WO2022262099A1 - 抗cd70内化的抗体、抗体偶联物及其应用 - Google Patents

抗cd70内化的抗体、抗体偶联物及其应用 Download PDF

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WO2022262099A1
WO2022262099A1 PCT/CN2021/112517 CN2021112517W WO2022262099A1 WO 2022262099 A1 WO2022262099 A1 WO 2022262099A1 CN 2021112517 W CN2021112517 W CN 2021112517W WO 2022262099 A1 WO2022262099 A1 WO 2022262099A1
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acid sequence
amino acid
antibody
heavy chain
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • 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
    • C07K2317/70—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

Definitions

  • the invention relates to the field of biotechnology, in particular to an anti-CD70 internalization antibody, antibody conjugates and applications thereof.
  • CD70 molecule Tumor Necrosis Factor Receptor Superfamily 7 Factor (TNFSF7), has a polypeptide chain length of 193 a.a. and a molecular weight of 21.1KD. It is a type II transmembrane glycoprotein (the soluble form has not yet been seen), and the C-terminal has 155 amino acids Located outside the membrane; the first, third, second, and fourth extracellular cysteines form disulfide bonds to maintain the spatial structure of the extracellular part; usually they exist on the membrane in the form of homotrimers.
  • TNFSF7 Tumor Necrosis Factor Receptor Superfamily 7 Factor
  • CD70 In normal tissues, the cellular expression profile of CD70 is narrow, mainly expressed in B cells in germinal centers (to induce antibody secretion) and T cells in local lymphoid tissues, and expressed at a low level in thymic medullary epithelial cells.
  • its expression can be greatly increased by antigen induction and decreased with the weakening of immune response, such as the high expression of CD70 in antigen-induced activated T and B cells.
  • cytokines such as IL1a, IL12, TNFa, and GM-CSF can up-regulate the expression of CD70, while IL4 and IL10 can down-regulate the expression of CD70.
  • CD70 is expressed under the mediation of TLR signaling pathway. These phenomena just indicate that CD70 is a co-stimulatory molecule required for the complete activation of naive T cells and plays an important role in regulating immune responses. It interacts with the ligand CD27, which not only acts as the second signal for T cell activation, but also regulates the proliferation and differentiation of immune cells such as B and NK.
  • CD70 which is transiently expressed in activated T and B lymphocytes and mature DC cells, is highly expressed in various tumor tissues under pathological conditions.
  • a large number of studies have shown that CD70 is highly expressed in various tumor tissues such as lymphoma, renal cancer, glioma, breast cancer, and hematogenous malignancies (such as Hodgkin's lymphoma, multiple myeloma, leukemia, etc.).
  • hematogenous malignancies such as Hodgkin's lymphoma, multiple myeloma, leukemia, etc.
  • CD70 interacts with CD27
  • CD27 is activated, TRAF2/5 binds to its intracellular segment, and further activates NF-KB and c-Jun kinase signaling pathways, causing cell proliferation, survival and differentiation.
  • CD70 and CD27 may also make the intracellular segment of CD27 bind to Siva, thereby causing Caspase-mediated apoptosis.
  • Cusatuzumab (Cusatuzumab, code-named ARGX) developed by Johnson & Johnson and arGEN-X is the fastest. -110), has completed Phase I clinical trials, and the results show that Kusatuzumab combined with Vidaza can significantly reduce leukemia stem cells in the bone marrow of AML patients, confirming the reliability of this target.
  • CD70/CD27 molecules In view of the important immune regulation function of CD70/CD27 molecules, the regulation of CD70 will inevitably cause a series of signal transmission and physiological responses of lymphocytes such as T cells, even B cells and NK cells, which is an ideal immunotherapy target. Proper application has the potential to mobilize a variety of cellular immunity.
  • the current CD70-targeting therapies include CART and naked antibody, with limited clinical effects, especially for solid tumors, these two methods are almost ineffective.
  • researchers generally believe that antibody-drug conjugate ADC will be one of the hopes for the future treatment of cancer.
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 1-16
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 17-32
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 33-48.
