。
SEQ ID NO:8
1 gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc
51 agtgaagatg tcctgcaaga cttctggcta cacctttact aggtacacga
101 tgcactgggt aaaacagagg cctggacagg gtctggaatg gattggatac
151 attaatccta gccgtggtta tactaattac aatcagaagt tcaaggacaa
201 ggccacattg actacagaca aatcctccag cacagcctac atgcaactga
251 gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat
301 gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt
351 ctcctcagtc gaaggtggaa gtggaggttc tggtggaagt ggaggttcag
401 gtggagtcga cgacattcag ctgacccagt ctccagcaat catgtctgca
451 tctccagggg agaaggtcac catgacctgc agagccagtt caagtgtaag
501 ttacatgaac tggtaccagc agaagtcagg cacctccccc aaaagatgga
551 tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc
601 agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga
651 agatgctgcc acttattact gccaacagtg gagtagtaac ccgctcacgt
701 tcggtgctgg gaccaagctg gagctgaaa
本发明的第三方面是包含了所述编码本发明的双特异性抗体蛋白的核苷酸序 列的基因工程载体。 所述载体可以是真核细胞载体或原核细胞载体， 只要所述载 体满足： （a)其编码序列包含复制起始的序列， 使得该载体能够在宿主细胞中得以 复制， （b)其包含有编码筛选标记的基因序列， 该基因编码的蛋白是该宿主细胞在 特定的选择培养基中生存和生长所必需的。 在宿主细胞如果没有被转染或转化包 含该基因的载体的情况下， 宿主细胞不能在特定选择培养基中生存。 典型的筛选 标记基因编码的蛋白包括具有对抗生素或毒素具有耐受性的蛋白， 抗生素或毒素 包括例如氨苄青霉素、 卡那霉素、 四环素、 新霉素、 潮霉素、 氨甲蝶呤等； 补偿 营养缺陷的相关蛋白， 供应培养基中不存在的关键营养成分， 例如编码 D-丙氨酸 消旋酶基因。 采用抗性筛选的例子包括， 通过转染包含新霉素抗性基因的外源载 体， 使宿主细胞获得在含有药物新霉素或 G418的培养基的情况下， 继续生存生 长。 另外一个例子是在哺乳动物细胞例如中国仓鼠卵巢细胞 （CHO ) 中使用二氢 叶酸还原酶 （DHFR) 筛选标记， 哺乳动物细胞宿主细胞是指 DHFR缺陷型的细 胞， 不含二氢叶酸还原酶基因， 不能合成核酸， 必须在含有 HT的培养基里生长。 在利用载体转染宿主细胞时， 可以通过上述培养基条件的选择筛选得到同时包含 目的基因和 DHFR基因的外源载体的阳性克隆。 （c ) 其编码序列包含启动子的序 列， （d) 表达载体还可能包括其它组成序列， 包括信号肽序列、 转录终止序列、 增强子序列等， 优选地， 本发明的载体为真核细胞载体。 优选地， 本发明的载体 为来自用于抗体真核表达的的 pH载体， 其包含了 CMV的启动子、 内部核糖体进 入位点序列 （Internal ribosome entry site, IRES ) 、 DHFR筛选标记等元件。 氨甲 喋呤 （MTX) 是 DHFR的抑制剂， 可阻碍其作用。 当细胞培养基内含有 MTX时， DHFR被抑制， 通过反馈调节， 使得该基因自我扩增， 连带其上下游基因都会扩 增， 如此目的基因也得到扩增， 即可提高目的蛋白的表达量。
本发明的第四方面涉及包含有上述基因工程载体的表达体系， 即涉及包含有 所述载体的宿主细胞， 用于表达所需的多功能抗体多肽。 与使用的载体相适应， 本发明的宿主细胞可以是任意的原核宿主细胞或真核宿主细胞。 真核宿主细胞， 包括酵母、 昆虫细胞、 植物细胞， 哺乳动物细胞等可以是优选的， 因为真核细胞 存在复杂的目的蛋白的翻译后修饰 （例如糖基化） ， 越来越多的被用于规模化的 培养。 常用的宿主细胞系包括猴肾细胞 （COS-7 ATCC CRL 1651 ) 、 人胚胎肾细 胞 293及其亚克隆细胞系， 幼仓鼠肾细胞 （BHK， ATCC CCL10) ， 中国仓鼠卵巢 细胞 （CHO ) 等。 优选地， 本发明的真核宿主细胞是中国仓鼠卵巢细胞。
