WO2022096020A1 - 抗胞外-5'-核苷酸酶的抗体序列 - Google Patents

抗胞外-5'-核苷酸酶的抗体序列 Download PDF

Info

Publication number
WO2022096020A1
WO2022096020A1 PCT/CN2021/131861 CN2021131861W WO2022096020A1 WO 2022096020 A1 WO2022096020 A1 WO 2022096020A1 CN 2021131861 W CN2021131861 W CN 2021131861W WO 2022096020 A1 WO2022096020 A1 WO 2022096020A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
chain variable
amino acid
seqidno
Prior art date
Application number
PCT/CN2021/131861
Other languages
English (en)
French (fr)
Inventor
廖高勇
丁海剑
王灵
张怡
Original Assignee
江苏中新医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏中新医药有限公司 filed Critical 江苏中新医药有限公司
Publication of WO2022096020A1 publication Critical patent/WO2022096020A1/zh
Priority to US17/827,630 priority Critical patent/US20220356267A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Extracellular 5 -nucleotidase ecto-55-nucleotidase, also referred to as CD73 hereinafter
  • CD73 Extracellular 5 -nucleotidase
  • CD73 monoclonal antibodies Although some therapeutic CD73 monoclonal antibodies have been developed, the monoclonal antibodies are secreted by a single clone of hybridoma cells and can only recognize antibodies of one epitope (antigenic determinant). A variety of different monoclonal antibodies can be prepared against different antigenic epitopes of the same biological macromolecule.
  • the purpose of the present invention is to provide a variety of effective anti-extracellular 5-nuclease (ie CD73) antibody sequences for the preparation of medicaments for the treatment of tumor-related diseases.
  • the present invention uses human CD73 protein to immunize mice, and obtains five anti-CD73 monoclonal antibodies respectively.
  • the heavy chain and light chain sequences of these monoclonal antibodies are completely new sequences, which have not been reported in the prior art.
  • the antigenic epitopes recognized by the four antibodies are also different from those of the prior art antibodies.
  • the CD73 protein used is the human CD73 protein expressed by the applicant, and the mouse used is BABL/C mouse.
  • the present invention completes the above-mentioned work by the following means:
  • a Human CD73 protein was used as an antigen to immunize BABL/C mice at 30pg/mouse, and the same dose was boosted after three weeks;
  • the five anti-CD73 monoclonal antibodies obtained in the present invention are named respectively: F2-2, E1-B6, 81-2-2, 06-6 and 9-4.
  • the molecular basis of the specificity of these antibodies mainly comes from its hypervariable regions CDR1, CDR2 and CDR3, which are key sites for antigen binding.
  • the CDR1, CDR2 and CDR3 in the heavy chain sum of each antibody are polypeptides with the following amino acid sequences respectively:
  • F2-2 SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; SEQIDNO.4, SEQIDNO.5 and SEQIDNO.6;
  • E1-B6 SEQ ID NO.9 > SEQ ID NO.10 and SEQ ID NO.1; SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14;
  • the "monoclonal antibody” described in the present invention should be construed to encompass antibodies with the desired specificity
  • Any specific binding factor of a specific binding domain can be monovalent or single-chain antibody, diabody, chimeric antibody, and derivatives, functional equivalents and homologues of the above-mentioned antibodies, including antibody fragments and containing Any polypeptide of an antigen binding domain.
  • Examples of monoclonal antibodies of the present invention are the immunoglobulin IgG subtype and its subtypes; although the molecular basis for antibody specificity mainly comes from its hypervariable regions CDR1, CDR2 and CDR3, in order to maintain preferred binding characteristics, the sequences of the above CDRs should be preserved as much as possible. However, even if individual amino acids are changed, it is possible to achieve the objectives of the present invention and even optimize the binding properties. However, changes in individual amino acids cannot depart from the concept and spirit of the present invention. Except for the hypervariable regions CDRLCDR2 and CDR3 in the heavy and light chains as described above, the others are framework regions. Framework regions can be replaced by other sequences without affecting the three-dimensional structure required for binding. Beneficial effects of the present invention: It has been confirmed by experiments that the five anti-CD73 monoclonal antibodies produced by the present invention have the following outstanding features:
  • FIG. 1 is a schematic diagram of the steps for obtaining five anti-CD73 monoclonal antibodies of the present invention.
  • Ka, Kd and KD are the binding constant, dissociation constant and affinity constant, respectively, Contbody is
  • FIG. 3 is the experimental result of Example 3 (F2-2, E1-B6, 81-2-2, 06-6 and 9-4 antibody antibody epitope analysis), wherein the epitope mapping sequence is followed All remaining antibodies were plotted for E1-B6, 81-2-2, 9-4, 06-4 and F2-2,
  • FIG. 5 is the experimental result of Example 4 (inhibition of CD73 enzymatic activity by F2-2, E1-B6, 81-2-2, 06-6 and 9-4 antibodies); wherein, Fig. 3 is the biochemical level, FIG. 4 is the cellular level (A549); FIG. 6 is the experimental result of Example 5 (F2-2, E1-B6, 81-2-2, 06-6 and 9-4 antibody animal efficacy) above
  • the F2-2, E1-B6, 81-2-2, 06-6 and 9-4 are respectively the names of the five anti-CD73 monoclonal antibodies obtained in the present invention.
  • SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are respectively the anti-CD73 monoclonal antibody F2-2
  • SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 are respectively the anti-CD73 monoclonal antibody F2-2
  • SEQ ID NO.7 and SEQ ID NO.8 are respectively the anti-CD73 monoclonal antibody F2-2 and the amino acid sequence;
  • SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11 respectively CDR1, CDR2 and CDR3 of anti-CD73 monoclonal antibodies E1-B6;
  • SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 are respectively anti-CD73 monoclonal antibodies E1-B6 of CDR1, CDR2 and CDR3;
  • SEQ ID NO.15 and SEQ ID NO.16 are respectively the anti-CD73 monoclonal antibody E1-B6 and the amino acid sequence;
  • SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.19 are CDR1, CDR2 and CDR3 of anti-CD73 monoclonal antibody 81-2-2, respectively;
  • SEQ ID NO.23 and SEQ ID NO.24 are respectively the anti-CD73 monoclonal antibody 81-2-2 and the amino acid sequence;
  • SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27 are CDR1, CDR2 and CDR3 of anti-CD73 monoclonal antibody 06-6, respectively;
  • SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30 respectively CDR1, CDR2 and CDR3 of anti-CD73 monoclonal antibody 06-6;
