WO2020088365A1 - 微环dna表达连接her2阳性细胞与效应细胞的桥接分子及其应用 - Google Patents

微环dna表达连接her2阳性细胞与效应细胞的桥接分子及其应用 Download PDF

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WO2020088365A1
WO2020088365A1 PCT/CN2019/113285 CN2019113285W WO2020088365A1 WO 2020088365 A1 WO2020088365 A1 WO 2020088365A1 CN 2019113285 W CN2019113285 W CN 2019113285W WO 2020088365 A1 WO2020088365 A1 WO 2020088365A1
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her2
cells
recombinant gene
cancer
variable region
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谌平
谢亦武
陈志英
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深圳新诺微环生物科技有限公司
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
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    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
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Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to a minicircle (MC) DNA vector, and more particularly to a microcircle DNA vector for expressing a bridging molecule connecting Her2 positive cells and effector cells in vivo.
  • MC minicircle
  • Her2 human epidermal growth factor receptor-2
  • Her2 / neu or ErbB2 is a member of the type I transmembrane tyrosine kinase receptor family and belongs to a type of transmembrane protein. Overexpression or mutation often induces homodimerization of Her2 or heterodimerization between Her2 and other members of the family, which in turn activates downstream RAS-MAPK and AKT signaling pathways targeting mTOR, leading to abnormal cell proliferation and Survive.
  • Her2 is highly expressed on the surface of a variety of cancer cells, including breast cancer, non-small cell lung cancer, salivary adenocarcinoma, gastric cancer, intestinal cancer, pancreatic cancer, bladder cancer, endometrial cancer, ovarian cancer, etc., is a broad spectrum Of cancer immunotherapy targets.
  • Her2 monoclonal antibody drugs are on the market, such as Trastuzumab (Herceptin) or Pertuzumab (Perjeta), which is used to treat breast cancer, gastric cancer and other cancers, and has achieved good clinical treatment effect.
  • Bispecific antibodies targeting Her2 have already entered clinical trials, such as Her2-TDB.
  • the currently reported Her2 bispecific antibodies (diabodies) are expressed in mammalian cells by traditional genetic engineering methods and then obtained by protein purification methods. In general, the traditional production preparation, purification, storage and transportation processes are complex, costly, and have a greater risk of drug contamination.
  • the double-antibody has a short half-life, which often requires continuous administration and increased dosage, which virtually increases the patient's economic burden, and there is a risk of drug tolerance.
  • the present invention expresses bispecific antibodies in vivo through the microcircle DNA gene carrier, and mediates effector cells (T cells or NK cells) to kill Her2 positive cancer cells (target cells).
  • T cells or NK cells mediates effector cells
  • target cells Her2 positive cancer cells
  • the invention avoids the problems in the prior art mentioned above, and is safe, efficient, low in cost and affordable for patients.
  • the technology closest to the present invention is the technology of microcircle DNA vector to express bispecific antibody MC.BsAb (CN201710245146.6).
  • the invention relates to a minicircle (MC) DNA vector expressing a bridging molecule connecting Her2 positive cells and effector cells in vivo, which is used to treat Her2 related cancer.
  • MC minicircle
  • the present invention provides a recombinant gene carrier expressing a bridge between Her2-positive cells and effector cells (Bridge between Her2-positive cells and Her2-BTEC).
  • the bridge molecule includes: target specificity with Her2-positive cells Part A that binds, and Part B that specifically binds to the target of effector cells.
  • the recombinant gene vector is selected from non-viral gene vectors, such as standard plasmids or other circular expression cassettes.
  • the recombinant gene vector is selected from microcircle DNA vectors.
  • the recombinant gene vector is selected from recombinant expression vectors.
  • the recombinant gene vector is selected from prokaryotic expression vector or eukaryotic expression vector. More preferably, the recombinant gene vector is selected from eukaryotic expression vectors. Particularly preferably, the recombinant gene vector is selected from recombinant expression vectors for mammalian cell expression.
  • the bridging molecule is selected from proteins or polypeptides.
  • the bridging molecule is selected from bispecific antibodies. More preferably, the bridging molecule is selected from a human-monkey cross-reactive Bispecific Antibody (hm-BsAb).
  • the part A and the part B are selected from protein molecules or polypeptide molecules, respectively.
  • the part A and part B are independently selected from Fab, Fab ', single chain antibodies (scFv), single domain antibodies (V H H), single-chain T-cell receptor (the scTCR), and others.
  • the Her2 positive cells are selected from Her2 positive cancer cells, Her2 overexpressing cells, or others.
  • the effector cells are selected from T cells, NK cells or others.
  • the target of the specific binding of Part A is selected from the various epitopes of Her2.
  • the target of specific binding of the part B is selected from CD3, CD16, CD28, 4-1BB, OX40, TCR, CD56, NKG2D, NCR or others.
  • the part A is selected from a single chain antibody (scFv), which comprises: a heavy chain variable region, and a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 1 and 3.
  • scFv single chain antibody
  • the portion B is selected from a single chain antibody (scFv), (1) which comprises: a heavy chain variable region and a light chain variable region, the amino acid sequence of which is selected from SEQ ID NO: 5 and 7, Or the group of 9 and 11; the target of the specific binding of the part B is selected from CD3; or (2) it includes: a heavy chain variable region and a light chain variable region, the amino acid sequence of which is SEQ ID NO : 13 and 15; the target of the specific binding of Part B is selected from CD16.
  • scFv single chain antibody
  • the heavy chain variable region included in Part A has an amino acid sequence that is at least 90%, 95%, 98%, or 99% homologous to the sequence shown in SEQ ID NO: 1, and the light chain included may The variable region has at least 90%, 95%, 98%, or 99% homology with the sequence shown in SEQ ID NO: 3, and has the same function as Part A described in the previous embodiment, that is, specific binding The same target.
