WO2023131193A1 - 靶向人b7-h3分子的人源化单克隆抗体及其应用 - Google Patents

靶向人b7-h3分子的人源化单克隆抗体及其应用 Download PDF

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WO2023131193A1
WO2023131193A1 PCT/CN2023/070469 CN2023070469W WO2023131193A1 WO 2023131193 A1 WO2023131193 A1 WO 2023131193A1 CN 2023070469 W CN2023070469 W CN 2023070469W WO 2023131193 A1 WO2023131193 A1 WO 2023131193A1
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cancer
monoclonal antibody
cdr
seq
hu4g4
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张学光
傅丰庆
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苏州旭光科星抗体生物科技有限公司
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C—CHEMISTRY; METALLURGY
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    • 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]

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  • the invention belongs to the technical field of biomedicine, and in particular relates to a humanized monoclonal antibody targeting human B7-H3 molecules and an application thereof.
  • B7-H3 protein is almost not expressed in normal tissues, but highly expressed in a variety of tumor tissues, including glioma, lung cancer, colon cancer, kidney cancer and prostate cancer tissues.
  • a monoclonal antibody (clone number: 376.96) that recognized tumor-associated antigens was identified as B7-H3, so B7-H3 was considered to be a marker widely expressed on the surface of tumor cells and was called "tumor-associated antigens”.
  • tumor-associated antigens A large number of studies have shown that the abnormal expression of B7-H3 is related to the poor prognosis of various tumors, including patient survival rate, disease progression and pathological grade, and promotes tumor growth, metastasis and recurrence.
  • B7-H3 After interfering with the expression of B7-H3 in tumor cells, it was found that B7-H3 molecules participated in the biological behavior, glycolysis ability and tumor angiogenesis of tumor cells. Although the receptors or interacting molecules of B7-H3 have not yet been clarified, and the role and mechanism of tumor cell expression of B7-H3 need further study, B7-H3 expressed by tumor cells can directly mediate the biological behavior and metabolism of tumor cells. Adjust. B7-H3 has become the most concerned immune checkpoint molecule in tumor immunotherapy, and it also provides a target of great clinical value for tumor targeted therapy.
  • the purpose of the present invention is to provide a humanized monoclonal antibody targeting human B7-H3 molecules and its application.
  • the provided monoclonal antibody can specifically recognize human B7-H3 molecules.
  • Animal experiments have confirmed the monoclonal antibody Conjugated iodine 131 can effectively inhibit the growth of tumors in mice, thus providing new therapeutic antibody candidate molecules for tumor cell immunotherapy.
  • the humanized monoclonal antibody hu4G4 specifically targeting human B7-H3 includes a heavy chain and a light chain, wherein the CDR region of the heavy chain includes CDR-H1, CDR-H2 and CDR-H3, Its sequence information is as follows:
  • CDR-H2 FIRNRVNGYTTEYTASVKG (SEQ ID NO: 2),
  • CDR-H3 DRGPGKHAIDY (SEQ ID NO: 3);
  • the light chain CDR region contains CDR-L1, CDR-L2 and CDR-L3, and its sequence information is as follows:
  • CDR-L2 HSARMDS (SEQ ID NO: 5),
  • CDR-L3 QNGYILPYT (SEQ ID NO: 6);
  • VH heavy chain variable region
  • VL light chain variable region
  • the monoclonal antibody hu4G4 provided by the present invention can be used to prepare products for detecting B7-H3 protein;
  • the monoclonal antibody hu4G4 provided by the present invention can also be used to prepare products for blocking B7-H3 protein;
  • the present invention also provides a pharmaceutical composition, which contains the monoclonal antibody hu4G4 of the present invention.
  • composition also includes pharmaceutically acceptable carriers and/or excipients.
  • the tumor is selected from acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, kidney tumor, bladder cancer, gastrointestinal tract cancer , prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell cancer Skin cancer, squamous cell skin cancer, dermatofibrosarcoma herniation, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.
  • the present invention provides a humanized monoclonal antibody against human B7-H3 molecule
  • the anti-human B7-H3 monoclonal antibody is hu4G4, including heavy chain and light chain, and the heavy chain variable region (VH ) is identical to SEQ ID NO.1
  • the amino acid sequence of the light chain variable region (VL) of hu4G4 is identical to SEQ ID NO.2
  • the humanized antibody can specifically recognize tumor cells in a mouse tumor-bearing model , coupled with iodine 131 can effectively inhibit tumor growth in glioma-bearing mice, and provide therapeutic antibody candidate molecules for tumor immunotherapy targeting B7-H3 molecules.
  • Figure 1 Anti-human B7-H3 monoclonal antibody hu4G4 protein electrophoresis staining identification diagram, in which heavy chain: H chain; light chain: L chain;
  • Figure 2 The antigen site map of the humanized B7-H3 monoclonal antibody hu4G4 competitively binding to the m4G4 monoclonal antibody, where the black line is the binding of hu4G4 to the antigen molecule after the gradient is added to the parental antibody m4G4; the red line is the gradient to the parental antibody After blocking by antibody m4G4, the binding of the control anti-human B7-H3 antibody to the antigen molecule.