本发明的第五方面涉及上述的 GPC3/CD3双特异性抗体在制备治疗或预防肿 瘤的药物中的应用。 所述肿瘤包括但不限于肝癌， 黑色素瘤， 卵巢透明细胞癌、 卵黄囊瘤、 神经母细胞瘤。
以下通过具体实施例来具体描述本发明的实质， 但需要注意的是下述实施例 不应该理解为对本发明范围的限制。 实施例 1 GPC3/CD3双特异性抗体的制备 1 ) 构建含有编码 GPC3/CD3双特异性抗体的核酸的表达载体
VLGPC 3和 VHGrc3核苷酸片段分别根据专利文献 US7919086B2 (以下称' 086专 利） 公布的 GC33抗体的对应 VL部分 （'086专利 SEQ ID NO: 91 ) 和 VH部分 （'086 专利 SEQ ID NO: 81 ) 的基因序列获得。 编码 GPC3单链抗体片段的核苷酸的序列 通过在 VLGPC3和 VHGPC3核苷酸片段之间引入编码 （Gly4Ser) 3 (简写作 （G4S)3) 氨基酸序列接头的核苷酸构成。
VLCD3和 VHCD3基因片段分别根据专利文献 US7112324B1公布的 SEQ ID NO:9 的第 857-1585位核苷酸序列获得， 其中该编码 CD3单链抗体片段的核苷酸序列通 过在 VLeD3和 VHeD3核苷酸片段之间引入编码氨基酸序列 VE(GGS)4GG接头的核苷 酸序列构成。
然后分别通过 5'GPC3-l/3'GPC3-2引物以 GPC3单链抗体的核苷酸片段为模本 PCR进行 15个循环， 以及通过 5'CD3-l/3 'CD3-2引物以 CD3单链抗体的核苷酸片段 为模本 PCR进行 15个循环， 收集 PCR产物混匀,取适量混合物作为模本,以 5'GPC3-1 和 3'CD3-2引物继续进行第二轮 PCR搭桥扩增进行 30个循环， 其中 5'GPC3-1含有 Nhel酶切位点， 3'CD3-2 含有 BamHI酶切位点， 通过 3'GPC3-2与 5'CD3-1搭桥， 同时在搭桥序列中引入 Gly4Ser的接头连接肽。 通过 PCR扩增， 获得编码如下序 列， 其结构域排列为 （ VLGPC3-(G4S)3-VHGPC3-(G4S)-VHCD3-VE(GGS)4GG-VLCD3)， 其序列为 SEQ ID NO: 9。 引物序列
SEQ ID NO: 1
5 ' GPC3 -1 CCCCGCTAGCTGATGTTGTGATGACTCAGTC
SEQ ID NO: 2
3 ' GPC3 -2 GGAGCCACCACCTCCTGAGGAGACGGTGACCAGGGTTCCCT
SEQ ID NO: 3
5 ' CD3 -1 TCCTCAGGAGGTGGTGGCTCCGATATCAAACTGCAGCA
SEQ ID NO: 4 扩增得到的序列 SEQ ID NO: 9用限制性内切酶 Nhel/Notl-HF同时酶切， 按照 酶供应商 （New England Biolabs, NEB ) 建议的反应条件在缓冲液 2中进行双酶 切。 表达 pH载体 （参见 WO/2011/035465实施例 7和图 15 ) 也用限制性内切酶 Nhel/Notl-HF进行同样的酶切，得到 pH/DHFR载体片段。 然后按照酶供应商 (NEB ) 建议的反应条件用 T4 DNA连接酶连接双酶切后 SEQ ID NO: 9的片段和 pH/DHFR载体片段,形成含有编码 GPC3/CD3双特异性抗体的核苷酸片段的表达载 体 pH-GPC3/CD,其详细连接结构如图 2所示。
2) GPC3/CD3双特异性抗体的表达和纯化
上述步骤得到的表达载体 PH-GPC3/CD3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-S ) 细胞中， 然 后根据 OptiCHO™ 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-S细胞的稳定克隆在摇瓶中 37°C， 130rpm的条件下 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° ( 。