  • the heavy chain variable region included in Part B has an amino acid sequence that is at least 90%, 95%, 98%, or 99% homologous to the sequence shown in SEQ ID NO: 5 or 9
  • the chain variable region has an amino acid sequence that is at least 90%, 95%, 98%, or 99% homologous to the sequence shown in SEQ ID NO: 7 or 11, and has the same function as Part B described in the previous embodiment, That is, it specifically binds to the same target.
  • the present invention provides a recombinant gene carrier expressing a bridge molecule of Her2 positive cells and effector cells, the recombinant gene carrier containing the gene encoding the bridge molecule.
  • the recombinant gene vector comprises: a part A coding gene, and / or a part B coding gene.
  • part A is selected from a single chain antibody (scFv), and its coding gene includes: heavy chain variable region and light chain variable region coding genes, and its nucleotide sequence is as SEQ ID NO: 2 and 4. Show.
  • scFv single chain antibody
  • coding gene includes: heavy chain variable region and light chain variable region coding genes, and its nucleotide sequence is as SEQ ID NO: 2 and 4. Show.
  • part B is selected from a single chain antibody (scFv), and its coding gene (1) comprises: heavy chain variable region and light chain variable region coding genes whose nucleotide sequence is selected from SEQ ID NO: 6 and 8, or 10 and 12, as shown in the group; or (2) contains: heavy chain variable region and light chain variable region encoding genes, the nucleotide sequence shown in SEQ ID NO: 14 and 16.
  • scFv single chain antibody
  • the recombinant gene vector comprises: a nucleotide sequence having at least 90%, 95%, 98% or 99% homology with the nucleotide sequence of the gene encoding the above-mentioned part A and / or part B
  • the encoded bridge molecule has the same function as the bridge molecule described in the previous embodiment. It is well known to those skilled in the art that, without changing the encoded amino acid, one or more codons in the coding gene sequence can be replaced synonymously, such as one or several codons, such as 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 codons.
  • the present invention provides a bridging molecule expressed by the recombinant gene vector described in the previous embodiment.
  • the recombinant gene vector is selected from non-viral gene vectors, such as standard plasmids or other circular expression cassettes.
  • the recombinant gene vector is selected from microcircle DNA vectors.
  • the present invention provides a method for preparing the recombinant gene vector described in the previous embodiment, and the specific steps include:
  • VL light chain variable region
  • VH heavy chain variable region
  • the Her2 positive cells are selected from Her2 positive cancer cells, Her2 overexpressing cells, or others.
  • the effector cells are selected from T cells, NK cells or others.
  • the recombinant gene vector is selected from non-viral gene vectors, such as standard plasmids or other circular expression cassettes.
  • the recombinant gene vector is selected from microcircle DNA vectors.
  • the bridging molecule is selected from proteins or polypeptides.
  • the bridging molecule is selected from bispecific antibodies.
  • the bridging molecule is selected from a human-monkey cross-reactive Bispecific Antibody (hm-BsAb).
  • the present invention provides a host cell comprising the recombinant gene vector described in the previous embodiment or the recombinant gene vector obtained by the preparation method described in the previous embodiment.
  • the host cell includes a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.
  • the present invention provides a method for preparing the bridge molecule described in the previous embodiment, and the specific steps include:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the recombinant gene vector described in the previous embodiment, or the recombinant gene vector obtained by the preparation method described in the previous embodiment, or the recombinant gene vector described in the previous embodiment Expressed bridging molecules, and pharmaceutically acceptable carriers.
  • the pharmaceutical composition can be made into a pharmaceutical preparation according to conventional methods. During the preparation process, it is preferable to mix or dilute the recombinant gene carrier or bispecific antibody with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier When the carrier serves as a diluent, it can be solid, semi-solid, or liquid.
  • the preparation is selected from the form of tablets, pills, powders, capsules, suspensions, emulsions, solutions, aerosols, solutions for injection and the like.
  • Suitable carriers, excipients or diluents include water, lactose, glucose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, polyvinylpyrrolidone, methyl hydroxybenzoate, propyl hydroxybenzoate, talc , Magnesium stearate and mineral oil.
  • the formulation may also include fillers, anticoagulants, lubricants, humectants, flavoring agents, emulsifiers, preservatives, and the like.
  • the present invention provides the use of the recombinant gene vector, the bridging molecule, the host cell or the pharmaceutical composition in the previous embodiment in the preparation of a medicament for treating Her2-related cancer.
  • the Her2-related cancer is selected from breast cancer, non-small cell lung cancer, salivary adenocarcinoma, gastric cancer, intestinal cancer, pancreatic cancer, bladder cancer, endometrial cancer, and ovarian cancer.
  • the Her2-related cancer is selected from breast cancer and gastric cancer.
  • the positive effects of the present invention include: the present invention discloses for the first time the design scheme of Her2 specific Her2 positive cells and effector cell bridging molecule microcircle DNA vector (MC.Her2-BTEC), which is suitable for the treatment of Her2 related cancers.
  • Her2-BTEC can greatly increase the killing effect of effector cells on Her2-positive cells.
  • the cytotoxic effect increases with the increase of Her2-BTEC concentration.
  • Her2-BTEC treatment can significantly reduce tumor burden and prolong the survival of tumor-bearing mice.
  • the Her2-BTEC of the present invention can also cross-recognize monkey antigens, which is beneficial to the pre-clinical safety and effectiveness of BTEC (BsAb, bispecific antibody) drugs based on non-human primate models sexual evaluation. It can be seen that the Her2-BTEC and microcircle DNA vectors of the present invention have good prospects for the prevention and treatment of Her2-related cancers, and provide new clinical ideas.
  • Figure 1 pMC. Her2-BTEC microcircle DNA mother plasmid vector map.
