WO2022262101A1 - 具有增强的adcc效应的抗cd70抗体及其应用 - Google Patents

具有增强的adcc效应的抗cd70抗体及其应用 Download PDF

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WO2022262101A1
WO2022262101A1 PCT/CN2021/112519 CN2021112519W WO2022262101A1 WO 2022262101 A1 WO2022262101 A1 WO 2022262101A1 CN 2021112519 W CN2021112519 W CN 2021112519W WO 2022262101 A1 WO2022262101 A1 WO 2022262101A1
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seq
acid sequence
amino acid
antibody
cdr1
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张军锋
郭志刚
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南京蓝盾生物科技有限公司
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    • 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
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • G01N2333/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153 or CD154

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  • the application belongs to biotechnology, and in particular relates to an anti-CD70 antibody with enhanced ADCC effect and its application.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • This application targets human CD70 to develop an antibody with strong ADCC effect.
  • the expression of CD70 in cancer cells is much higher than that in normal cells, and the ADCC effect mainly acts on cancer cells, thus playing the role of treating cancer.
  • not all antibodies have ADCC, and even antibodies with very similar structures do not necessarily have ADCC effects.
  • the specific spatial structure of antibody variable regions has an important impact on ADCC effects. Starting from the ADCC effect, increasing the types of antibody action mechanisms is a way to improve the therapeutic effect of antibodies. At present, the ADCC effect of antibodies in clinical trials is still weak, and it is still insufficient to enhance the therapeutic effect.
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 1-14, and the amino acid sequence of CDR2 is selected from SEQ ID NO: 15-28, the amino acid sequence of CDR3 is selected from SEQ ID NO: 29-42.
  • the anti-CD70 antibody with enhanced ADCC effect described in the present application comprises any of the following heavy chain variable regions:
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 1
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 15
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 29;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 2
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 16
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 30;
  • amino acid sequence of CDR1 is selected from SEQ ID NO: 3
  • amino acid sequence of CDR2 is selected from SEQ ID NO: 17
  • amino acid sequence of CDR3 is selected from SEQ ID NO: 31;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 4
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 18
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 32;
  • amino acid sequence of CDR1 is selected from SEQ ID NO:5
  • amino acid sequence of CDR2 is selected from SEQ ID NO:19
  • amino acid sequence of CDR3 is selected from SEQ ID NO:33;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO:6, the amino acid sequence of CDR2 is selected from SEQ ID NO:20, and the amino acid sequence of CDR3 is selected from SEQ ID NO:34;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 7
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 21
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 35;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 8
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 22
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 36;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 9
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 23
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 37;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 10
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 24
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 38;
  • amino acid sequence of CDR1 is selected from SEQ ID NO: 11
  • amino acid sequence of CDR2 is selected from SEQ ID NO: 25
  • amino acid sequence of CDR3 is selected from SEQ ID NO: 39;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 12
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 26
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 40;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 13
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 27
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 41;
  • the amino acid sequence of CDR1 is selected from SEQ ID NO: 14
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 28
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 42.
  • SEQ ID NO: 1-42 is shown in the following table:
  • the present application also discloses a DNA molecule encoding the above-mentioned anti-CD70 VHH antibody with enhanced ADCC effect, the coding nucleotide sequence corresponding to the amino acids described in SEQ ID NO: 1-42 of the heavy chain variable region is shown in sequence as SEQ ID NO :43-84.
  • SEQ ID NO: 43-84 are shown in the table below:
  • the present application also discloses the application of the anti-CD70 with enhanced ADCC effect for detecting CD70 molecules.
  • the application of the anti-CD70 antibody with enhanced ADCC effect in the preparation of drugs for treating tumors is also within the protection scope of the present application.
  • the anti-CD70 antibody with enhanced ADCC effect of the present application its variable region can target and bind to a specific antigen, and the Fc region can bind to the Fc receptor (FcR) on the surface of immune cells, thereby activating the killing function of immune cells, Cells expressing the antigen recognized by the variable region are killed.
  • FcR Fc receptor
  • This application uses commercialized recombinant human CD70 biotin-tagged protein and phage display antibody library as starting materials.
  • the CD70 biotin-labeled protein was co-incubated with streptavidin-coated magnetic beads, CD70 was immobilized on the magnetic beads through the interaction between streptavidin and biotin, incubated with the antibody library, and washed away Bind/weakly bind phages, elute phages bound to CD70 protein. This was repeated 3 times, changing the amount of CD70 biotin-labeled protein and washing conditions each time, eliminating weakly binding phages and retaining as many strong binding phages as possible.
  • the coding sequences of the signal peptide, anti-CD70 antibody and human IgG1Fc are linked and read in-frame to construct a mammalian expression vector.
  • the 293F cells were transfected, and the supernatant was harvested after shaking culture for 5 days.