  • SEQ ID NO.31 and SEQ ID NO.32 are respectively the anti-CD73 monoclonal antibody 06-6 and the amino acid sequence;
  • SEQ ID NO.33, SEQ ID NO.34 and SEQ ID NO.35 are CDR1, CDR2 and CDR3 of anti-CD73 monoclonal antibody 9-4, respectively;
  • SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38 respectively CDR1, CDR2 and CDR3 of anti-CD73 monoclonal antibody 9-4;
  • SEQ ID NO.39, SEQ ID NO.40 are anti-CD73 monoclonal antibody 9-4 and amino acid sequence respectively;
  • SEQ ID NO. 41 is the amino acid sequence of CD73 protein constructed and expressed by the inventors of the present application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the present invention provides the following five specific heavy chains and sequences of anti-CD73 monoclonal antibodies.
  • the monoclonal antibody is expressed by a corresponding monoclonal cell line obtained by immunizing BABL/c mice with CD73 protein through hybridoma screening; the monoclonal antibody type is IgG type.
  • the antigen used in the following examples is the self-expressed human CD73 protein, and the C-terminus of the protein contains a 6xHis tag; the immune adjuvant used is Beijing Boaolong 5-week rapid immune adjuvant, which is boosted once 21 days after the initial immunization.
  • Cell fusion can be performed by using antigen shock immunization once before fusion; the fusion method used is electrofusion method, the electrofusion apparatus is ECM2001 of BTX company, and the fusion buffer used is the original cell fusion solution; the cells after fusion grow out
  • the expression of supernatant antibody was detected by ELISA after the cell pellet, and the ELISA plate was coated with CD73 protein or His protein, and His protein was used to exclude the false positive wells of Anti-His; rounds to finally obtain a positive monoclonal cell line;
  • the positive monoclonal was purified after ascites preparation to obtain the corresponding monoclonal antibody.
  • the monoclonal antibody was further used for affinity determination, enzyme activity inhibition experiment and animal efficacy evaluation.
  • Example 1 Antigen immunization, cell fusion, screening of positive clones, and preparation and purification of ascites antibodies
  • Example 2 Affinity kinetic analysis of five anti-CD73 monoclonal antibodies and recombinant human CD73 Experimental Objective: The Biacore T200 system was used to detect the affinity kinetic constant of each antibody. Reagents and Methods:
  • Mouse Antibody Capture Kit is a commercial kit purchased from GE.
  • the anti-mouse Fc IgG is immobilized on the CM5 sensor chip by amino-coupling method, and the tested antibody is captured by the conjugated anti-mouse Fc IgG, and then injected A series of concentration gradients of human CD73 protein were then used to regenerate the detection system of pH 1.7 Glycine-HCl included in the kit.
  • Running buffer was HBS-EP+ (10mM HEPES, pH7.4, 150mM Nach, 3mM EDTA and 0.05% P20), the measurement temperature was 25 °C; the control substance selected in the experiment was MEDI-9447; MEDI-9447 was the CD73 antibody of Medlmmune Company, and we synthesized, expressed and purified according to the sequence of its patent (US2016/0194407 A1) to obtain the control substance.
  • Association (Ka) and dissociation (Kd) rate constants and equilibrium constants (KD) were calculated using Biacore T200 evaluation software to fit the data according to a 1:1 binding model.
  • Mouse Antibody Capture Kit is a commercial kit purchased from GE.
  • the anti-mouse Fc IgG was immobilized on the CM5 sensor chip by amino-coupling method, and the primary antibody was captured by the conjugated anti-mouse Fc IgG.
  • the redundant sites were blocked with 100 Pig/mL Mouse IgG, and then CD73 and secondary antibody were injected sequentially, and the similarities and differences of the antigenic epitopes recognized by each antibody were analyzed by sensorgram.
  • the control substance selected in the experiment is MEDI-9447 o Experimental results: The specific experimental results are shown in Figure 3; according to the analysis results, the epitopes of each antibody are summarized in the following table: According to the epitope mapping results, among the five antibodies, F2-2 recognizes an independent epitope, E1-B6, 06-6, and 9-4 recognize the same epitope, and the four antibodies recognize different epitopes from MED 1-9447. 81-2-2 and MED 1-9447 recognize the same epitope, but the amino acid sequences of the CDR regions of 81-2-2 and MED 1-9447 are different. Therefore, 81-2-2 is also different from MED 1-9447. 9447's new antibody.
  • Cell method F2-2, E1-B6, 81-2-2, 06-6, 9-4 and MED 1-9447 antibodies were added to the bottom of the 96-well plate at a suitable concentration gradient, and A549 cells were digested and resuspended.
  • Example 5 Animal efficacy evaluation experiments of five antibodies Objective: In vivo experiments were conducted to test the effect of five antibodies in inhibiting the growth of tumor cells. Experimental method: 90 B-NDG mice were taken and reared adaptively for at least one week. The A549 cells were cultured, passaged every other day, and finally the cells were collected. The final cell concentration was adjusted to 5*107/mL with PBS, and 0.1 mL of each was inoculated into the right shoulder of the mouse subcutaneously. About 10 days after inoculation, mice with a tumor volume of 20-30 mm 3 were selected for grouping, 10 mice in each group, and 7 groups in total.
  • the PBMC cells were recovered on the day of the grouping, the cell concentration was adjusted to 25 million/mL with PBS, and each mouse was iv injected with 200 ⁇ L (5millon) PBMC, and then administered iv, and the tumor volume was measured twice a week after administration; the administration frequency was Q3D, 10 times in total.
  • the tumor growth curves of each group were drawn with the tumor volume as the ordinate and the administration time as the abscissa; each drug group and the control group were analyzed by one way ANOVA to compare the differences between the two groups. Experimental results: See Figure 6.
  • the experimental results showed that, compared with the control group IgG, F2-2, E1-B6, 81-2-2, 06-6 and 9-4 antibodies all showed a significant effect of inhibiting the growth of tumor cells. Among them, 81-2-2 has the same effect as 9-4, F2-2 has the same effect as E1-B6, and 06-6 has the strongest effect.
  • the five anti-CD73 monoclonal antibodies of the present invention all have the effect of significantly inhibiting the growth of tumor cells.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本发明用人胞外-5'-核苷酸酶(CD73)蛋白免疫小鼠，所得到的多个单克隆抗体的重链和轻链序列均是全新的氨基酸序列，可用于制备CD73酶活抑制剂和抗肿瘤药物。