  • Figure 2 MC.Her2-BTEC microcircle preparation and BTEC expression; (left) agarose gel electrophoresis to detect MC; (right) SDS-PAGE to detect BTEC protein expression; Lane A: cell supernatant; Lane B: flow-through solution ; Lane C: 20mM imidazole eluent; Lane D: 50mM imidazole eluent; Lane E: 200mM imidazole eluent; Lane F: 500mM imidazole eluent.
  • FIG. 3 Her2-BTEC mediates the cytotoxic effect of effector cells (T cells) on target cells (human ovarian cancer SKOV3 cells); T cells and Her2-positive ovarian cancer SKOV3 cells were incubated for 12 hours with an effective target ratio of 10: 1 , LDH release method to detect Her2-BTEC-mediated cell killing rate.
  • Figure 4 Results of treatment experiments on mice transplanted with human ovarian cancer.
  • Her2-BTEC Design Her2-BTEC and construct the corresponding microcircle DNA expression vector.
  • the Her2-BTEC expression box includes a variety of structures, which are listed as follows:
  • SP is a signal peptide (Signal Peptide);
  • Linker is a linking sequence.
  • V L represents the light chain variable region, and V H represents the heavy chain variable region.
  • the anti-Her2scFv and antiCD3 (or antiCD16) scFv positions are interchangeable.
  • the amino acid sequence of antiHer2.V H is shown in SEQ ID NO: 1
  • the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2
  • the amino acid sequence of antiHer2.V L is shown in SEQ ID NO: 3
  • the nucleotide sequence of the coding gene is shown in SEQ ID NO: 4
  • the amino acid sequence of antiCD3.V H is shown in SEQ ID NO: 5 or 9
  • the nucleotide sequence of the coding gene is shown in SEQ ID NO: 6 or 10.
  • the amino acid sequence of antiCD3.V L is shown in SEQ ID NO: 7 or 11, the nucleotide sequence of the coding gene is shown in SEQ ID NO: 8 or 12; the amino acid sequence of antiCD16.V H is shown in SEQ ID NO: 13, the nucleotide sequence encoding a gene as SEQ ID NO: 14 shown; L antiCD16.V an amino acid sequence as SEQ ID NO: 15, the nucleotide sequence encoding a gene, such as SEQ ID NO: 16 Suo Show.
  • the DART expression frame After translation and shearing, the DART expression frame forms two chains (Chain 1, Chain 2); the two chains form a stable heterodimer through paired E / K-coli.
  • the positions of Chain1 and Chain2 are interchangeable.
  • SP is a signal peptide (Signal)
  • Linker is a linking sequence
  • Furin is a furin protease cleavage site
  • 2A is a 2A self-cleavage site.
  • the amino acid sequence of the Furin cleavage site is RX- [R / K] -R (such as RRKR)
  • X refers to any kind of amino acid
  • E / Kcoli is a paired helical structure with opposite electrical properties
  • Kcoli and E The interchangeable positions of coli
  • 2A includes E2A, F2A, P2A and T2A.
  • amino acid sequence of each element of antiHer2.V H , antiHer2.V L , antiCD3.V H , antiCD3.V L , antiCD16.V H , antiCD16.V L and their coding genes are the same as in (1) above.
  • the DART-Fc expression frame is translated and sheared to form two chains (Chain 1, Chain 2); Chain 1 and Chain 2 form stable differences through the paired E / K-coli and Fc "knob-into-hole" structure Dimer.
  • the "knob-into-hole” design not only avoids the production of homodimers, but also increases the half-life.
  • the positions of Chain1 and Chain2 are interchangeable.
  • SP is a signal peptide (Signal)
  • Linker is a linking sequence
  • Furin is a furin protease cleavage site
  • 2A is a 2A self-cleavage site.
  • the amino acid sequence of the Furin cleavage site is RX- [R / K] -R (such as RRKR), X refers to any kind of amino acid
  • E / Kcoli is a paired helical structure with opposite electrical properties
  • Fc-knob and Fc-hole are IgG CH2-CH3 mutants of Fc segment, respectively, and they can also exchange positions
  • 2A includes E2A, F2A, P2A and T2A.
  • amino acid sequence of each element of antiHer2.V H , antiHer2.V L , antiCD3.V H , antiCD3.V L , antiCD16.V H , antiCD16.V L and their coding genes are the same as in (1) above.
  • microcircle DNA vector pMC.Her2-BTEC is transformed into E. coli E. coli.ZYCY10P3S2T, and the microcircle MC.Her2-BTEC is obtained according to the standard microcircle preparation method.
  • microcircle DNA empty vector pMC.BESPX engineering bacteria E. coli.ZYCY10P3S2T
  • preparation method of microcircle see reference Nat Biotechnol.2010,28: 1287-1289
  • Figure 2 shows the results of agarose gel electrophoresis prepared by MC.Her2-BTEC microcircles. It can be seen from the figure that the microcircle MC.Her2-BTEC can be prepared by the construction method of Her2-BTEC microcircle DNA vector.
  • the supercircle DNA transfection kit (Invitrogen) was used to transfect the above-mentioned microcircle DNA into 293T cells. After cultured in serum-free medium for three days, 293T cell culture supernatants were collected.
  • Her2-BTEC uses His-Tag affinity resin (cOmplete His-Tag Purification Resin, Roche) for purification.
  • the purified protein is qualitatively detected by PAGE or Western Blot, and the protein concentration is quantitatively detected by Bradford method.
  • FIG. 2 shows the results of SDS-PAGE detection of Her2-BTEC expression. It can be seen from the figure that Her2-BTEC can be prepared by the methods of cell transfection, protein expression, and purification.
  • Target cells Her2 positive cells, such as SKOV3 cells
  • effector cells T cells or NK cells
  • Each experimental group was assigned 1 ⁇ 10 5 target cells and effector cells, grouped as follows:
  • T cells or NK cells effector cells
  • target cells Her2 positive cells, such as human ovarian cancer SKOV3 cells
  • Her2-BTEC Her2 positive cells and effector cell bridging molecules
  • the amount of cell inoculation is 2 ⁇ 10 4 / well; at the same time set up grouping (as shown in Table 1; 3 replicate wells per group).