  • the anti-CD70 internalization antibody described in the present application includes any of the following heavy chain variable regions:
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 1
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 17
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 33;
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 2
  • 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: 34;
  • amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 3
  • amino acid sequence of CDR2 is selected from SEQ ID NO: 19
  • amino acid sequence of CDR3 is selected from SEQ ID NO: 35;
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 4, 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: 36;
  • the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO: 5
  • 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: 37;
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 6, the amino acid sequence of CDR2 is selected from SEQ ID NO: 22, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 38;
  • the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO: 7
  • 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: 39;
  • the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO: 8
  • 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: 40;
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 9
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 25
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 41;
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 10
  • 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: 42;
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 11
  • 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: 43;
  • the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO: 12
  • 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: 44;
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 13
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 29
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 45;
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 14
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 30
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 46;
  • the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO: 15
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 31
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 47;
  • the amino acid sequence of CDR1 of the heavy chain variable region is selected from SEQ ID NO: 16
  • the amino acid sequence of CDR2 is selected from SEQ ID NO: 32
  • the amino acid sequence of CDR3 is selected from SEQ ID NO: 48.
  • SEQ ID NO: 1-48 is shown in the following table:
  • the present application also discloses a DNA molecule encoding the above-mentioned anti-CD70 internalization antibody, the nucleotide sequence corresponding to the amino acids described in SEQ ID NO: 1-48 of the heavy chain variable region is shown in sequence as SEQ ID NO: 49-96 .
  • SEQ ID NO: 49-96 are shown in the table below:
  • the application also discloses the application of the anti-CD70 internalization antibody for detecting CD70 molecules.
  • the present application also discloses the application of the anti-CD70 internalization antibody in the preparation of drugs for treating tumors.
  • the tumor is preferably renal clear cell adenocarcinoma.
  • the present application also discloses an antibody conjugate, which includes the above-mentioned anti-CD70 internalization antibody.
  • 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. Cultivate the target cells, incubate the antibody with them at 4°C, and do two groups in parallel. One group was incubated at 4°C and then transferred to 37°C for further incubation, and the other group was incubated at 4°C.
  • a fluorescently labeled detection antibody incubate at 4°C and detect the fluorescent antibody by flow cytometry, that is, the anti-CD70 antibody captured by CD70 on the cell surface.
  • flow cytometry that is, the anti-CD70 antibody captured by CD70 on the cell surface.
  • This application takes human CD70 as the target, and successfully develops an antibody (fusion protein) that can bind to human CD70 molecules.
  • the antibody has good specificity and high affinity, binds to human CD70 expressed on the cell surface, and It can be efficiently transported into the cell by cells; because the antibody has internalization function, it can carry toxin molecules into the cell to kill the cell, that is, using the antibody drug conjugate (antibody-drug conjugate, ADC) pathway, CD70 in cancer cells
  • ADC antibody-drug conjugate
  • the expression level is much higher than that of normal cells, and the ADC mainly targets cancer cells, thereby playing a role in treating cancer. Therefore, the antibody has novel characteristics and drug development direction, and 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 a test of the internalization performance of the purified antibody
  • Figure 7 is a comparison of the internalization performance of the purified antibody with that of ARGX-110;
  • Figure 8 is the detection of the killing efficiency of the purified antibody internalization-mediated toxin on cells
  • Figure 9 is a comparison of the cell-killing efficacy of the purified antibody internalization-mediated toxin with that of ARGX-110.
  • 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 of the binding 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: 97, and the nucleic acid sequence is shown in SEQ ID NO: 98; the full-length amino acid sequence of the variable region of the antibody number 2 is shown in SEQ ID NO: 99 As shown, the nucleic acid sequence is shown in SEQ ID NO: 100; the full-length amino acid sequence of the variable region of the antibody number 3 is shown in SEQ ID NO: 101, and the nucleic acid sequence is shown in SEQ ID NO: 102.
  • 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 phage flow pattern is shown in Figure 2.
  • the left and right column graphs in the figure represent the differences with wild-type CHO-K1 and CD70 respectively.