培养上清中的 GPC3/CD3蛋白通过固定化金属亲和层析 (； immobilized metal affinity chromatography (IMAC))技术富集， 采用组氨酸亲和层析柱 （His Trap HP column, 购自 GE Healthcare)进行蛋白纯化。 按照层析柱厂商提供的方法步骤， 用 缓冲液 A (20mM 磷酸盐 pH 7.4, 0.4M NaCl)平衡， 然后将 PBS透析后的细胞培养 上清 （500 mL上清)加入到层析柱上 （10 mL；)， 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 20mM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°C 下操作。 纯化的蛋白经 PBS透析后测定 280nm波长光谱吸收， 以 0.53mg/OD的系数 计算蛋白浓度。 纯化的 GPC3/CD3双特异性抗体蛋白经 SDS-PAGE电泳分析， 如图 3所示， 其分子量在 55kD左右。 实施例 2. GPC3/CD3双特异性抗体与靶细胞结合特异性的分析
2.1 实验材料：
Figure imgf000014_0001
*用 Ficoll (来自 B iochrom)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。 PBMC中 含 T细胞。
2.2 实验方法：
通过荧光激活细胞分选仪 (FACS , 通常又称为流式细胞仪 FACScalibur, 由 BD公司提供)分析双特异性抗体 GPC3/CD3的结合能力。
具体方法如下：
1.取对数生长期的如表 1所列各肿瘤细胞接种到 6cm平皿中， 接种细胞密度约 为 90%， 37°C孵箱过夜培养。
2.使用 10mM的 EDTA消化细胞， 200gx5min离心收集细胞。 以 1 χ 106〜 l x l07/mL的浓度重悬于 1%含小牛血清的磷酸盐缓冲液 （NBS PBS)中， 按 lOOul/管的量加入流式专用管中。
3. 200gx5min离心， 弃上清。
4.两个实验组分别加入待测抗体 7B3/CD3和 806/CD3 , 同时一个对照组加入 抗体 NGR/CD3作为阴性对照， 另一个对照组为不加抗体的 PBS空白对 照。 各抗体的终浓度均为 2(^g/ml， 每管加入 100ul。 冰浴， 45分钟。
5.每管加入 2ml 1%NBS PBS， 以 200gx5min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 200gx5min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 200gx5min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。
每个样品至少采用流式细胞分析仪分析 10,000个细胞。
2.3 实验结果：
如图 4A-C所示的流式细胞仪分析结果， 本发明的 GPC3/CD3双特异抗体可以 与表达 GPC3的肝癌细胞系 Huh7和 HepG2结合， 而基本不与不表达 GPC3的 CHO- K1细胞系结合。 如图 4D和 E所示， GPC3/CD3抗体与 Jurkat和 PBMC细胞有效地结 合， 显示 GPC3/CD3双特异抗体对表面表达有 CD3的 T细胞的结合特异性。 综合以 上图 4A-E的结果可知,本发明的 GPC3/CD3双特异抗体具有同时特异性识别 GPC3 和 CD3的能力。 实施例 3. GPC3/CD3双特异性抗体介导的对肿瘤细胞的细胞毒杀伤作用
3.1 实验材料： 两种表达 GPC3有关的肿瘤细胞 （Huh-7和 HepG2细胞系） 和一种不表达 GPC3 的对照细胞 （CHO-K1细胞系） 被用来分别分析 GPC3/CD3双特异性介导的 T细胞 对肿瘤细胞的杀伤能力。
3.2 实验方法：
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 7 X 105/mL o 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 7 X 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 75 μ L/孔的体积加到 96孔板中。 然后各孔分别添加 25 L从 1000ng/mL到 0.1ng/mL的十倍系列梯度稀释的 GPC3/CD3单链双功能抗体。 在 37 °C ， 5% CO2的培养箱中孵育 40小时后， 根据生产商的操作说明， 用 CytoTox96®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 96®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 96®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 （foraiazan) 。 生成的红 色产物的量与裂解的细胞数成正比。