  • Cell killing rate (experiment-spontaneous effector cell-spontaneous target cell) / (maximum target cell-spontaneous target cell) x 100%
  • FIG. 3 shows the cytotoxic effect of Her2-BTEC-mediated effector cells (T cells) on target cells (human ovarian cancer SKOV3 cells). It can be seen from the figure that the cytotoxic effect increases continuously with the increase of Her2-BTEC concentration.
  • the immunodeficient NOD / SCID mice are inoculated with firefly luciferase (luc) -labeled Her2 positive tumor cells (such as SKOV3-luc).
  • luc firefly luciferase
  • mice 7 days after inoculation of tumor cells, record the fluorescence intensity of luciferase and monitor the tumor formation of mice with In Vivo Imaging System (IVIS); set up groups at the same time, including control group, control group, T cell group and experiment There are three groups with 5 mice in each group.
  • IVIS In Vivo Imaging System
  • the control group does not apply any treatment
  • the T cell group injects human T cells at the prescribed time point
  • the experimental group injects MC.Her2-BTEC microcircle DNA and human T cells at the prescribed time point.
  • Figure 4 shows the results of treatment experiments on mice transplanted with human ovarian cancer. It can be seen from the figure that compared with untreated tumor-bearing control mice, Her2-BTEC treatment can significantly reduce tumor burden and prolong the survival period of tumor-bearing mice.

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Abstract

提供了一种能同时结合Her2阳性细胞以及T细胞或NK细胞的双特异性抗体，及其编码该双特异性抗体的微环DNA表达载体。该双特异性抗体能够介导T细胞或NK细胞杀灭Her2阳性癌细胞，可用于治疗Her2相关癌症。

Description

微环DNA表达连接HER2阳性细胞与效应细胞的桥接分子及其应用 技术领域
本发明属于生物医药领域，具体涉及一种微环(Minicircle，MC)DNA载体，更具体为一种用于在体内表达连接Her2阳性细胞与效应细胞的桥接分子的微环DNA载体。
背景技术
Her2(human epidermal growth factor receptor-2，人表皮生长因子受体2)，又称Her2/neu或ErbB2，为Ⅰ型跨膜酪氨酸激酶受体家族的成员，属于跨膜蛋白的一种，其超量表达或突变时往往诱导Her2同二聚化或Her2与家族其他成员间异二聚化的产生，进而激活下游的RAS-MAPK、靶向mTOR的AKT信号通路，导致细胞的异常增殖和存活。目前已发现Her2在多种癌症细胞表面高表达，包括乳腺癌、非小细胞肺癌、唾液腺癌、胃癌、肠癌、胰腺癌、膀胱癌、子宫内膜癌、卵巢癌等，是一种广谱的癌症免疫治疗靶点。
目前已有Her2单抗药物上市，比如Trastuzumab曲妥珠单抗(Herceptin赫赛汀)或Pertuzumab帕妥珠单抗(Perjeta)，其用于治疗乳腺癌、胃癌等癌症，并取得了良好的临床治疗效果。不过，近年来理论研究表明双特异性抗体对于癌症/肿瘤的治疗疗效更佳，已有靶向Her2的双特异性抗体进入临床试验阶段，比如Her2-TDB。现有报道的Her2双特异性抗体(双抗)均通过传统的基因工程方法在哺乳动物细胞中表达，之后通过蛋白纯化方法得到。总体而言，传统的生产制备、纯化以及储存运输等过程均很复杂，成本很高，并且存在药品污染的较大风险。另外，双抗的半衰期较短，往往需要持续给药，增加用量，这无形中增加了患者的经济负担，并且存在药物耐受性的风险。
本发明通过微环DNA基因载体，在体内表达双特异性抗体，介导效应细胞(T细胞或NK细胞)杀灭Her2阳性癌细胞(靶细胞)。这种以基因投递的方式在体内产生Her2双特异性抗体的研究在现有技术中未见相关报道。本发明避免了上述现有技术中存在的问题，而且安全、高效、成本较低、患者可负担。与本发明最接近的技术是微环DNA载体表达双特异性抗体的技术MC.BsAb(CN201710245146.6)。
发明内容
本发明涉及微环(Minicircle，MC)DNA载体在体内表达连接Her2阳性细胞与效应细胞的桥接分子，用于治疗Her2相关癌症。
本发明提供了一种表达Her2阳性细胞与效应细胞桥接分子(Bridge between Her2-positive cells and effector cells，Her2-BTEC)的重组基因载体，所述桥接分子包含：与Her2阳性细胞作用靶点特异性结合的部分A，和与效应细胞作用靶点特异性结合的部分B。
在一个方面中，所述重组基因载体选自非病毒基因载体，如标准质粒或其它环状表达盒子。优选的，所述重组基因载体选自微环DNA载体。