  • the original data of the binding signal of the overexpressed CHO-K1 cell line are shown in Table 2, where the values are the median fluorescence intensity (MFI) and its ratio.
  • the flow pattern of the purified antibody is shown in Figure 5, where ARGX-110 is the positive control, hIgG1 is the negative control, and the original data are shown in Table 3, where the value is the median fluorescence intensity (MFI).
  • step 3 Dilute the antibody to a 10-fold EC50 value, add FabFc-ZAP human reagent at a concentration of 45nM (the working concentration is 4.5nM; purchased from Advanced Targeting Systems), take 10ul of the mixture, and add it to step 2) In the cells, mix well.

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Abstract

一种抗CD70内化的抗体、抗体偶联物及其应用。以人源CD70为靶点，成功开发得到可结合人源CD70分子的抗体，该抗体特异性好，亲合力较高，结合细胞表面表达的人源CD70，并可被细胞高效转运至胞内；由于该抗体具有内化功能，进而可以携带毒素分子进入细胞杀死该细胞，起到治疗癌症的作用。

Description

抗CD70内化的抗体、抗体偶联物及其应用 技术领域
本发明涉及生物技术领域，尤其涉及一种抗CD70内化的抗体、抗体偶联物及其应用。
背景技术
CD70分子，即肿瘤坏死因子受体超家族7因子(TNFSF7)，多肽链长193个a.a.，分子量21.1KD，是一种Ⅱ型跨膜糖蛋白(尚未见可溶形式)，C端155个氨基酸位于膜外；胞外的第一、三及第二、四个半胱氨酸之间形成二硫键，维持胞外部分的空间结构；通常其以同源三聚体形式存在于膜上。在正常组织中，CD70的细胞表达谱较窄，主要表达于生发中心的B细胞(诱导分泌抗体)和局部淋巴组织的T细胞，在胸腺髓质上皮细胞中低水平表达。但其表达可被抗原诱导大量增加并随着免疫应答的减弱而减少，比如抗原诱导活化的T、B细胞中高表达CD70。同时其表达受到细胞因子的调控，如IL1a、IL12、TNFa、GM-CSF可上调CD70表达，IL4、IL10下调CD70表达。另外在成熟的DCs中，在TLR信号通路的介导下CD70表达。这些现象正好表明CD70为初始T细胞完全活化所需的一种协同刺激分子，在调控免疫应答中发挥重要作用。其与配体CD27相互作用，不仅起着T细胞活化第二信号的作用，而且二者之间的互作还可以调节B、NK等免疫细胞的增殖和分化。
短暂表达于活化的T和B淋巴细胞及成熟DC细胞中的CD70，在病理状态下却高表达于多种肿瘤组织中。大量研究表明，CD70在淋巴瘤、肾癌、胶质细胞瘤、乳腺癌、血源性恶性肿瘤(如霍奇金淋巴瘤、多发性骨髓瘤、白血病等)等多种肿瘤组织中高水平表达。同时观察到CD27在这些组织中的表达与CD70同步上升或下降。可能二者作为配体/受体对，形成了反馈环，促进淋巴细胞的增殖与分化，反而起到介导疾病发生的作用。CD70与CD27互作后，CD27活化，TRAF2/5结合到其胞内段，进一步激活NF-KB和c-Jun激酶信号通路，引起细胞增殖、生存和分化等。另外，亦有人认为CD70与CD27结合后也可能使CD27胞内段结合Siva从而引起Caspase介导的细胞凋亡。
鉴于以上现象，以CD70为靶点制备特异性抗体，开展肿瘤免疫治疗引起人 们浓厚兴趣。目前针对CD70靶点，CART治疗、抗体药和抗体偶联药物三种应用方式均在开发中，进展最快的为强生与arGEN-X合作开发的库萨图珠单抗(Cusatuzumab，研发代号ARGX-110)，目前已完成I期临床，结果显示库萨图珠单抗联合维达扎可显著降低AML患者骨髓中的白血病干细胞，证实该靶点的可靠性。
鉴于CD70/CD27分子对重要免疫调节功能，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，是一个很理想的免疫治疗靶点，合理应用有可能调动多种细胞免疫。目前靶向CD70的疗法有CART及裸抗，临床效果有限，特别是对实体瘤来说，这两种方法几乎无效。研发人员普遍认为，抗体药物偶联物ADC将是未来治疗癌症的希望之一。
发明内容
发明目的：针对上述现有技术，本申请提供了一种抗CD70内化的抗体、抗体偶联物及其应用。
技术方案：本申请所述的一组抗CD70内化的抗体，其重链可变区CDR1的氨基酸序列选自SEQ ID NO：1-16，CDR2的氨基酸序列选自SEQ ID NO：17-32，CDR3的氨基酸序列选自SEQ ID NO：33-48。
进一步优选的，本申请所述抗CD70内化的抗体，包括以下任一所示重链可变区：