肿瘤细胞的杀伤率 （即， 细胞毒性％) 是根据 CytoTOX96® 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：
,→ . 实 ¾-效应细鹿自发靶细鹿自发
· ¾、 ¾、、 、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、，
, ^ > ,· . , , 八腦
¾细藤最大 ~ie细鹿自发
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" 指的是只含有效应细胞的对照孔自发产生的 LDH释放。 本 发明中是指效应细胞的对照孔
"靶细胞自发" 是指只含有靶细胞的对照孔在靶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处理后靶细胞使其完全裂解对对照孔所 产生的 LDH释放，
"靶细胞最大-靶细胞自发" 代表着靶细胞受外界处理后完全裂解所产生的 LDH释放。
3.3 实验结果：
GPC3/CD3双特异性抗体在不同浓度下对各肿瘤的细胞毒性结果如下。 （建 议根据图 5提供下表的具体数据）
Figure imgf000017_0001
从图 5和上表共同显示的结果可知， 不同浓度的 GPC3/CD3双特异性抗体对肝 癌细胞株 Huh-7和 HepG2均表现出比非肝癌细胞株 （即， CHO-K1 ) 明显强烈的细 胞毒杀伤作用。 这些结果表明表达 GPC3的肝癌细胞系可以被 GPC3/CD3双特异抗 体再靶向的人 T细胞特异性地有效裂解。
据上表和图 5的细胞杀伤％数据和所使用的双功能抗体的浓度， 采用 GraphPad Prism 5软件 （GraphPad Software inc., San Diego, USA) 分析程序计算得到双功能 抗体针对肿瘤细胞杀伤的 EC5Q值。 GPC3/CD3双特异性抗体对 HepG2细胞株的 EC5Q 值分别是 14.22ng/ml， 对 Huh-7细胞株的为 656.7ng/ml。
所有本说明书中列出的出版物的内容都包含在本说明书中。 此外， 本领域技 术人员可以理解， 在不背离权利要求书中所述的技术范围和实质内容的情况下， 对本发明进行多种不同的修饰和改变是可能的。 本发明还包括上述这些修饰和改 变。

Claims

权 利 要 求 书
1. 一种双特异性抗体， 其包含：
特异性识别磷脂酰肌醇蛋白多糖 -3的第一功能域，
特异性识别人 T细胞抗原 CD3的第二功能域， 和
连接上述功能域的接头。
2. 权利要求 1所述的抗体， 其中第一功能域特异性识别 GPC3的 C末端肽。
3. 权利要求 1所述的抗体， 其中第一和 /或第二功能域选自完整抗体， 单链抗 体 (scFV)， Fab片段， Fd片段， Fv片段， F(ab')2片段和其衍生物。
4. 权利要求 1所述的抗体， 其中第一和 /或第二功能域各自为包含一个重链可 变区和一个轻链可变区的单链抗体。
5. 权利要求 1的抗体， 其中第一功能域的氨基酸序列为， SEQ ID NO: 5或与 该氨基酸序列相似性为 95%及以上的氨基酸序列。
6. 权利要求 1 的抗体， 其中第二功能域的氨基酸序列为， SEQ ID NO: 7 或 与该氨基酸序列相似性为 95%及以上的氨基酸序列。
7. 权利要求 1所述的抗体， 其中第一和 /或第二功能域是人源化的， 嵌合的或 鼠源的。
8. 编码权利要求 1-7任一所述的抗体的核苷酸序列。
9. 包含有权利要求 8所述核苷酸序列的载体。
10.权利要求 9的载体，其为 pH载体。
1 1.包含有权利要求 9所述的载体的真核宿主细胞或原核宿主细胞。
12.权利要求 1 1的真核宿主细胞， 其为中国仓鼠卵巢细胞。
13.权利要求 1-7任一所述的抗体在制备治疗或预防肿瘤的药物中的应用。
PCT/CN2014/077521 2014-05-14 2014-05-14 针对磷脂酰肌醇蛋白多糖-3和t细胞抗原的双特异性抗体 WO2015172341A1 (zh)

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