在一个方面中，所述重组基因载体选自重组表达载体。优选的，所述重组基因载体选自原核表达载体或真核表达载体。更优选的，所述重组基因载体选自真核表达载体。特别优选的，所述重组基因载体选自用于哺乳动物细胞表达的重组表达载体。
在一个方面中，所述桥接分子选自蛋白或多肽。优选的，所述桥接分子选自双特异性抗体。更优选的，所述桥接分子选自人-猴交叉性双特异性抗体(Human-Monkey cross-reactive Bispecific Antibody，hm-BsAb)。
在一个方面中，所述部分A和所述部分B分别选自蛋白分子或多肽分子。优选的，所述部分A和部分B分别选自Fab、Fab’、单链抗体(scFv)、单域抗体(V HH)、单链T细胞受体(scTCR)和其它。
在一个方面中，所述Her2阳性细胞选自Her2阳性癌细胞、Her2过表达细胞或其它。所述效应细胞选自T细胞、NK细胞或其它。
在一个方面中，所述部分A特异性结合的作用靶点选自Her2的各个抗原表位。所述部分B特异性结合的作用靶点选自CD3、CD16、CD28、4-1BB、OX40、TCR、CD56、NKG2D、NCR或其它。
在一个方面中，所述部分A选自单链抗体(scFv)，其包含：重链可变区，和轻链可变区，其氨基酸序列如SEQ ID NO：1和3所示。
在一个方面中，所述部分B选自单链抗体(scFv)，(1)其包含：重链可变区和轻链可变区，其氨基酸序列如选自SEQ ID NO：5和7，或9和11的组所示；所述部分B特异性结合的作用靶点选自CD3；或者(2)其包含：重链可变 区和轻链可变区，其氨基酸序列如SEQ ID NO：13和15所示；所述部分B特异性结合的作用靶点选自CD16。
在一个方面中，所述部分A包含的重链可变区具有与SEQ ID NO：1所示序列至少90％、95％、98％或99％同源性的氨基酸序列，包含的轻链可变区与SEQ ID NO：3所示序列具有至少90％、95％、98％或99％同源性的氨基酸序列，且具有与之前实施方案中所述部分A具有相同功能，即特异性结合相同的作用靶点。
在一个方面中，所述部分B包含的重链可变区具有与SEQ ID NO：5或9所示序列至少90％、95％、98％或99％同源性的氨基酸序列，包含的轻链可变区具有与SEQ ID NO：7或11所示序列至少90％、95％、98％或99％同源性的氨基酸序列，且具有与之前实施方案中所述部分B具有相同功能，即特异性结合相同的作用靶点。
本发明提供了一种表达Her2阳性细胞与效应细胞桥接分子的重组基因载体，所述重组基因载体包含所述桥接分子的编码基因。
在一个方面中，所述重组基因载体包含：部分A的编码基因，和/或包含部分B的编码基因。
在一个方面中，部分A选自单链抗体(scFv)，其编码基因包含：重链可变区和轻链可变区的编码基因，其核苷酸序列如SEQ ID NO：2和4所示。
在一个方面，部分B选自单链抗体(scFv)，其编码基因(1)包含：重链可变区和轻链可变区的编码基因，其核苷酸序列如选自SEQ ID NO：6和8、或10和12的组所示；或者(2)包含：重链可变区和轻链可变区的编码基因，其核苷酸序列如SEQ ID NO：14和16所示。
在一个方面中，所述重组基因载体包含：与上述部分A和/或部分B的编码基因的核苷酸序列具有至少90％、95％、98％或99％同源性的核苷酸序列，且编码得到的桥接分子具有与之前实施方案中所述桥接分子相同的功能。本领域技术人员公知，在不改变所编码的氨基酸的情况下，所述编码基因序列中的一个或多个密码子可以进行等义替换，如一个或几个密码子，如1、2、3、4、5、6、7、8、9、10、15、20、30、40、50个密码子。
本发明提供了一种由之前实施方案中所述重组基因载体所表达的桥接分子。
在一个方面中，所述重组基因载体选自非病毒基因载体，如标准质粒或其它 环状表达盒子。优选的，所述重组基因载体选自微环DNA载体。
本发明提供了一种之前实施方案中所述重组基因载体的制备方法，具体步骤包括：
(1)从现有技术中分别获取Her2抗体、CD3抗体及CD16抗体的轻链可变区(VL)序列、重链可变区(VH)序列；
(2)根据上述(1)中VH、VL序列设计成多种组合形式的桥接分子，并构建表达所述桥接分子的重组基因载体；
任选的，
(3)在体内外鉴定所述重组基因载体的表达水平，并检测表达产物介导效应细胞对Her2阳性细胞的杀伤效果。
在一个方面中，所述Her2阳性细胞选自Her2阳性癌细胞、Her2过表达细胞或其它。所述效应细胞选自T细胞、NK细胞或其它。
在一个方面中，所述重组基因载体选自非病毒基因载体，如标准质粒或其它环状表达盒子。优选的，所述重组基因载体选自微环DNA载体。
在一个方面中，所述桥接分子选自蛋白或多肽。优选的，所述桥接分子选自双特异性抗体。特别优选的，所述桥接分子选自人-猴交叉性双特异性抗体(Human-Monkey cross-reactive Bispecific Antibody，hm-BsAb)。
本发明提供了一种宿主细胞，包含之前实施方案中所述重组基因载体，或由之前实施方案中所述制备方法所得到的重组基因载体。
在一个实施方案中，所述宿主细胞包括细菌细胞、酵母菌细胞、昆虫细胞或哺乳动物细胞。
本发明提供了一种之前实施方案中所述桥接分子的制备方法，具体步骤包括：
(1)构建所述重组基因载体；
(2)将所述重组基因载体导入宿主细胞；
(3)在适合表达的条件下，培养宿主细胞，诱导表达，分离纯化，获得所述桥接分子。
本发明提供了一种药物组合物，包含之前实施方案中所述重组基因载体，或由之前实施方案中所述制备方法所得到的重组基因载体，或由之前实施方案中所述重组基因载体所表达的桥接分子，以及药学上可接受的载体。
在一个方面中，所述药物组合物可根据常规方法制成药物制剂。制剂过程中， 优选将重组基因载体或双特异性抗体与药学上可接受的载体混合或用载体稀释。当载体作为稀释剂时，其可以为固体、半固体或液体。制剂选自片剂、丸剂、粉剂、胶囊、混悬剂、乳剂、溶液剂、气溶胶、注射用溶液等形式。合适的载体、赋形剂或稀释剂包括水、乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、硅酸钙、纤维素、聚乙烯吡咯烷酮、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石粉、硬脂酸镁和矿物油等。制剂还可以包括填充剂、抗凝血剂、润滑剂、保湿剂、调味剂、乳化剂、防腐剂等。
本发明提供了之前实施方案中所述重组基因载体、所述桥接分子、所述宿主细胞或所述药物组合物在制备用于治疗Her2相关癌症的药物中的用途。优选的，所述Her2相关癌症选自乳腺癌、非小细胞肺癌、唾液腺癌、胃癌、肠癌、胰腺癌、膀胱癌、子宫内膜癌、卵巢癌。特别优选的，所述Her2相关癌症选自乳腺癌、胃癌。