Description

抗 胞外 -5，-核昔酸酶的抗体序列 技术领域： 本发 明涉及基因工程抗体， 特别涉及多种抗胞外 5 -核昔酸酶的抗体序列。 背景技术： 胞外 5-核昔酸酶 （ ecto- 55 -nucleotidase , 以下亦简称为 CD73）, 是多功能的胞外核酸 酶。 尽管 目前已经有人研发了一些治疗型 CD73单克隆抗体，但是， 单克隆抗体是由单个 克隆的杂交瘤细胞分泌的， 只能识别一种表位（抗原决定簇）的抗体。 而针对同一个生物 大分子的不同抗原表位可以制备出多种不同的单克隆抗体， 即使抗原表位相同，获得的单 抗序列也有可能不同。不同的序列以及靶位可能带来更优的药效或者更低的毒性以及更为 广泛的抗瘤谱， 开发出更多的抗胞外 5 -核昔酸酶的抗体序列， 才有可能解决更多的临床 问题。 发明内容 本发 明的目的是提供多种有效的抗胞外 5 -核昔酸酶 （即 CD73） 的抗体序列， 以用 于制备治疗肿瘤相关疾病的药物。 本发 明用人源 CD73蛋白免疫小鼠， 分别获得了五个抗 CD73单克隆抗体， 这些单克 隆抗体的重链和轻链序列， 均是全新的序列， 现有技术未见报道过。 本发 明获得的五个抗 CD73单克隆抗体中，其中四个抗体与现有技术抗体识别的抗原 表位也不同。 所用 的 CD73蛋白为申请人自主表达的人源 CD73蛋白，所用的小鼠为 BABL/C小鼠。 具体 的， 本发明通过如下手段完成了上述工作：
A 人源 CD73蛋白作为抗原以 30pg/只免疫 BABL/C小鼠,三周后同样剂量加强免疫;
B ELISA法测定免疫后小 鼠血清中的抗体滴度， 达到理想效果后以 50瞄剂量冲击 免疫；
C 取免疫成功的小鼠脾细胞与 SP2/0细胞进行融合， 待细胞长成细胞团后进行上清 滴度检测， 通过三轮亚克隆获得阳性单克隆细胞株;
D 单克隆细胞株扩大培养后进行小鼠腹腔注射制备腹水， 并从收集的腹水纯化得到 相应的抗体；
E 利用 SPR技术进行单克隆抗体的亲和动力学测定；
F 测定单克隆抗体对于 CD73酶活性的抑制作用；
G 测定单克隆抗体对于移植瘤模型的肿瘤抑制作用。 本 发明获得的五个抗 CD73单克隆抗体分别命名为： F2-2、 E1-B6、 81-2-2、 06-6和 9-4。 这些抗体特异性的分子基础主要来自于它的高度可变区 CDR1、 CDR2 和 CDR3, 这些区域是与抗原结合的关键部位。 本 发明获得的五个抗 CD73单克隆抗体中， 每个抗体的重链和中的 CDR1、 CDR2和 CDR3 分别是如下所示氨基酸序列的多肽：
F2-2： SEQIDNO.l、 SEQ ID NO.2和 SEQ ID NO.3； SEQIDN0.4、 SEQIDN0.5和 SEQIDN0.6 ；
E1-B6： SEQ ID NO.9 > SEQ ID NO.10和 SEQIDNO.il； SEQ ID NO.12、 SEQ ID NO.13 和 SEQ ID NO.14；
81-2-2： SEQ ID NO.17 > SEQ ID NO.18和 SEQIDNO.19； SEQIDNO.20、 SEQ ID NO.21和 SEQIDNO.22；
06-6： SEQ ID NO.25、 SEQ ID NO.26和 SEQ ID NO.27； SEQ ID NO.28、 SEQ ID NO.29 和 SEQIDNO.30；
9-4： SEQIDNO.33、 SEQ ID NO.34和 SEQ ID NO.35； SEQIDNO.36、 SEQ ID NO.37 和 SEQIDNO.38o 本 发明所述 “单克隆抗体 ”应该解释为涵盖具有所需特异性的结合结构域的任意特异 性结合因子， 可以是单价的或是单链抗体、 双链抗体、 嵌合抗体以及上述抗体的衍生物、 功能等同物和同源物， 也包括抗体片段和含有抗原结合结构域的任何多肽。 本 发明所述单克隆抗体的实例是免疫球蛋白 IgG亚型及其亚型亚类； 尽 管抗体特异性的分子基础主要来自于它的高度可变区 CDR1、 CDR2 和 CDR3, 为了维持优选的结合特性， 上述 CDR的序列应尽可能保留。 然而， 尽管如果有个别氨基 酸的改变， 也有可能能够达到本发明目的， 甚至使结合特性更为优化。但是， 个别氨基酸 的改变并不能脱离本发明的构思和发明精神。 除了如上所述重链和轻链中的高度可变区 CDRLCDR2 和 CDR3夕卜，其它为框架区。 框架区可在结合所需的三维结构不受影响的条件下被其他序列置换。 本发 明的有益效果： 经实验证实， 本发明所生产的五个抗 CD73单克隆抗体具有以下突出特点：