  • the fusion protein was purified from the supernatant by protein A magnetic bead affinity chromatography, and its binding properties to CD70 were identified.
  • the target cells are cultured, and the cells with the luciferase reporter gene that can display ADCC signal transduction are added in a certain proportion, and incubated with a series of gradient concentrations of antibodies, and the reporter gene is activated to varying degrees. Ultimately expressed through reporter gene activity.
  • PBMC peripheral blood mononuclear cells
  • the present application successfully prepared an antibody (fusion protein) that can bind to human CD70 molecules.
  • the antibody has good specificity and high affinity, and can bind to human CD70 expressed on the cell surface. Binding to CD70 not only activates the signal transduction required for the ADCC effect, but ultimately kills the target cells.
  • the anti-human CD70 monoclonal antibody is a potential drug for tumor immunotherapy.
  • Figure 1 is the ELISA detection result of the combination of phage crude culture solution and human CD70;
  • Figure 2 is the FACS detection result of the combination of phage crude culture solution and human CD70
  • Figure 3 is a schematic diagram of the CD70 antibody expression vector structure, MCS is a multiple cloning site, and the target gene is inserted into this position;
  • Figure 4 is the SDS-PAGE electrophoresis of the purified antibody
  • Figure 5 is the detection of the binding performance of the purified antibody to CD70 on the cell surface
  • Figure 6 is the detection of purified antibody ADCC reporter gene activation
  • Fig. 7 is the detection of the killing effect of purified antibody ADCC.
  • Activation of host bacteria TG1 prepare a mini agar medium plate [1 ⁇ M9 salt, 2% glucose, 2mM MgSO 4 , 0.1mM CaCl 2 , 1mM vitamin B1], culture TG1 overnight in a 37°C incubator by streaking.
  • Phage binding place the magnetic beads on the magnetic stand and remove the liquid. Add the blocked library to magnetic beads, resuspend and incubate with rotation for 1 hour, and remove the liquid.
  • Phage precipitation centrifuge at 10,000 rpm for 15 minutes to remove bacteria, add 1/5 volume of 2.5M NaCl/20% PEG8000 to the supernatant, and ice-bath for 2 hours. Centrifuge at 10,000rpm for 10 minutes to obtain phage precipitate, remove the residual liquid, add 0.2ml 0.01M PBS (pH7.4) to resuspend the precipitate, and measure the titer as above.
  • Table 1 ELISA detection results of the combination of phage crude culture medium and human CD70
  • the full-length amino acid sequence of the variable region of the antibody number 1 is shown in SEQ ID NO: 85, and the nucleic acid sequence is shown in SEQ ID NO: 86; the full-length amino acid sequence of the variable region of the antibody number 2 is shown in SEQ ID NO: 87 As shown, the nucleic acid sequence is shown in SEQ ID NO: 88; the full-length amino acid sequence of the variable region of the antibody number 3 is shown in SEQ ID NO: 89, and the nucleic acid sequence is shown in SEQ ID NO: 90.
  • step (3) 4Add 100 ⁇ l of the phage supernatant in step (2)2) or 100ul 10ug/ml of the purified antibody in step (3)9), mix well, and incubate at room temperature for 30 minutes to 1 hour.
  • phage detection add 100ul of anti-M13 phage antibody (purchased from Beijing Sino Biological) diluted 1000 times with PBS, incubate for 30min, wash once with PBS, add 100ul of fluorescent marker (such as FITC, APC) diluted 100 times with PBS
  • fluorescent marker such as FITC, APC
  • the flow cytometry of the purified antibody is shown in Figure 5.
  • Flow cytometry was used in the detection, wherein ARGX-110 was used as a positive control, and hIgG1 was used as a negative control. Its raw data are shown in Table 3, where the value is MFI.
  • the antibody was diluted with RPMI-1640 medium to make a series of mother solutions with 5 times the working concentration, and 10ul/well was added to the cells.
  • the working concentration of ARGX-110 starts from 0.2ug/ml
  • the working concentration of the detection antibody starts from 0.2ug/ml
  • the working concentration of the detection antibody starts from 0.2ug/ml
  • the working concentration of the negative control IgG1 starts from 0.2ug/ml, 4x down 7 gradients.
  • the effector cell PBMC (purchased from Rubai Biotechnology) was washed with PBS, collected by centrifugation at 1000 g for 10 minutes, suspended in RPMI-1640 medium containing 10% bovine serum, and tested for activity rate (above 90%) by trypan blue staining. Take 20ul/well, and make sure to take 50,000/well, and add it to a 384-well plate.
  • the antibody was diluted with RPMI-1640 medium containing 10% bovine serum to make a series of mother solutions of 5 times the working concentration, and 10ul/well was added to the cells.
  • the working concentration of ARGX-110 starts from 2ug/ml, which is reduced by 10 gradients by 3 times;
  • the working concentration of the detection antibody starts from 2ug/ml, and is reduced by 10 gradients by 4 times;
  • the working concentration of the negative control IgG1 starts from 20ug/ml, which is reduced by 4 times 10 gradients.