其重链可变区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：17，CDR3的氨基酸序列选自SEQ ID NO：33；
其重链可变区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：18，CDR3的氨基酸序列选自SEQ ID NO：34；
其重链可变区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：19，CDR3的氨基酸序列选自SEQ ID NO：35；
其重链可变区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：20，CDR3的氨基酸序列选自SEQ ID NO：36；
其重链可变区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：21，CDR3的氨基酸序列选自SEQ ID NO：37；
其重链可变区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：22，CDR3的氨基酸序列选自SEQ ID NO：38；
其重链可变区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：23，CDR3的氨基酸序列选自SEQ ID NO：39；
其重链可变区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：24，CDR3的氨基酸序列选自SEQ ID NO：40；
其重链可变区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：25，CDR3的氨基酸序列选自SEQ ID NO：41；
其重链可变区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：26，CDR3的氨基酸序列选自SEQ ID NO：42；
其重链可变区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：27，CDR3的氨基酸序列选自SEQ ID NO：43；
其重链可变区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：28，CDR3的氨基酸序列选自SEQ ID NO：44；
其重链可变区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：29，CDR3的氨基酸序列选自SEQ ID NO：45；
其重链可变区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：30，CDR3的氨基酸序列选自SEQ ID NO：46；
其重链可变区CDR1的氨基酸序列选自SEQ ID NO：15，CDR2的氨基酸序列选自SEQ ID NO：31，CDR3的氨基酸序列选自SEQ ID NO：47；
其重链可变区CDR1的氨基酸序列选自SEQ ID NO：16，CDR2的氨基酸序列选自SEQ ID NO：32，CDR3的氨基酸序列选自SEQ ID NO：48。
SEQ ID NO：1-48如下表所示：
Figure PCTCN2021112517-appb-000001
Figure PCTCN2021112517-appb-000002
本申请还公开了编码上述抗CD70内化的抗体的DNA分子，其重链可变区SEQ ID NO：1-48所述氨基酸对应的核苷酸序列依次如SEQ ID NO：49-96所示。
SEQ ID NO：49-96如下表所示：
Figure PCTCN2021112517-appb-000003
Figure PCTCN2021112517-appb-000004
本申请还公开了所述抗CD70内化的抗体用于检测CD70分子的应用。
进一步的，本申请还公开了所述抗CD70内化的抗体在制备***的药物中的应用。所述肿瘤优选为肾透明细胞腺癌。
本申请还公开了一种抗体偶联物，其包括上述的抗CD70内化的抗体。
所述抗体偶联物在制备***的药物中的应用也在本申请的保护范围内。
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，于4℃将抗体与其共孵育，平行做两组。一组经4℃孵育后转移至37℃继续孵育，另一组继续于4℃孵育。孵育结束后，加入荧光标记检测抗体，4℃孵育并用流式细胞术检测荧光抗体，即获知细胞表面被CD70捕获的抗CD70抗体。将靶细胞铺于96孔板中，过夜培养使细胞充分贴壁。加入抗体和/或毒素试剂后进行细胞培养并观察细胞生长特性。培养结束后，检测细胞活率。
技术效果：本申请以人源CD70为靶点，成功开发得到可结合人源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，并可被细胞高效转运至胞内；由于该抗体具有内化功能，进而可以携带毒素分子进入细胞杀死该细胞，即利用抗体药物偶联物(antibody-drug conjugate，ADC)途径，癌细胞中CD70表达量远高于正常细胞，该ADC主要地靶向癌细胞，从而起到治疗癌症的作用。因此，该抗体具有新颖的特性及药物开发方向，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***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是纯化抗体内化性能检测；