1、 与人 CD73具有高亲和力 （详见实施例 2）；
2、 与现有技术抗 CD73单克隆抗体识别的抗原表位不同 （详见实施例 3）；
3、 生化水平实验和细胞水平实验都显示具有高效的 CD73酶活抑制作用 （详见实施 例 4）；
4、 可显著抑制人免疫重构鼠移植瘤的生长 （详见实施例 5）。 附图说明 图 1 本发明的 5个抗 CD73单克隆抗体获得步骤的示意图； 图 2为实施例 2 （F2-2、 E1-B6、 81-2-2, 06-6和 9-4抗体与人 CD73的亲和力） 的实 验结果， 图中 Ka、 Kd和 KD分别为结合常数、 解离常数以及亲和力常数， Contbody为
MED I -9447； 图 3为实施例 3（F2-2、 E1-B6、 81-2-2、 06-6和 9-4抗体抗体表位分析）的实验结果, 其中表位作 图顺序依次为 E1-B6、 81-2-2、 9-4、 06-4以及 F2-2对其余所有抗体作图，
Contbody为 MEDI-9447； 图 5为实施例 4 （F2-2、 E1-B6、 81-2-2、 06-6和 9-4抗体对 CD73酶活性的抑制） 的 实验结果； 其中， 图 3是生化水平， 图 4是细胞水平 （A549）； 图 6为实施例 5 （F2-2、 E1-B6、 81-2-2、 06-6和 9-4抗体动物药效） 的实验结果 以上所述 F2-2、 E1-B6、 81-2-2、 06-6和 9-4, 分别是本发明得到的五个抗 CD73单克 隆抗体的名称。 序列信息：
SEQ ID NO.1、 SEQ ID NO.2和 SEQ ID NO.3 , 分别是抗 CD73单克隆抗体 F2-2的
CDR1 、 CDR2和 CDR3；
SEQ ID NO.4、 SEQ ID NO.5和 SEQ ID NO.6, 分别是抗 CD73单克隆抗体 F2-2的
CDR1 、 CDR2和 CDR3；
SEQ ID NO.7、 SEQ ID NO.8, 分别是抗 CD73单克隆抗体 F2-2和氨基酸序列；
SEQ ID NO.9、 SEQ ID NO.10和 SEQ ID NO.il , 分别是抗 CD73单克隆抗体 E1-B6 的 CDR1、 CDR2和 CDR3；
SEQ ID NO.12、 SEQ ID NO.13和 SEQ ID NO.14, 分别是抗 CD73单克隆抗体 E1-B6 的 CDR1、 CDR2和 CDR3；
SEQ ID NO.15、 SEQ ID NO.16分别是抗 CD73单克隆抗体 E1-B6和氨基酸序列；
SEQ ID NO.17、 SEQ ID NO.18和 SEQ ID NO.19分别是抗 CD73单克隆抗体 81-2-2 的 CDR1、 CDR2和 CDR3；
SEQ ID NO.20、 SEQ ID NO.21和 SEQ ID NO.22, 分别是抗 CD73单克隆抗体 81-2-2 的 CDR1、 CDR2和 CDR3；
SEQ ID NO.23、 SEQ ID NO.24分别是抗 CD73单克隆抗体 81-2-2和氨基酸序列；
SEQ ID NO.25、 SEQ ID NO.26和 SEQ ID NO.27分别是抗 CD73单克隆抗体 06-6的 CDR1、 CDR2和 CDR3；
SEQ ID NO.28、 SEQ ID NO.29和 SEQ ID NO.30, 分别是抗 CD73单克隆抗体 06-6 的 CDR1、 CDR2和 CDR3；
SEQ ID NO.31、 SEQ ID NO.32分别是抗 CD73单克隆抗体 06-6和氨基酸序列；
SEQ ID NO.33、 SEQ ID NO.34和 SEQ ID NO.35分别是抗 CD73单克隆抗体 9-4的 CDR1、 CDR2和 CDR3；
SEQ ID NO.36、 SEQ ID NO.37和 SEQ ID NO.38, 分别是抗 CD73单克隆抗体 9-4的 CDR1、 CDR2和 CDR3；
SEQ ID NO.39、 SEQ ID NO.40分别是抗 CD73单克隆抗体 9-4和氨基酸序列；