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Abstract

本发明公开了一种具有增强的ADCC效应的抗CD70抗体及其应用。本申请成功制备了可结合人源CD70分子的抗体(融合蛋白)，该抗体特异性好，亲合力较高，结合细胞表面表达的人源CD70。结合CD70后不仅激活了ADCC效应所需信号转导，而且最终将靶细胞杀死。该抗人CD70单克隆抗体是肿瘤免疫治疗的潜在药物。

Description

具有增强的ADCC效应的抗CD70抗体及其应用 技术领域
本申请属于生物技术，特别是涉及一种具有增强的ADCC效应的抗CD70抗体及其应用。
背景技术
鉴于CD70/CD27分子具有重要免疫调节功能，对CD70进行调控必然会引起一系列的T细胞、甚至B细胞及NK细胞等淋巴细胞的信号传递及生理反应，是一个理想的免疫治疗靶点，合理应用有可能调动多种细胞免疫。抗体依赖细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity,ADCC)是抗体的重要特性，一般由自然杀伤细胞(natural killer cell，NK)完成杀伤，也可通过巨噬细胞进行杀伤。通过ADCC效应，不仅可以进行直接杀伤，被杀伤的细胞释放抗原进而激活获得性免疫。因此ADCC不仅能够直接杀伤细胞，而且调动了天然免疫的调理作用，作用机制更多样，对于实体瘤的治疗具有更重要的实际意义。因此，多数抗体药均采用具有ADCC效应的抗体分子。
本申请以人源CD70为靶点，开发具有强烈ADCC效应的抗体。癌细胞中CD70表达量远高于正常细胞，ADCC效应主要地作用于癌细胞，从而起到治疗癌症的作用。但并非任一抗体均具有ADCC，甚至结构很相似的抗体也并不一定有ADCC效应，抗体可变区的具体空间结构对ADCC效应有重要影响。从ADCC效应入手，增加抗体作用机制的类型，是提高抗体治疗效果的一种方式。目前，临床实验中的抗体的ADCC效应仍较弱，对于增强治疗效果仍有欠缺。
发明内容
发明目的：针对上述现有技术中存在的技术问题，本申请提供了一组具有增强的ADCC效应的抗CD70抗体及其应用。
技术方案：本申请所述的一组具有增强的ADCC效应的抗CD70抗体，其重链可变区CDR1的氨基酸序列选自SEQ ID NO：1-14，CDR2的氨基酸序列选自SEQ ID NO：15-28，CDR3的氨基酸序列选自SEQ ID NO：29-42。
优选的，本申请所述的具有增强的ADCC效应的抗CD70抗体，包括以下任一所示重链可变区：
CDR1的氨基酸序列选自SEQ ID NO：1，CDR2的氨基酸序列选自SEQ ID NO：15，CDR3的氨基酸序列选自SEQ ID NO：29；
CDR1的氨基酸序列选自SEQ ID NO：2，CDR2的氨基酸序列选自SEQ ID NO：16，CDR3的氨基酸序列选自SEQ ID NO：30；
CDR1的氨基酸序列选自SEQ ID NO：3，CDR2的氨基酸序列选自SEQ ID NO：17，CDR3的氨基酸序列选自SEQ ID NO：31；
CDR1的氨基酸序列选自SEQ ID NO：4，CDR2的氨基酸序列选自SEQ ID NO：18，CDR3的氨基酸序列选自SEQ ID NO：32；
CDR1的氨基酸序列选自SEQ ID NO：5，CDR2的氨基酸序列选自SEQ ID NO：19，CDR3的氨基酸序列选自SEQ ID NO：33；
CDR1的氨基酸序列选自SEQ ID NO：6，CDR2的氨基酸序列选自SEQ ID NO：20，CDR3的氨基酸序列选自SEQ ID NO：34；
CDR1的氨基酸序列选自SEQ ID NO：7，CDR2的氨基酸序列选自SEQ ID NO：21，CDR3的氨基酸序列选自SEQ ID NO：35；
CDR1的氨基酸序列选自SEQ ID NO：8，CDR2的氨基酸序列选自SEQ ID NO：22，CDR3的氨基酸序列选自SEQ ID NO：36；
CDR1的氨基酸序列选自SEQ ID NO：9，CDR2的氨基酸序列选自SEQ ID NO：23，CDR3的氨基酸序列选自SEQ ID NO：37；
CDR1的氨基酸序列选自SEQ ID NO：10，CDR2的氨基酸序列选自SEQ ID NO：24，CDR3的氨基酸序列选自SEQ ID NO：38；
CDR1的氨基酸序列选自SEQ ID NO：11，CDR2的氨基酸序列选自SEQ ID NO：25，CDR3的氨基酸序列选自SEQ ID NO：39；
CDR1的氨基酸序列选自SEQ ID NO：12，CDR2的氨基酸序列选自SEQ ID NO：26，CDR3的氨基酸序列选自SEQ ID NO：40；