图7是纯化抗体内化性能与ARGX-110对比；
图8是纯化抗体内化介导毒素对细胞的杀伤效能检测；
图9是纯化抗体内化介导毒素对细胞的杀伤效能与ARGX-110对比。
具体实施方式
下面结合具体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那霉素重悬，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那霉素重悬，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T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
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 0.4176 1.8039 4.3197 9 0.3736 2.5193 6.7433
2 0.3653 1.1169 3.0575 10 0.3661 2.5015 6.8328
3 0.4211 1.6144 3.8338 11 1.4312 2.5900 1.8097
4 0.3643 1.2439 3.4145 12 0.5815 1.6496 2.8368
5 0.5043 0.9839 1.9510 13 0.3834 0.6903 1.8005
6 0.3660 1.2861 3.5139 14 0.3945 0.5332 1.3516
7 0.3159 1.9717 6.2415 15 0.2688 1.2027 4.4743
8 0.3344 0.7223 2.1599 16 0.2917 0.6650 2.2797
(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***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取)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，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1-16的抗体：
Figure PCTCN2021112517-appb-000005
Figure PCTCN2021112517-appb-000006
Figure PCTCN2021112517-appb-000007
其中，编号1抗体的可变区全长氨基酸序列如SEQ ID NO：97所示，核酸序列如SEQ ID NO：98所示；编号2抗体的可变区全长氨基酸序列如SEQ ID NO：99所示，核酸序列如SEQ ID NO：100所示；编号3抗体的可变区全长氨基酸序列如SEQ ID NO：101所示，核酸序列如SEQ ID NO：102所示。
SEQ ID NO：97：
Figure PCTCN2021112517-appb-000008
SEQ ID NO： 98：
Figure PCTCN2021112517-appb-000009
SEQ ID NO： 99：
Figure PCTCN2021112517-appb-000010
SEQ ID NO： 100：
Figure PCTCN2021112517-appb-000011
SEQ ID NO： 101：
Figure PCTCN2021112517-appb-000012
SEQ ID NO： 102：
Figure PCTCN2021112517-appb-000013
Figure PCTCN2021112517-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，其中数值为荧光强度中值(MFI)及其比值。纯化抗体流式如图5所示，其中ARGX-110为阳性对照，hIgG1为阴性对照，其原始数据如表3，其中数值为荧光强度中值(MFI)。
表2：phage粗培液与人源CD70结合FACS检测结果
Figure PCTCN2021112517-appb-000015
Figure PCTCN2021112517-appb-000016
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 7
5 89.0 12418.8 8609.0 11987.1 13689.4 3432.6 9041 10485.9 10746.8
1.25 64.9 12599.1 9067.2 13122.4 14076.3 2999.1 11379.5 11309.3 9939.8
0.3125 53.4 10454.3 7496.8 13127.1 11707.1 1580.4 10484.4 11518.5 6865.7
0.0781 140.8 7363.1 4512.0 10307.0 10255.8 686.3 7695.9 9669.2 3319.6
0.0195 112.4 3930.7 3641.6 6087.3 4576.2 149.1 3786 5428 1109.3
0.0049 100.1 1132.5 697.9 4267.7 2820.6 76.1 957.6 875.3 383.9