本发明的积极效果包括：本发明首次公开Her2特异性的Her2阳性细胞与效应细胞桥接分子的微环DNA载体(MC.Her2-BTEC)设计方案，适用于Her2相关癌症的治疗。Her2-BTEC能够大幅度提高效应细胞对Her2阳性细胞的杀伤作用。细胞毒性作用随Her2-BTEC浓度的增加而不断增大。与未治疗的荷瘤对照小鼠相比，Her2-BTEC治疗能够显著降低瘤负荷并延长荷瘤小鼠的生存期。本发明的Her2-BTEC除了结合人的抗原分子之外，还能交叉识别猴的抗原，有利于BTEC(BsAb，双特异性抗体)药物基于非人灵长类动物模型的临床前安全性与有效性评价。可见，本发明的Her2-BTEC、微环DNA载体对于预防和治疗Her2相关癌症具有很好的前景，并提供了新的临床思路。
附图说明
图1：pMC.Her2-BTEC微环DNA母质粒载体图谱。
图2：MC.Her2-BTEC微环制备与BTEC表达；(左)琼脂糖凝胶电泳检测MC；(右)SDS-PAGE检测BTEC蛋白表达；Lane A：细胞上清；Lane B：流穿液；Lane C：20mM咪唑洗脱液；Lane D：50mM咪唑洗脱液；Lane E：200mM咪唑洗脱液；Lane F：500mM咪唑洗脱。
图3：Her2-BTEC介导效应细胞(T细胞)对靶细胞(人卵巢癌SKOV3细胞)的细胞毒性作用；T细胞和Her2阳性的卵巢癌SKOV3细胞共孵育12小时， 效靶比10:1，LDH释放法检测Her2-BTEC介导的细胞杀伤率。
图4：人卵巢癌移植瘤小鼠治疗实验的结果。
具体实施方式
下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。在不背离本发明精神的情况下，本领域技术人员结合公知技术，可以对本发明做出许多修改，这样的修改也落入本发明的范围。
实施例1、Her2阳性细胞与效应细胞桥接分子(Her2-BTEC)的构建
设计Her2-BTEC并构建相应的微环DNA表达载体，Her2-BTEC表达框包括多种结构，分列如下：
(1)scFv-scFv结构：
Figure PCTCN2019113285-appb-000001
其中，SP为信号肽(Signal Peptide)；Linker为连接序列。V L代表轻链可变区，V H代表重链可变区。anti-Her2scFv、antiCD3(或antiCD16)scFv位置可互换。
其中，antiHer2.V H的氨基酸序列如SEQ ID NO：1所示，编码基因的核苷酸序列如SEQ ID NO：2所示；antiHer2.V L的氨基酸序列如SEQ ID NO：3所 示，编码基因的核苷酸序列如SEQ ID NO：4所示；antiCD3.V H的氨基酸序列如SEQ ID NO：5或9所示，编码基因的核苷酸序列如SEQ ID NO：6或10所示；antiCD3.V L的氨基酸序列如SEQ ID NO：7或11所示，编码基因的核苷酸序列如SEQ ID NO：8或12所示；antiCD16.V H的氨基酸序列如SEQ ID NO：13所示，编码基因的核苷酸序列如SEQ ID NO：14所示；antiCD16.V L的氨基酸序列如SEQ ID NO：15所示，编码基因的核苷酸序列如SEQ ID NO：16所示。
(2)双特异性抗体DART(Dual-Affinity Re-Targeting antibody)结构：
Figure PCTCN2019113285-appb-000002
DART表达框经翻译、剪切后形成两条链(Chain 1、Chain 2)；这两条链通过配对的E/K-coli形成稳定的异二聚体。Chain1、Chain2位置可互换。
其中，SP为信号肽(Signal Peptide)；Linker为连接序列，Furin为furin蛋白酶剪切位点，2A为2A自剪切位点。Furin剪切位点的氨基酸序列为R-X-[R/K]-R(比如RRKR)，X指代任一种氨基酸；E/K coli为电性相反、配对的螺旋结构，且K coli和E coli可互换位置；2A包括E2A、F2A、P2A和T2A等。
其中，antiHer2.V H、antiHer2.V L、antiCD3.V H、antiCD3.V L、antiCD16.V H、antiCD16.V L各元件的氨基酸序列及其编码基因与上述(1)中的一致。
(3)双特异性抗体DART-Fc结构：
Figure PCTCN2019113285-appb-000003
DART-Fc表达框经翻译、剪切后形成两条链(Chain 1、Chain 2)；Chain 1 和Chain 2通过配对的E/K-coli以及Fc“knob-into-hole”结构形成稳定的异二聚体。“knob-into-hole”设计既可避免同二聚体的产生，还能增加半衰期。Chain1、Chain2位置可互换。
其中，SP为信号肽(Signal Peptide)；Linker为连接序列，Furin为furin蛋白酶剪切位点，2A为2A自剪切位点。Furin剪切位点的氨基酸序列为R-X-[R/K]-R(比如RRKR)，X指代任一种氨基酸；E/K coli为电性相反、配对的螺旋结构，且K coli和E coli可互换位置；Fc-knob和Fc-hole分别为IgG Fc段CH2-CH3突变体，它们也可互换位置；2A包括E2A、F2A、P2A和T2A等。
其中，antiHer2.V H、antiHer2.V L、antiCD3.V H、antiCD3.V L、antiCD16.V H、antiCD16.V L各元件的氨基酸序列及其编码基因与上述(1)中的一致。
实施例2、Her2-BTEC微环DNA载体的构建及微环(MC)的制备
一、实验方法
1、合成Her2-BTEC的编码基因，为方便BTEC蛋白纯化与检测，在C端插入6xHis标签。
2、选择合适的位点双酶切线性化微环DNA空载体pMC.BESPX。
3、使用seamless克隆或者传统克隆方法(如，酶切-连接)将Her2-BTEC的编码基因插入线性化的空载体pMC.BESPX中，构建成微环DNA载体pMC.Her2-BTEC。
4、微环DNA载体pMC.Her2-BTEC转化大肠杆菌E coli.ZYCY10P3S2T，按标准微环制备方法得到微环MC.Her2-BTEC。
其中，微环DNA空载体pMC.BESPX、工程菌E coli.ZYCY10P3S2T，以及微环制备方法见参考文献Nat Biotechnol.2010,28:1287-1289.