CDR1的氨基酸序列选自SEQ ID NO：13，CDR2的氨基酸序列选自SEQ ID NO：27，CDR3的氨基酸序列选自SEQ ID NO：41；
CDR1的氨基酸序列选自SEQ ID NO：14，CDR2的氨基酸序列选自SEQ ID NO：28，CDR3的氨基酸序列选自SEQ ID NO：42。
SEQ ID NO：1-42如下表所示：
Figure PCTCN2021112519-appb-000001
Figure PCTCN2021112519-appb-000002
本申请还公开了编码上述具有增强的ADCC效应的抗CD70的VHH抗体的DNA分子，其重链可变区SEQ ID NO：1-42所述氨基酸对应的编码核苷酸序列依次如SEQ ID NO：43-84所示。
SEQ ID NO：43-84如下表所示：
Figure PCTCN2021112519-appb-000003
Figure PCTCN2021112519-appb-000004
本申请还公开了上述具有增强的ADCC效应的抗CD70用于检测CD70分子的应用。
进一步的，所述具有增强的ADCC效应的抗CD70抗体在制备治疗肿瘤的药物中的应用也在本申请的保护范围内。
本申请具有增强的ADCC效应的抗CD70抗体，其可变区能够靶向所识别并结合特定的抗原，Fc区能够结合免疫细胞表面的Fc受体(FcR)，进而激活免疫细胞的杀伤功能，将表达可变区所识别的抗原的细胞杀死。
本申请以商品化的重组人CD70生物素标签蛋白和噬菌体展示抗体库为起始材料。将CD70生物素标签蛋白与链霉亲和素包被磁珠共孵育，通过链霉亲和素与生物素之间的互作将CD70固定在磁珠上，用抗体库与其孵育，洗去未结 合/结合弱的噬菌体，将与CD70蛋白结合的噬菌体洗脱下来。如此反复3次，每次改变CD70生物素标签蛋白的使用量和洗涤条件，逐次淘汰掉结合弱的噬菌体并尽可能保留更多的结合强的噬菌体。用强结合的噬菌体侵染宿主菌，涂板并过夜培养，获得单克隆菌落，即将可结合CD70的噬菌体抗体进行单克隆化。利用单克隆培养上清进行ELISA筛选，并对单克隆进行核酸序列测定。
将信号肽、抗CD70抗体、人源IgG1Fc的编码序列连接，同框阅读，构建哺乳动物表达载体。转染293F细胞，振荡培养5天后收获上清，利用蛋白A磁珠亲和层析从上清中纯化获得融合蛋白，并对其与CD70结合特性进行鉴定。
培养靶细胞，按一定比例加入可显示ADCC信号转导的荧光素酶报告基因的细胞，与一系列梯度浓度的抗体共孵育，报告基因不同程度被激活。最终通过报告基因活性表现出来。
培养超表达荧火虫荧光素酶的靶细胞，按一定比例加入外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)，与一系列梯度浓度的抗体共孵育，激活PBMC中效应细胞的ADCC功能，将靶细胞杀死。抗体的ADCC效应越强，存活的靶细胞越少，荧光素酶活性越低。最终通过检测靶细胞的荧光素酶总活性获知抗体的ADCC效应。
技术效果：本申请成功制备了可结合人源CD70分子的抗体(融合蛋白)，该抗体特异性好，亲合力较高，结合细胞表面表达的人源CD70。结合CD70后不仅激活了ADCC效应所需信号转导，而且最终将靶细胞杀死。该抗人CD70单克隆抗体是肿瘤免疫治疗的潜在药物。
附图说明
图1是phage粗培液与人源CD70结合ELISA检测结果；
图2是phage粗培液与人源CD70结合FACS检测结果；
图3是CD70抗体表达载体结构示意图，MCS为多克隆位点，靶基因插入此位置；
图4是纯化抗体SDS-PAGE电泳；
图5是纯化抗体与细胞表面CD70结合性能检测；
图6是纯化抗体ADCC报告基因激活检测；
图7是纯化抗体ADCC杀伤效应检测。
具体实施方式
下面结合实施例对本申请作出详细说明。
(1)噬菌体展示抗体文库淘选
1)宿主菌TG1活化：制备mini agar培养基平板[1×M9盐，2％葡萄糖，2mM MgSO 4，0.1mM CaCl 2，1mM维生素B1]，划线法过夜培养TG1于37℃培养箱。
2)磁珠洗涤及封闭：吸取50ul磁珠(购自Invitrogen)，置于磁力架上，吸附后吸去液体，1ml PBS重悬、洗涤两次，用1ml 1.5％脱脂奶粉+1.5％BSA的封闭剂(第二轮、第三轮淘选时封闭剂浓度逐渐增加)封闭1小时，去除液体。
3)抗原结合：按16ug/ml的浓度将人源CD70蛋白(购自Acro Biosystem，以后各轮淘选时抗原浓度逐渐降低)用PBS中(pH7.2-7.4)稀释至1ml，重悬磁珠，旋转孵育1小时。
4)库封闭：与抗原结合至磁珠同步，取10 11pfu噬菌体病毒颗粒(来自于原始抗体库或淘选扩增产物)，用1ml 1.0％脱脂奶粉+1.0％BSA的封闭剂旋转孵育1小时。
5)噬菌体结合：将磁珠置于磁力架上，去除液体。将封闭后的库加入磁珠，重悬并旋转孵育1小时，去除液体。