SEQ ID NO.41是本申请发明人自主构建表达的 CD73蛋白氨基酸序列。 具体实施方式 本发 明提供以下五条特异性的抗 CD73单克隆抗体的重链和序列。所述单克隆抗体由 CD73 蛋白免疫 BABL/c小鼠后经杂交瘤筛选得到的对应的单克隆细胞株表达； 所述单克 隆抗体型别为 IgG型。 以下实施例中所用的抗原为自主表达的人源 CD73蛋白， 蛋白 C端包含 6xHis标签; 所使用 的免疫佐剂为北京博奥龙 5周快速免疫佐剂，初次免疫 21天后加强免疫一次, 细胞融合前使用抗原冲击免疫一次即可进行细胞融合； 所使用 的融合方法为电融合法， 电融合仪为 BTX公司的 ECM2001型， 所用融合缓 冲液为原厂细胞融合液； 融合后 的细胞长出细胞团后采用 ELISA检测上清抗体表达， 酶标板包被 CD73蛋白 或者 His蛋白， His蛋白用来排除 Anti-His的假阳性孔； 阳性孔采用有限稀释法进行亚克隆，亚克隆共进行三轮，最终获得阳性单克隆细胞株; 阳性单克隆进行腹水制备后纯化，获得对应的单克隆抗体，单克隆抗体进一步用于亲 和力测定、 酶活抑制实验以及动物药效评价。 实施例 1 抗原免疫、 细胞融合以及阳性克隆的筛选和腹水抗体的制备纯化 实验 目的： 采用 自主表达的人源 CD73蛋白作为抗原制备单克隆抗体。 实验方法： 使用杂交瘤技术制备抗人 CD73单克隆抗体。 具体方法为： 使用 30曲蛋白对 4-6周的雌性 BALB/c小鼠进行免疫。 首次免疫后第 21天采用同样方法加强免疫一次。 首次免疫后第 35天通过眼眦采血分离血清进行血清滴度的 ELISA测定。 抗体滴度达到要求后采用 50瞄 CD73蛋白进行抗原冲击免疫。 冲击免疫 3天后取脾细胞与 SP2/0细胞进行融合， 待细胞成团后 ELISA检测杂交瘤 上清液中的抗 CD73 抗体。 实验结果： 经过三轮亚克隆，通过亲和力以及酶活抑制双重筛选， 最终获得了 5株 CD73抗体高 表达单克隆细胞株， 分别命名为 F2-2、 E1-B6、 81-2-2、 06-6和 9-4。 将单克隆细胞扩增 后进行腹水制备纯化抗体， 用于后续亲和力测定、 酶活抑制实验以及动物药效测定。 经验证，所得 5个单克隆抗体均具有高亲和力，同时可以有效抑制 CD73酶活，同时， 动物药效显示了理想的抑瘤效果。 实施例 2 五个抗 CD73单克隆抗体与重组人 CD73亲和动力学分析 实验 目的： 采用 Biacore T200系统检测各抗体的亲和动力学常数。 试剂和方法：
Mouse Antibody Capture Kit为购自 GE公司的商品化试剂盒，采用氨基偶联的方法将 抗小鼠 Fc IgG固定在 CM5传感器芯片上， 通过偶联的抗小鼠 Fc IgG捕获被测抗体， 然 后注射一系列浓度梯度的人 CD73蛋白， 之后采用试剂盒自带的 pH 1.7的 Glycine-Hcl再 生检测系统。 运行缓冲液为 HBS-EP+ ( lOmM HEPES, pH7.4, 150mM Nach 3mM EDTA和 0.05% P20), 测定温度 25 °C； 实验 中选用的对照物为 MEDI-9447； MEDI-9447为 Medlmmune公司的 CD73抗体， 我们依据其专利 ( US2016/0194407 A1 ) 的序列合成表达并纯化得到对照物。 使用 Biacore T200评估软件， 根据 1： 1结合模型拟合数据， 计算结合 (Ka)和解离 (Kd) 速率常数以及平衡常数 (KD)。 实验结果： 根据检测结果， 各抗体亲和力数据的具体结果见图 2和下表：
Figure imgf000008_0001
结果显示， 5个抗 CD73单克隆抗体均与重组人 CD73具有高亲和力，其中 F2-2亲和 力优于其它 4个抗体， 同时 F2-2抗体亲和力优于 MEDI-9447, 其余四个抗体亲和力稍弱 于 MEDI-9447 o 实验结论： 本发明获得的 5个抗 CD73单克隆抗体与人 CD73均具有高亲和力。 实施例 3 五个抗 CD73单克隆抗体的抗原表位分析 实验 目的： 分析各抗体抗原表位 的异同。 试剂和方法： 利用 Biacore T200系统分析各抗体抗原表位的异同。