0.0012 119.1 622.5 629.7 1486.6 465.3 66.3 393.3 250.7 144.3
0.0003 96.4 283.5 1263.6 485.2 173.1 343.6 197.4 272 116.5
浓度ug/ml 8 9 10 11 12 13 14 15 16
5 17027.1 5318.8 18997.2 18658.2 16308.4 7303.1 9995.4 11034.7 8213.7
1.25 19287.1 6009.8 18780.9 18599.9 16708.3 7267.5 9000.9 10878.6 8076.9
0.3125 19126.1 6170.7 16032.5 16648.0 17391.7 5319.8 8216.2 9521.7 7723.0
0.0781 8412.4 3523.3 9042.7 6333.4 17250.2 4247.4 2866.7 3027.9 6113.4
0.0195 2577.6 1156 2964 1942.3 12055.9 1298.6 1653.0 1237.6 3240.5
0.0049 875.1 306.5 937.5 604.2 4218.5 942.7 1800.8 937.1 1091.9
0.0012 309.2 96.3 347.2 231.5 1590.8 498.2 1216.1 336.8 352.4
0.0003 137.7 44.8 140.2 110.6 848.1 267.4 399.2 272.7 255.7
(4)抗体内化功能检测
1)0.25％Typsin-EDTA消化786-0细胞，离心弃上清，用完全培养基重悬。
2)将75ul细胞悬液加至96孔板中，并使得每孔有1.5，并使 5个细胞。
3)按抗体EC80的工作浓度(即饱和浓度的80％)加入25ul，混匀。每个抗体分两板，每板各做一孔。4℃、孵育1小时。
4)加入PBS至终体积250ul，500g常温离心5min，甩去上清保留细胞沉淀，用吸水纸吸净残液，轻拍实验板使细胞分散，PBS再洗一次，分散细胞。此步去掉多余未结合的抗体。
5)用100ul基础培养基重悬细胞，其中一板于37℃孵育2小时，另一板于4℃孵育2小时。37℃孵育过程中抗体会转入细胞，而4℃则抑制了内化。
6)加入100ul用PBS稀释的荧光二抗(加入前二抗浓度为工作浓度的2倍)，4℃孵育0.5小时。
7)500g常温离心5min，甩去上清保留细胞沉淀，用吸水纸吸净残液，轻拍实验板使细胞分散，PBS再洗一次，分散细胞。
8)加入100ul PBS重悬，流式细胞仪上机检测，Graphpad Prism处理数据。如图6所示，抗体与细胞结合后，通过荧光二抗检测结合于细胞表面的待测抗体，图6原始数据如表4，其中数值为荧光强度中值(MFI)。图7是将检测样品即本文中的seq No.ID于37℃测得的MFI除以4℃测得的MFI定义为数值A，将ARGX-110于37℃测得的MFI除以4℃测得的MFI定义为数值B，则A/B值即为相对内化。
表4：纯化抗体内化性能检测
样品/seq No.ID 37℃ 4℃ 样品/seq No.ID 37℃ 4℃
ARGX-110 6335.0 7365.6 9 1296.7 3418.8
1 1149.3 2160.8 10 6910.7 13886.0
2 1003.9 7450.1 11 8750.5 13581.0
3 1561.3 3420.7 12 6870.6 9654.2
4 370.3 774.1 13 3774.3 5686.8
5 716.8 2580.4 14 3595.1 4961.0
6 1884.7 3071.9 15 2505.8 3528.0
7 1492.8 2544.3 16 4025.4 6003.4
8 6032.4 13007.9      
(5)抗体内化介导毒素对细胞的杀伤效能检测
1)0.25％Typsin-EDTA消化786-0细胞，离心弃上清，用完全培养基重悬。
2)将90ul细胞悬液加至96孔板中，并使得每孔有2500个细胞。
3)将抗体稀释至10倍的EC50值，向其中加入浓度为45nM的FabFc-ZAP human试剂(其工作浓度为4.5nM；购自Advanced Targeting Systems)，取该混合液10ul，加入到步骤2)中的细胞中，混匀。
4)37℃，5％CO 2培养箱培养72小时。
5)将CCK8试剂盒室温平衡15min。取出96孔细胞培养板，加入CCK8试剂，于37℃培养箱避光孵育1至3小时，酶标仪读取OD450值。如图8所示，抗体、毒素和细胞共孵育后，CCK8法测得的细胞活性低于仅抗体和细胞共孵育相比。其原始数据如表5所示，其中数值为450nm的吸光值(A450)。将抗体(ARGX-110或Seq No.ID所指抗体)与毒素同时处理细胞后测得的450nm吸光值除以仅用抗体(ARGX-110或Seq No.ID所指抗体)处理细胞后测得的450nm 吸光值定义为数值A，将毒素处理细胞后测得的450nm吸光值除以未处理的细胞测得的450nm吸光值定义为数值B，则A/B值即为相对杀伤。如图9所示。表5：纯化抗体内化介导毒素对细胞的杀伤效能检测
Seq No.ID 抗体+毒素 抗体 Seq No.ID 抗体+毒素 抗体
ARGX-110 2.3615 2.8350 9 1.0498 2.2105