6)洗涤：用1ml PBST[0.01M PBS(pH7.4)，0.1％Tween-20(第二、三轮Tween-20浓度分别用0.2％、0.3％)]洗涤，再用0.01M PBS(pH7.4)洗涤。
7)洗脱：吸去液体，用300ul 0.2M甘氨酸-盐酸(pH2.2)洗脱10分钟，加入20ul中和液[1M Tris-Cl(pH9.0)]混匀，暂时保存于4℃。
8)测滴度：取2ul、0.2ul(原液用2×YT培养基稀释10倍后取2ul)、0.02ul(原液用2×YT培养基稀释100倍后取2ul)洗脱液，与0.2ml对数中期(OD600＝0.5)的TG1混匀，室温孵育30分钟，均匀涂于2×YT-GA100[含2％葡萄糖，100ug/ml氨苄青霉素]平板上，37℃过夜培养，计数约50个克隆的平板上的克隆数，根据稀释倍数计算滴度。
9)噬菌体扩增：在淘选进行的同时，挑取mini agar平板上的TG1单克隆，接种于10ml 2×YT培养液中，37℃下，250rpm振荡培养至对数中期(OD600＝0.5)。加入200ul淘选获得的洗脱产物，37℃孵育30分钟。加入辅助噬菌体M13KO7，37℃再孵育30分钟，37℃下，250rpm振荡培养1小时。离心去上清，用20ml 含工作浓度100ug/ml氨苄青霉素和工作浓度50ug/ml卡那霉素的2×YT重悬，30℃，220rpm振荡培养过夜。
10)噬菌体沉淀：10000rpm离心15分钟去除菌体，上清中加入1/5体积的2.5M NaCl/20％PEG8000，冰浴2小时。10000rpm离心10分钟得到噬菌体沉淀，将残液去除干净，加入0.2ml 0.01M PBS(pH7.4)液重悬沉淀，如上文测滴度。
11)重复步骤2)-10)两或三次，获得结合力强的噬菌体展示抗体。
(2)单克隆ELISA
1)0.3ug/ml链霉亲和素4℃过夜包被酶标板，2％BSA/PBS封闭液处理2h，并用PBS洗涤3次。
2)从2×YT-GA100平板上挑取的单克隆菌落，振荡培养至对数中期，加入辅助噬菌体M13KO7，37℃孵育30分钟。37℃，220rpm振荡培养1小时，4000rpm离心15分钟。用400ul含工作浓度100ug/ml氨苄青霉素和工作浓度50ug/ml卡那霉素的2×YT重悬，30℃，220rpm振荡培养过夜。4000rpm离心15分钟，沉淀菌体。
3)取50ul 4％BSA/PBS加入到酶标板中，同时取50ul噬菌体上清，混匀，孵育1小时。
4)去除液体，0.1％PBST洗5次，PBS洗3次，去除液体。
5)将HRP标记抗M13噬菌体抗体(购自北京义翘神州)用2％BSA稀释3000倍，取100ul加入到酶标板中，孵育1小时，去除液体，0.1％PBST洗3次，拍干残液。
6)加入100ul TMB显色液，37℃孵育10min或至蓝色充分显现，加入100ul 1M硫酸终止反应，在酶标仪上读取OD450。结果如图1所示，在噬菌体淘选过程中，用含有phage病毒颗粒的2毒颗粒培养基50ul做检测。其原始数据如表1所示，其中数值为450nm处的吸光值及其比值。
表1：phage粗培液与人源CD70结合ELISA检测结果
No ID 空白组 实验组 实验/空白 No ID 空白组 实验组 实验/空白
1 0.3153 2.2647 7.1827 8 0.6853 2.3980 3.4992
2 0.2934 2.4846 8.4683 9 0.7504 2.5394 3.3841
3 0.3926 1.6513 4.2061 10 0.7109 2.4764 3.4835
4 1.3320 2.3823 1.7885 11 0.3947 3.0286 7.6732
5 1.0969 2.1793 1.9868 12 0.5849 2.0823 3.5601
6 0.5647 2.6997 4.7806 13 0.5835 1.8947 3.2473
7 0.6547 1.9861 3.0336 14 0.7350 2.7793 3.7814
(3)人源CD70单抗制备及流式检测
1)从2×YT-GA100平板上挑取的单克隆菌落，液体振荡培养过夜，按质粒提取法提取噬菌粒(phagemid)。
2)合成引物，PCR扩增噬菌体所展示的抗体基因编码区。
3)将以上核酸片段按序插入到真核表达载体Abexp-uIgG1的MCS区(图3)，以编码N端为信号肽、中段为抗体、C端为Fc标签的融合蛋白。
4)制备无菌无内毒素的质粒。取23ug，用0.75mL的稀释液(如OPM-293CD05培养基)稀释，同时取70基)的转染试剂(如PEI溶液)加入到0.75mL的稀释液中(如OPM-293CD05培养基)轻柔混匀。将PEI稀释液加入质粒稀释液中，立即用枪轻柔混匀，室温静置15min，避免扰动。