二、实验结果
图2(左)显示了MC.Her2-BTEC微环制备的琼脂糖凝胶电泳检测结果。由图中可知，通过Her2-BTEC微环DNA载体的构建方法，能够制备得到微环MC.Her2-BTEC。
实施例3、Her2-BTEC的表达、纯化
一、Her2-BTEC在293T细胞中的表达
采用superfect质粒转染试剂盒(Invitrogen公司)将上述微环DNA转染293T细胞，在无血清培养基中培养三天后分别收集293T细胞培养上清。
二、Her2-BTEC的纯化
1、收集细胞培养上清液进行低温超速离心，取上清。
2、Her2-BTEC采用His-Tag亲和树脂(cOmplete His-Tag Purification Resin， Roche)纯化。
3、纯化后的蛋白使用PAGE或Western Blot定性检测，并使用Bradford法定量检测蛋白浓度。
4、纯化产物置于-20℃或-80℃长期保存。
三、实验结果
图2(右)显示了Her2-BTEC表达的SDS-PAGE检测结果。由图中可知，通过细胞转染、蛋白表达、纯化的方法，能够制备得到Her2-BTEC。
实施例4、Her2-BTEC的结合实验
利用流式细胞术检测Her2-BTEC与靶细胞(Target，T)、效应细胞(Effector，E)的结合活性，包括如下步骤：
1、细胞培养：靶细胞(Her2阳性细胞，比如SKOV3细胞)；效应细胞(T细胞或NK细胞)。
2、收集细胞：贴壁细胞使用胰酶消化，加含血清培养基中和后离心弃上清得细胞；悬浮细胞直接离心收集。
3、两种细胞均用预冷PBS洗涤2次，200g离心4min，收集细胞，分别计数。
4、每个实验组分别分配1×10 5个靶细胞和效应细胞，分组如下：
空白组(不加Her2-BTEC)和Her2-BTEC组
5、加入Her2-BTEC溶液100μL/组，冰上孵育30min。
6、加入1mL预冷PBS洗涤，200g离心4min，收集细胞，加事先预冷并混好anti-flag抗体的PBS 100μL，冰上孵育30min。
7、加入1mL预冷PBS洗涤，200g离心4min，收集细胞，加入100μl PBS重悬，上流式观察结合情况。
实施例5、Her2-BTEC介导效应细胞杀伤靶细胞
一、实验分组
利用LDH(乳酸脱氢酶)释放法测定Her2-BTEC介导效应细胞(Effector，E)对靶细胞(Target，T)的杀伤作用。
实验材料：效应细胞(T细胞或NK细胞)，靶细胞(Her2阳性细胞，比如人卵巢癌SKOV3细胞)，Her2-BTEC(Her2阳性细胞与效应细胞桥接分子)。
表1 Her2-BTEC介导效应细胞杀伤靶细胞的不同分组
Figure PCTCN2019113285-appb-000004
Figure PCTCN2019113285-appb-000005
二、实验方法
1、提前1天将靶细胞接种于96孔板上，细胞接种量为2×10 4/孔；同时设置分组(如表1所示；每组3个复孔)。
2、第二天更换新鲜opti-MEM培养基，100μL/孔。
3、效应细胞(E)计数并重悬于opti-MEM培养基中，按照T∶E＝1∶10的比例，根据(a)中分组加入相应数量的效应细胞(2.0×10 5/孔，100μL/孔)。
4、根据分组，在相应的孔中每孔加入相应浓度的Her2-BTEC，混匀；在靶细胞最大组对应孔中还需加入细胞裂解液破碎细胞，以便充分释放LDH。
5、37℃细胞培养箱孵育12小时。
6、根据LDH试剂盒(Promega，美国)说明书，检测LDH释放量并计算细胞杀伤率，公式如下：
细胞杀伤率＝(实验-效应细胞自发-靶细胞自发)/(靶细胞最大-靶细胞自发)x100％
二、实验结果
图3显示了Her2-BTEC介导效应细胞(T细胞)对靶细胞(人卵巢癌SKOV3细胞)的细胞毒性作用。由图中可知，细胞毒性作用随Her2-BTEC浓度的增加而不断增大。
实施例6、Her2阳性移植瘤小鼠治疗实验
一、实验方法
1、免疫缺陷的NOD/SCID小鼠接种带萤火虫荧光酶(firefly luciferase，luc)标记的Her2阳性肿瘤细胞(例如SKOV3-luc)。
2、接种肿瘤细胞7天后，用小动物活体成像系统(In Vivo Imaging System，IVIS)记录luciferase荧光强度、监测小鼠成瘤情况；同时设置分组，包括对照组(Control)、T细胞组和实验组三组，每组5只小鼠。
3、对照组不施加任何治疗、T细胞组在规定时间点注射人T细胞、实验组在规定时间点注射MC.Her2-BTEC微环DNA并注射人T细胞。
4、按分组实施不同治疗处理后，定期监测生存情况并用IVIS系统跟踪肿瘤生长情况(记录荷瘤小鼠luciferase荧光强度)。
二、实验结果
图4显示了人卵巢癌移植瘤小鼠治疗实验的结果。由图中可知，与未治疗的荷瘤对照小鼠相比，Her2-BTEC治疗能够显著降低瘤负荷并延长荷瘤小鼠的生 存期。
Figure PCTCN2019113285-appb-000006
Figure PCTCN2019113285-appb-000007
Figure PCTCN2019113285-appb-000008