1 1.0227 2.1778 10 1.1626 3.1110
2 0.8658 2.2844 11 1.0138 3.0691
3 0.7365 2.2092 12 1.2944 2.9997
4 1.0377 2.2920 13 1.7668 2.5112
5 0.6969 2.2609 14 1.0145 2.1882
6 0.8546 2.3082 15 1.8259 2.5652
7 0.9212 2.8220 16 1.5359 2.5258
8 0.9660 2.3672 - - -
毒素+细胞 2.1185 - 细胞 2.4601 -

Claims (8)

  1. 一组抗CD70内化的抗体，其特征在于，其重链可变区CDR1的氨基酸序列选自SEQ ID NO：1-16，CDR2的氨基酸序列选自SEQ ID NO：17-32，CDR3的氨基酸序列选自SEQ ID NO：33-48。
  2. 根据权利要求1所述的抗CD70内化的抗体，其特征在于，包括以下任一所示重链可变区：
    其重链可变区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：17，CDR3的氨基酸序列选自SEQ ID NO：33；
    其重链可变区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：18，CDR3的氨基酸序列选自SEQ ID NO：34；
    其重链可变区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：19，CDR3的氨基酸序列选自SEQ ID NO：35；
    其重链可变区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：20，CDR3的氨基酸序列选自SEQ ID NO：36；
    其重链可变区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：21，CDR3的氨基酸序列选自SEQ ID NO：37；
    其重链可变区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：22，CDR3的氨基酸序列选自SEQ ID NO：38；
    其重链可变区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：23，CDR3的氨基酸序列选自SEQ ID NO：39；
    其重链可变区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：24，CDR3的氨基酸序列选自SEQ ID NO：40；
    其重链可变区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：25，CDR3的氨基酸序列选自SEQ ID NO：41；
    其重链可变区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：26，CDR3的氨基酸序列选自SEQ ID NO：42；
    其重链可变区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：27，CDR3的氨基酸序列选自SEQ ID NO：43；
    其重链可变区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：28，CDR3的氨基酸序列选自SEQ ID NO：44；
    其重链可变区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：29，CDR3的氨基酸序列选自SEQ ID NO：45；
    其重链可变区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：30，CDR3的氨基酸序列选自SEQ ID NO：46；
    其重链可变区CDR1的氨基酸序列选自SEQ ID NO：15，CDR2的氨基酸序列选自SEQ ID NO：31，CDR3的氨基酸序列选自SEQ ID NO：47；
    其重链可变区CDR1的氨基酸序列选自SEQ ID NO：16，CDR2的氨基酸序列选自SEQ ID NO：32，CDR3的氨基酸序列选自SEQ ID NO：48。
  3. 编码权利要求1所述抗CD70内化的抗体的DNA分子，其特征在于，其重链可变区SEQ ID NO：1-48所述氨基酸对应的核苷酸序列依次如SEQ ID NO：49-96所示。
  4. 权利要求1或2所述抗CD70内化的抗体用于检测CD70分子的应用。
  5. 权利要求1或2所述抗CD70内化的抗体在制备***的药物中的应用。
  6. 一种抗体偶联物，其特征在于，包括权利要求1或2所述的抗CD70内化的抗体。
  7. 权利要求6所述抗体偶联物在制备***的药物中的应用。
  8. 根据权利要求5或7所述应用，其特征在于，所述肿瘤为肾透明细胞腺癌。
PCT/CN2021/112517 2021-06-17 2021-08-13 抗cd70内化的抗体、抗体偶联物及其应用 WO2022262099A1 (zh)

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