5)加入到25ml 293F细胞及其培养液中，80rpm，37℃，5％CO 2条件下培养24小时，再加入25mL新鲜生长培养基(如OPM-293CD05)，80rpm，37℃，5％CO 2继续培养72小时。
6)10000rpm离心10分钟，取上清。与平衡后的proteinA亲和磁珠旋转孵育1小时，置于磁力架上，去除上清。
7)用30ml PBS洗杂3次，加入5ml 0.1M甘氨酸(pH 3.0)洗脱10分钟，置于磁力架上，吸取上清立即用1M Tris-HCl缓冲液(pH 8.5)至中性，即获得纯化的抗体。
8)SDS-PAGE检测纯化抗体，同时进行浓度测定。采用还原电泳，抗体分子中的二硫键被打开，分子以伸展的单肽链状态进行电泳迁，移结果如图4所示。
对筛选所得纯化抗体测序，得到编号下表所示的编号1-14的抗体：
Figure PCTCN2021112519-appb-000005
Figure PCTCN2021112519-appb-000006
Figure PCTCN2021112519-appb-000007
其中，编号1抗体的可变区全长氨基酸序列如SEQ ID NO：85所示，核酸序列如SEQ ID NO：86所示；编号2抗体的可变区全长氨基酸序列如SEQ ID NO：87所示，核酸序列如SEQ ID NO：88所示；编号3抗体的可变区全长氨基酸序列如SEQ ID NO：89所示，核酸序列如SEQ ID NO：90所示。
SEQ ID NO：85：
Figure PCTCN2021112519-appb-000008
SEQ ID NO：86：
Figure PCTCN2021112519-appb-000009
SEQ ID NO：87：
Figure PCTCN2021112519-appb-000010
Figure PCTCN2021112519-appb-000011
SEQ ID NO：88：
Figure PCTCN2021112519-appb-000012
SEQ ID NO：89：
Figure PCTCN2021112519-appb-000013
SEQ ID NO：90
Figure PCTCN2021112519-appb-000014
9)流式细胞术检测纯化抗体的细胞结合能力
①将表达CD70的细胞株(如786-0)用0.25％的胰酶充分消化，血清终止消化，离心收集细胞，PBS轻轻吹打制备单细胞悬液。
②10ml PBS洗涤细胞1次，1000rpm离心5min,再用1ml PBS悬浮细胞，细胞计数。
③取2.5×10 5个细胞于96孔细胞培养板中，离心收集细胞。
④加入100μl步骤(2)2)中的phage上清或100ul 10ug/ml步骤(3)9)中 的纯化抗体，混匀，室温孵育30分钟至1小时。
⑤离心收集细胞，用300ul PBS洗涤细胞1次。
⑥若为phage检测，加入100ul用PBS稀释1000倍的抗M13噬菌体抗体(购自北京义翘神州)，孵育30min，PBS洗一次，加入100ul用PBS稀释100倍的荧光标记(如FITC、APC)抗体，室温下避光反应20min；若为纯化抗体检测，加入100μl用PBS稀释200倍的荧光标记(如FITC、APC)抗体，室温下避光反应20min。
⑦用1ml PBS洗涤细胞1次，1000rpm离心8min，去除上清液。
⑧加入100μl PBS重悬成单细胞悬液，用流式细胞仪上机检测。phage流式如图2所示，图中左侧、右侧柱形图分别代表与野生型CHO-K1、CD70超表达CHO-K1细胞株结合信号。其原始数据如表2所示，其中数值为荧光强度中值(MFI)及其比值。
表2：phage粗培液与人源CD70结合FACS检测结果
No.ID 对照组 实验组 实验/对照 No.ID 对照组 实验组 实验/对照
1 978.80 19548.80 19.97 8 543.20 7898.20 14.54
2 522.60 5463.60 10.45 9 538.80 7974.30 14.80
3 820.50 6673.90 8.13 10 544.50 9048.30 16.62
4 545.10 8022.20 14.72 11 572.70 3829.30 6.69
5 551.10 7572.80 13.74 12 614.50 4377.20 7.12
6 527.80 7748.60 14.68 13 551.80 5303.90 9.61
7 539.90 7151.70 13.25 14 547.80 7865.30 14.36
纯化抗体流式如图5所示，流式细胞术运用于该检测，其中ARGX-110为阳性对照，hIgG1为阴性对照。其原始数据如表3所示，其中的数值为MFI。
表3：纯化抗体与细胞表面CD70结合性能检测