Mouse Antibody Capture Kit为购自 GE公司的商品化试剂盒，采用氨基偶联的方法将 抗小鼠 Fc IgG固定在 CM5传感器芯片上， 通过偶联的抗小鼠 Fc IgG捕获第一抗体， 然 后以 1 OOpig/mL的 Mouse IgG封闭多余位点， 之后依次进样 CD73和第二抗体， 通过传感 图分析各抗体识别抗原表位的异同。 实验 中选用的对照物为 MEDI-9447 o 实验结果： 具体实验结果见图 3； 根据分析结果， 各抗体表位归纳汇总如下表:
Figure imgf000009_0001
根据表位作 图结果， 五个抗体中 F2-2识别独立表位， E1-B6、 06-6、 9-4识别同一表 位， 四个抗体与 MED 1-9447识别表位均不相同， 81-2-2与 MED 1-9447识别同一抗原表位, 但是 81-2-2与 MED 1-9447的 CDR区氨基酸序列均不相同 ,应此 , 81-2-2也是异于 MED 1-9447 的全新抗体。 实验结论： 本发 明获得的 5个抗 CD73单克隆抗体，其中有 4个抗体与现有技术抗 CD73单克隆 抗体识别的抗原表位不同。 实施例 4 五个抗体对 CD73酶活性的抑制 实验 目的： 分别从生化水平和细胞水平检测 F2-2、 E1-B6、 81-2-2、 06-6以及 9-4抗体抑制 CD73 酶活的能力。 实验方法：
1、 生化方法 将 F2-2、 E1-B6、 81-2-2、 06-6、 9-4以及 MED 1-9447抗体以合适的浓度梯度加至 96 孔白板底部， 然后每孔加 CD73蛋白至终浓度为 0.25ng/mL, 37°C孵育 15min, 孵育结束 后分别添加 AMP和 ATP至终浓度为 500pmol/L和 100pnol/L, 37°C孵育 30min。 最后， 每孔加入等体积的 Cell titer Gio。 酶标仪化学发光法检测各孔信号值， 计算并处理数据；
2、 细胞方法 将 F2-2、 E1-B6、 81-2-2、 06-6、 9-4以及 MED 1-9447抗体以合适的浓度梯度加至 96 孔板底部， A549细胞消化重悬后计数， 以 1 ><105个 /孔的密度接种至对应的孔， 二氧化碳 培养箱孵育 15min, 孵育结束后将 AMP加至各孔， 终浓度为 500pmol/L, 二氧化碳培养 箱继续孵育 24h； 24h后将 96孔板取出 lOOOrpm离心 5min, 每孔取 50[iL上清至新的 96 孔白板对应位置， 之后每孔加入 ATP溶液至终浓度为 100|imol/L, 然后每孔加入等体积 的 Cell titer Gio。 酶标仪化学发光检测各孔信号值， 计算并处理数据。 实验结果： 见图 4、 图 5 活抑制效应 EC50汇总见下表， 其中对照物为 MEDI-9447：
Figure imgf000010_0001
五种单克隆抗体 中， F2-2生化和细胞水平 CD73酶活抑制作用均优于其余四个抗体 和 MED 1-9447, 其余四个抗体对 CD73酶活抑制作用比 MED 1-9447稍弱。 实验结论： 生化水平实验和细胞水平实验都显示，本发明获得的 5个抗 CD73单克隆抗体具有高 效的 CD73酶活抑制作用。 实施例 5 五个抗体的动物药效评价 实验 目的： 体内实验测试五个抗体抑制肿瘤细胞生长效果。 实验方法： 取 90只 B-NDG小鼠， 至少适应性饲养一周。 培养 A549细胞，隔天传一次代，最后收集细胞， PBS将细胞终浓度调整到 5*107/mL, 每只 O. lmL接种到小鼠右侧肩部皮下。 接种后 10天左右， 选取肿瘤体积 20-30mm3的小鼠进行分组， 每组 10只， 共 7组。 分组当天复苏 PBMC 细胞， PBS将细胞浓度调整到 25million/mL, 每只小鼠 i.v.注射 200|iL ( 5millon) PBMC,然后 i.v.给药，给药后每周测量 2次肿瘤体积；给药频率为 Q3D, 共 10次。 以肿瘤体积为纵坐标、给药时间为横坐标绘制各组肿瘤生长曲线；各药物组与对照组 进行 one way ANOVA分析， 比较两组之间的差异。 实验结果： 见图 6。 实验结果表明， 与对照组 IgG相比， F2-2、 E1-B6、 81-2-2、 06-6 以及 9-4抗体均显示出显著的抑制肿瘤细胞生长的效果。其中 81-2-2与 9-4效果相当, F2-2 与 E1-B6效果相当， 06-6药效最强。 实验结论： 本发明的五个抗 CD73单克隆抗体均具有显著抑制肿瘤细胞生长的作用。