Figure PCTCN2019113285-appb-000009
Figure PCTCN2019113285-appb-000010

Claims (10)

  1. 一种表达Her2阳性细胞与效应细胞桥接分子(Bridge between Her2-positive cells and effector cells，Her2-BTEC)的重组基因载体，其特征在于，所述桥接分子包含：与Her2阳性细胞作用靶点特异性结合的部分A，和与效应细胞作用靶点特异性结合的部分B；
    优选的，所述重组基因载体选自非病毒基因载体，如标准质粒或其它环状表达盒子；更优选的，所述重组基因载体选自微环DNA载体；
    再优选的，所述桥接分子选自蛋白或多肽；更优选的，所述桥接分子选自双特异性抗体；特别优选的，所述桥接分子选自人-猴交叉性双特异性抗体(Human-Monkey cross-reactive Bispecific Antibody，hm-BsAb)；
    更优选的，所述部分A和所述部分B分别选自蛋白分子或多肽分子；特别优选的，所述部分A和部分B分别选自Fab、Fab’、单链抗体(scFv)、单域抗体(V HH)、单链T细胞受体(scTCR)和其它。
  2. 如权利要求1所述重组基因载体，所述Her2阳性细胞选自Her2阳性癌细胞、Her2过表达细胞或其它；所述效应细胞选自T细胞、NK细胞或其它；
    优选的，所述部分A特异性结合的作用靶点选自Her2的各个抗原表位；优选的，所述部分B特异性结合的作用靶点选自CD3、CD16、CD28、4-1BB、OX40、TCR、CD56、NKG2D、NCR或其它。
  3. 如权利要求1或2所述重组基因载体，所述部分A选自单链抗体(scFv)，其包含重链可变区和轻链可变区，其氨基酸序列如SEQ ID NO：1和3所示；
    和/或，所述部分B选自单链抗体(scFv)，(1)其包含：重链可变区和轻链可变区，其氨基酸序列如选自SEQ ID NO：5和7，或9和11的组所示；所述部分B特异性结合的作用靶点选自CD3；或者(2)其包含：重链可变区和轻链可变区，其氨基酸序列如SEQ ID NO：13和15所示；所述部分B特异性结合的作用靶点选自CD16。
  4. 如权利要求1-3中任一项所述重组基因载体，所述重组基因载体包含所述桥接分子的编码基因；
    优选的，所述重组基因载体包含：部分A的编码基因，和/或包含部分B的 编码基因；
    更优选的，部分A选自单链抗体(scFv)，其编码基因包含：重链可变区和轻链可变区的编码基因，其核苷酸序列如SEQ ID NO：2和4所示；
    更优选的，部分B选自单链抗体(scFv)，其编码基因(1)包含：重链可变区和轻链可变区的编码基因，其核苷酸序列如选自SEQ ID NO：6和8、或10和12的组所示；或者(2)包含：重链可变区和轻链可变区的编码基因，其核苷酸序列如SEQ ID NO：14和16所示。
  5. 如权利要求1-4中任一项所述重组基因载体所表达的桥接分子。
  6. 如权利要求1-4中任一项所述重组基因载体的制备方法，具体步骤包括：
    (1)从现有技术中分别获取Her2抗体、CD3抗体及CD16抗体的轻链可变区(VL)序列、重链可变区(VH)序列；
    (2)根据上述(1)中VH、VL序列设计成多种组合形式的桥接分子，并构建表达所述桥接分子的重组基因载体；
    任选的，
    (3)在体内外鉴定所述重组基因载体的表达水平，并检测表达产物介导效应细胞对Her2阳性细胞的杀伤效果。
  7. 如权利要求6所述制备方法，所述Her2阳性细胞选自Her2阳性癌细胞、Her2过表达细胞或其它。所述效应细胞选自T细胞、NK细胞或其它；
    优选的，所述重组基因载体选自非病毒基因载体，更优选的，所述重组基因载体选自微环DNA载体；
    再优选的，所述桥接分子选自蛋白或多肽；更优选的，所述桥接分子选自双特异性抗体；特别优选的，所述桥接分子选自人-猴交叉性双特异性抗体(Human-Monkey cross-reactive Bispecific Antibody，hm-BsAb)。
  8. 一种宿主细胞，其包含权利要求1-4中任一项所述重组基因载体，或由权利要求6或7所述制备方法所得到的重组基因载体；
    优选的，所述宿主细胞包括细菌细胞、酵母菌细胞、昆虫细胞或哺乳动物细 胞。
  9. 一种药物组合物，其包含如权利要求1-4中任一项所述重组基因载体，或如权利要求5所述桥接分子，或由权利要求6或7所述制备方法所得到的重组基因载体，以及药学上可接受的载体。
  10. 如权利要求1-4中任一项所述重组基因载体，由权利要求6或7所述制备方法所得到的重组基因载体，如权利要求5所述桥接分子，如权利要求8所述宿主细胞，或如权利要求9所述药物组合物在制备用于治疗Her2相关癌症的药物中的用途；
    优选的，所述Her2相关癌症选自乳腺癌、非小细胞肺癌、唾液腺癌、胃癌、肠癌、胰腺癌、膀胱癌、子宫内膜癌、卵巢癌；特别优选的，所述Her2相关癌症选自乳腺癌、胃癌。
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