浓度ug/ml hIgG1 ARGX-110 1 2 3 4 5 6
5 315.7 12445.0 9686.1 5934.6 11608.7 10591.2 16539.4 13577.3
1.25 196.4 12937.3 9846.9 6662.0 11220.8 11507.3 18004.6 14487.2
0.3125 78.7 11559.2 8482.3 5726.3 9657.4 10124.0 17724.0 14889.3
0.0781 48.7 4427.4 4573.2 3035.0 5229.8 7693.6 16539.4 12980.2
0.0195 45.8 1297.5 3187.4 910.2 1901.7 2465.1 10136.9 11233.2
0.0049 55.3 531.9 2005.1 278.9 635.0 767.9 3982.9 5214.8
0.0012 62.1 171.8 1900.5 121.0 222.2 639.2 1517.5 2111.7
0.0003 93.7 91.8 717.7 66.7 84.0 254.5 658.3 903.7
浓度ug/ml 7 8 9 10 11 12 13 14
5 17261.0 18828.5 17332.9 19839.9 15865.3 12612.2 15948.1 13997.5
1.25 17558.5 20055.7 19106.1 20939.5 16123.7 13229.9 15392.4 15273.4
0.3125 18732.2 19581.7 18991.1 20800.2 15063.2 12363.3 15633.9 13140.2
0.0781 17098.1 18074.2 13016.7 18196.8 13769.2 8101.3 9790.2 6624.2
0.0195 8687.8 8928.6 5121.3 7497.8 2900.2 3968.2 3397.8 1765.2
0.0049 3351.5 3549.1 1983.9 3476.3 1942.2 1622.9 1034.3 678.6
0.0012 1342.5 1440.1 857.2 1282.0 717.7 809.0 361.1 172.1
0.0003 638.6 631.7 460.7 645.2 495.6 593.9 160.1 217.2
(4)纯化抗体ADCC报告基因激活检测
1)培养靶细胞raji和效应细胞(ADCC报告基因细胞)ADCC Fc测，用流式细胞仪上机检测。胞，温静置取法提取噬菌粒(重悬Cell Line(购自吉满生物)。
2)将靶细胞用RPMI-1640培养基悬浮，取20ul/孔，并确保取20000个/孔，加入到384孔板中。
3)将效应细胞用RPMI-1640培养基悬浮，取20ul/孔，并确保取50000个/孔，加入到384孔板中。
3)抗体用RPMI-1640培养基稀释成一系列工作浓度5倍的母液，取10ul/孔加入到细胞中。其中ARGX-110工作浓度从0.2ug/ml开始，2倍降低8个梯度；检测抗体工作浓度从0.2ug/ml开始，4倍降低8个梯度；阴性对照IgG1工作浓度从0.2ug/ml开始，4倍降低7个梯度。
4)置于5％CO 2培养箱中，37℃孵育6小时。
5)将One-Lite Luciferase Assay System(购自诺唯赞)提前恢复至室温，按按30ul/孔加入。
6)反应3分钟，用酶标仪读数(15分钟内完成)。结果如图6所示，运用荧光素酶报告基因系统，若抗体激活了ADCC信号转导，报告基因转录激活，表达出荧光素酶，其中ARGX-110为阳性对照，hIgG1为阴性对照。其原始数据如表4所示，其中的数值为MFI。
表4：纯化抗体ADCC报告基因激活检测
Figure PCTCN2021112519-appb-000015
Figure PCTCN2021112519-appb-000016
(5)纯化抗体ADCC杀伤效应检测
1)培养靶细胞-超表达荧火虫荧光素酶的raji细胞。用含10％牛血清的RPMI-1640培养基悬浮，取20ul/孔，并确保取20000个/孔，加入到384孔板中。
2)将效应细胞PBMC(购自儒佰生物)用PBS洗涤，1000g离心10分钟收集，含10％牛血清的RPMI-1640培养基悬浮，台酚蓝染色测活率(90％以上)。取20ul/孔，并确保取50000个/孔，加入到384孔板中。
3)抗体用含10％牛血清的RPMI-1640培养基稀释成一系列工作浓度5倍的母液，取10ul/孔加入到细胞中。其中ARGX-110工作浓度从2ug/ml开始，3倍降低10个梯度；检测抗体工作浓度从2ug/ml开始，4倍降低10个梯度；阴性对照IgG1工作浓度从20ug/ml开始，4倍降低10个梯度。