Claims

权 利 要 求 书
1、 以下氨基酸序列：
SEQIDNO.l、 SEQIDNO.2、 SEQIDNO.3、 SEQIDNO.4、 SEQIDNO.5、 SEQ ID NO.6、 SEQIDNO.9、 SEQ ID NO.10、 SEQIDNO.11、 EQ ID NO.12、 SEQ ID NO.13、 SEQ IDNO.14、 SEQ ID NO.17. SEQIDNO.18、 SEQ ID NO.19. SEQIDNO.20、 SEQ ID NO.21、 SEQ ID NO.22、 SEQIDNO.25、 SEQ ID NO.26、 SEQ ID NO.27、 SEQIDNO.28、 SEQ ID NO.29、 SEQIDNO.30、 SEQIDNO.33、 SEQ ID NO.34、 SEQ ID NO.35、 SEQIDNO.36、 SEQ ID NO.37和 SEQ ID NO.38 o
2、权利要求 1所述氨基酸序列中的一个或多个在制备抗 CD73单克隆抗体中的应用。
3、 权利要求 1所述氨基酸序列中的一个或多个在制备 CD73酶活抑制剂中的应用。
4、 权利要求 1所述氨基酸序列中的一个或多个在制备抗肿瘤药物中的应用。
5、 以下各组氨基酸中一组或两组在制备抗 CD73单克隆抗体中的应用，每组的氨基 酸序列如下所示:
SEQIDN0.1、 SEQ ID NO.2和 SEQ ID NO.3；
SEQIDN0.4、 SEQ ID NO.5和 SEQ ID NO.6；
SEQIDN0.9、 SEQ ID NO.10和 SEQIDNO.il；
SEQ ID NO.12、 SEQ ID NO.13和 SEQ ID NO.14；
SEQIDNO.17、 SEQ ID NO.18和 SEQ ID NO.19；
SEQIDNO.20、 SEQ ID NO.21和 SEQ ID NO.22；
SEQIDNO.25、 SEQ ID NO.26和 SEQ ID NO.27；
SEQIDNO.28、 SEQ ID NO.29和 SEQ ID NO.30；
SEQIDNO.33、 SEQ ID NO.34和 SEQ ID NO.35；
SEQIDNO.36、 SEQ ID NO.37和 SEQ ID NO.38。
6、 权利要求 5所述组中一组或两组氨基酸序列在制备抗肿瘤药物中的应用。
7、 含有权利要求 5所述组中一组或两组氨基酸序列的单克隆抗体。
8、 以下五个抗 CD73单克隆抗体， 每个抗体的重链和轻链可变区中的 CDR1、 CDR2 和 CDR3分别是下述氨基酸序列： 抗 体 F2-2： 重链可变区 SEQIDNO.l、 SEQ ID NO.2和 SEQ ID NO.3； 轻链可变区 SEQIDNO.4 、 SEQ ID NO.5和 SEQIDNO.6； 抗体 E1-B6： 重链可变区 SEQIDN0.9、 SEQIDNO.10和 SEQIDNO.il； 轻链可变 区 SEQIDN0.12、 SEQIDN0.13和 SEQIDN0.14； 抗体 81-2-2： 重链可变区 SEQIDN0.17、 SEQIDN0.18和 SEQIDN0.19； 轻链可 变区 SEQ ID NO.20、 SEQ ID NO.21和 SEQ ID NO.22； 抗体 06-6： 重链可变区 SEQIDNO.25、 SEQ ID NO.26和 SEQIDNO.27； 轻链可变 区 SEQ ID NO.28、 SEQ ID NO.29和 SEQ ID NO.30； 抗体 9-4： 重链可变区 SEQIDNO.33、 SEQ ID NO.34和 SEQIDNO.35； 轻链可变区 SEQIDNO.36 、 SEQ ID NO.37和 SEQIDNO.38。
9、 权利要求 7或 8所述的单克隆抗体在制备抗肿瘤药物中的应用。
10、 权利要求 7或 8所述的单克隆抗体在制备 CD73酶活抑制剂中的应用。
PCT/CN2021/131861 2020-11-09 2021-11-19 抗胞外-5'-核苷酸酶的抗体序列 WO2022096020A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/827,630 US20220356267A1 (en) 2020-11-09 2022-05-27 Sequences of anti-ecto-5'-nucleotidase antibodies