4)置于5％CO 2培养箱，37℃孵育48小时，取出384孔板，使其降至室温。
5)将One-Lite Luciferase Assay System(购自诺唯赞)提前恢复至室温，按按30ul/孔加入。
6)反应3分钟，用酶标仪读数(15分钟内完成)。结果如图7所示，PBMC做为效应细胞，表达荧光素酶报告基因的raji细胞为靶细胞。加抗体处理的效应细胞和靶细胞组合为检测组，未加抗体的效应细胞和靶细胞组合为参比组。测定活细胞的荧光值，用参比组的荧光值减去检测组的荧光值即为因相应浓度抗体的加入造成的荧光值的下降，以此值对抗体浓度对数做图。其原始数据如表5所示， 其中的数值为MFI。
表5：纯化抗体ADCC杀伤效应检测
Figure PCTCN2021112519-appb-000017

Claims (5)

  1. 一组具有增强的ADCC效应的抗CD70的抗体，其特征在于，其重链可变区CDR1的氨基酸序列选自SEQ ID NO：1-14，CDR2的氨基酸序列选自SEQ ID NO：15-28，CDR3的氨基酸序列选自SEQ ID NO：29-42。
  2. 根据权利要求1所述的具有增强的ADCC效应的抗CD70的抗体，其特征在于，包括以下任一所示重链可变区：
    CDR1的氨基酸序列选自SEQ ID NO：1，CDR2的氨基酸序列选自SEQ ID NO：15，CDR3的氨基酸序列选自SEQ ID NO：29；
    CDR1的氨基酸序列选自SEQ ID NO：2，CDR2的氨基酸序列选自SEQ ID NO：16，CDR3的氨基酸序列选自SEQ ID NO：30；
    CDR1的氨基酸序列选自SEQ ID NO：3，CDR2的氨基酸序列选自SEQ ID NO：17，CDR3的氨基酸序列选自SEQ ID NO：31；
    CDR1的氨基酸序列选自SEQ ID NO：4，CDR2的氨基酸序列选自SEQ ID NO：18，CDR3的氨基酸序列选自SEQ ID NO：32；
    CDR1的氨基酸序列选自SEQ ID NO：5，CDR2的氨基酸序列选自SEQ ID NO：19，CDR3的氨基酸序列选自SEQ ID NO：33；
    CDR1的氨基酸序列选自SEQ ID NO：6，CDR2的氨基酸序列选自SEQ ID NO：20，CDR3的氨基酸序列选自SEQ ID NO：34；
    CDR1的氨基酸序列选自SEQ ID NO：7，CDR2的氨基酸序列选自SEQ ID NO：21，CDR3的氨基酸序列选自SEQ ID NO：35；
    CDR1的氨基酸序列选自SEQ ID NO：8，CDR2的氨基酸序列选自SEQ ID NO：22，CDR3的氨基酸序列选自SEQ ID NO：36；
    CDR1的氨基酸序列选自SEQ ID NO：9，CDR2的氨基酸序列选自SEQ ID NO：23，CDR3的氨基酸序列选自SEQ ID NO：37；
    CDR1的氨基酸序列选自SEQ ID NO：10，CDR2的氨基酸序列选自SEQ ID NO：24，CDR3的氨基酸序列选自SEQ ID NO：38；
    CDR1的氨基酸序列选自SEQ ID NO：11，CDR2的氨基酸序列选自SEQ ID NO：25，CDR3的氨基酸序列选自SEQ ID NO：39；
    CDR1的氨基酸序列选自SEQ ID NO：12，CDR2的氨基酸序列选自SEQ ID NO：26，CDR3的氨基酸序列选自SEQ ID NO：40；
    CDR1的氨基酸序列选自SEQ ID NO：13，CDR2的氨基酸序列选自SEQ ID NO：27，CDR3的氨基酸序列选自SEQ ID NO：41；
    CDR1的氨基酸序列选自SEQ ID NO：14，CDR2的氨基酸序列选自SEQ ID NO：28，CDR3的氨基酸序列选自SEQ ID NO：42。
  3. 编码权利要求1所述具有增强的ADCC效应的抗CD70抗体的DNA分子，其特征在于，其重链可变区SEQ ID NO：1-42所述氨基酸对应的编码核苷酸序列依次如SEQ ID NO：43-84所示。
  4. 权利要求1或2所述具有增强的ADCC效应的抗CD70抗体用于检测CD70分子的应用。
  5. 权利要求1或2所述具有增强的ADCC效应的抗CD70抗体在制备治疗肿瘤的药物中的应用。
PCT/CN2021/112519 2021-06-17 2021-08-13 具有增强的adcc效应的抗cd70抗体及其应用 WO2022262101A1 (zh)

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