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011239238.1 2020-11-09
CN202011239238.1A CN114456268B (zh) 2020-11-09 2020-11-09 抗胞外-5’-核苷酸酶的抗体序列

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/827,630 Continuation US20220356267A1 (en) 2020-11-09 2022-05-27 Sequences of anti-ecto-5'-nucleotidase antibodies

Publications (1)

Publication Number Publication Date
WO2022096020A1 true WO2022096020A1 (zh) 2022-05-12

Family

ID=81405044

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/131861 WO2022096020A1 (zh) 2020-11-09 2021-11-19 抗胞外-5'-核苷酸酶的抗体序列

Country Status (3)

Country Link
US (1) US20220356267A1 (zh)
CN (5) CN117164713A (zh)
WO (1) WO2022096020A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023201267A1 (en) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Combination therapy for treating trop-2 expressing cancers
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107001472A (zh) * 2014-11-10 2017-08-01 免疫医疗有限公司 对cd73具有特异性的结合分子及其用途
CN107001474A (zh) * 2014-11-21 2017-08-01 百时美施贵宝公司 抗cd73抗体及其用途
CN110785187A (zh) * 2017-06-22 2020-02-11 诺华股份有限公司 针对cd73的抗体分子及其用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103003424B (zh) * 2010-05-18 2015-05-13 株式会社医学生物学研究所 结合转化生长因子α且对Ras基因突变的癌具有增殖抑制活性的抗体
MX2019006897A (es) * 2016-12-13 2019-08-22 Astellas Pharma Inc Anticuerpo anti cd73 humana.
JP2020536495A (ja) * 2017-08-30 2020-12-17 フェインズ セラピューティクス，インコーポレーテッド 抗ｌａｇ−３抗体及びその使用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107001472A (zh) * 2014-11-10 2017-08-01 免疫医疗有限公司 对cd73具有特异性的结合分子及其用途
CN107001474A (zh) * 2014-11-21 2017-08-01 百时美施贵宝公司 抗cd73抗体及其用途
CN110785187A (zh) * 2017-06-22 2020-02-11 诺华股份有限公司 针对cd73的抗体分子及其用途

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023201267A1 (en) 2022-04-13 2023-10-19 Gilead Sciences, Inc. Combination therapy for treating trop-2 expressing cancers
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

Also Published As

Publication number Publication date
CN114456268B (zh) 2023-08-18
CN117164713A (zh) 2023-12-05
CN117126281A (zh) 2023-11-28
US20220356267A1 (en) 2022-11-10
CN114456268A (zh) 2022-05-10
CN117264060A (zh) 2023-12-22
CN117285630A (zh) 2023-12-26

Similar Documents

Publication Publication Date Title
CN111051347B (zh) Tigit抗体、其抗原结合片段及医药用途
CN110914304B (zh) Cd96抗体、其抗原结合片段及医药用途
WO2018177393A1 (zh) B7-h3抗体、其抗原结合片段及其医药用途
WO2016173558A1 (zh) 抗诺如病毒gii.4型鼠源单克隆抗体的制备和应用
CN111744007A (zh) 一种抗tigit抗体药物组合物及其用途
WO2022096020A1 (zh) 抗胞外-5&#39;-核苷酸酶的抗体序列
US11370836B2 (en) Monoclonal antibody binding to human IL-5, preparation method therefor and use thereof
CN111744013A (zh) 抗tigit抗体联合pd-1抑制剂治疗疾病的方法和药物组合
US20230138315A1 (en) Anti-angptl3 antibody and use thereof
WO2016173559A1 (zh) 抗诺如病毒gi.1型鼠源单克隆抗体的制备和应用
CN117062838A (zh) 一种靶向bcma的纳米抗体及其应用
WO2019096219A1 (zh) 一种人源化抗il-13抗体及其制备方法和应用
WO2019109947A1 (zh) 抗人il6单克隆抗体及其制备方法和用途
CN108727488B (zh) 抗诺如病毒gii.17单克隆抗体的制备和应用
CN113817052A (zh) 抗SARS-CoV-2核衣壳蛋白单克隆抗体及其制备方法和用途
CN111051343A (zh) Il-6r抗体、其抗原结合片段及医药用途
CN114195888B (zh) 一种抗新冠抗体药物的核心序列
CN114685667B (zh) 间皮素结合分子及其应用
WO2020239014A1 (zh) 抗cgrp抗体及其应用
WO2023206242A1 (zh) 无钩状效应的抗人cd73单克隆抗体
CN114907480B (zh) 无钩状效应的抗人cd73单克隆抗体
WO2021218574A1 (zh) 结合人ngf的抗体、其制备方法和用途
WO2023083327A1 (zh) 抗cldn18.2单克隆抗体及其应用
TW202325739A (zh) 新穎Nav1.7單株抗體
CN115746144A (zh) 抗tigit-pd1双特异性抗体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21888712

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21888712

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 21888712

Country of ref document: EP

Kind code of ref document: A1