  • Figure 3 The antigen site map of the humanized B7-H3 monoclonal antibody hu4G4 competitively binding to the cell surface of m4G4 monoclonal antibody, in which the dotted line peak is the binding of mouse parental antibody m4G4 to B7-H3 transgenic cells; the solid line peak is the addition of 10ug Binding of m4G4 to B7-H3 transgenic cells after blocking by humanized antibody hu4G4; the gray peak is the control.
  • Figure 4 Affinity detection diagram of humanized B7-H3 monoclonal antibody hu4G4, in which ka binding constant, kd dissociation constant, KD affinity constant.
  • the main reagents used in the specific examples of the present invention are: human antibody B7-H3 (hu4G4), mouse antibody B7-H3 (m4G4); PBS, 1640 medium, fetal bovine serum, PE-goat anti-human Fc Anti-, PE-mouse anti-human Fc secondary antibodies, B7-H3 antigen protein, 89Zr and 131I are all commercially available products, and in addition, the reagents, operating tools and instruments used in the following specific examples are all Conventional reagents, operating tools and instruments in this field will not be repeated here.
  • the L929/B7-H3 cell line was selected as the detection cell, the hu4G4 purified antibody was collected as the primary antibody, and the PE-labeled goat anti-human Fc fragment antibody was used as the secondary antibody to detect it by flow cytometry.
  • CHO eukaryotic cells were used to express hu4G4 monoclonal antibody, the initial cell concentration was adjusted to 2 ⁇ 106/ml, and the expression supernatant was collected after 7 days of culture, concentrated by ultrafiltration and concentration system, and Protein G affinity layer was used according to the protocol provided by Phamacia Company After column separation and purification, it was quantified by the Lowy method, and the antibody was sterilized by filtration and stored in -20°C.
  • the purified antibody was analyzed by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie brilliant blue overnight, decolorized with the decolorizing solution, imaged and photographed, and the molecular weight was compared with the standard protein as the molecular weight control.
  • hu4G4 recognizes B7-H3 molecules on the surface of U87 cells
  • Human glioma U87 cells with high expression of B7-H3 in the logarithmic growth phase were incubated with hu4G4 monoclonal antibody and corresponding isotype control IgG at 4°C for 40 minutes, after washing, the secondary antibody was added and incubated at 4°C for 20 minutes, washed with PBS buffer After 2 times, the cells were resuspended and the expression of B7-H3 on the cell surface was detected by flow cytometry.
  • hu4G4 targets and recognizes tumor cells in vivo
  • glioma-bearing mouse model Construction of a glioma-bearing mouse model.
  • the antibody was labeled with 89Zr, injected into the mice through the tail vein, and performed dynamic PET imaging at 24h, 72h, and 168h to observe the accumulation of nuclides in the animal organs, bones and tumor sites.
  • glioma-bearing mouse model Construction of a glioma-bearing mouse model. Antibodies were labeled with the therapeutic nuclide iodine 131 and injected into mice via the tail vein, and tumor size and growth curves were monitored.
  • Example 1 Enzyme-linked immunosorbent assay (ELISA) flow cytometry identification humanized B7-H3 monoclonal antibody competes with parental mouse antibody for epitope recognition
  • the purified hu4G4 SDS-PAGE electrophoresis showed the position and size of the heavy chain and light chain of the human B7-H3 antibody as shown in Figure 1.
  • CDR-H2 FIRNRVNGYTTEYTASVKG (SEQ ID NO: 2),
  • CDR-H3 DRGPGKHAIDY (SEQ ID NO: 3);
  • the light chain CDR region contains CDR-L1, CDR-L2 and CDR-L3, and its sequence information is as follows:
  • CDR-L2 HSARMDS (SEQ ID NO: 5),
  • CDR-L3 QNGYILPYT (SEQ ID NO: 6);
  • VH heavy chain variable region
  • VL light chain variable region
  • Collect L929/B7-H3 transgenic cells wash with PBS, resuspend to adjust the cell concentration to 5 ⁇ 105/tube, use immunofluorescence labeling competition experiment, add 10ug/ml human B7-H3 monoclonal antibody hu4G4, act for 40min at 4°C and use After washing twice with PBS, add mouse B7-H3 monoclonal antibody m4G4 (1.0 ⁇ g/tube) for 40 minutes at 4°C, wash twice with PBS, use PE-labeled goat anti-mouse IgG antibody as a secondary antibody for 20 minutes at 4°C, and wash twice with PBS Post-flow cytometry analysis.
  • Example 2 Humanized B7-H3 monoclonal antibody and antigen binding ability and affinity detection
  • the B7-H3 antigen protein was formulated into 7 concentrations of 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM and 1.56nM, and sampled on a 96-well plate.
  • Human Protein G probe was selected, and the stationary phase was antigen.
  • the mobile phase was human B7-H3 monoclonal antibody hu4G4, and the binding time was set to 180s and the dissociation time was set to 300s according to the experimental protocol.
  • Example 4 Anti-human B7-H3 humanized antibody coupled to iodine 131 inhibits tumor growth
  • the hu4G4 antibody was labeled with a therapeutic nuclide 131I, and its antitumor efficacy was studied in a glioma model.
  • the results of the study showed that after 7 days of 131I-hu4G4 treatment (radioactive dose 400-500 ⁇ Ci/monkey), the tumor volume was significantly lower than that of the PBs and 131I-IgG control group (p ⁇ 0.05); The tumor inhibition rates were all >80%, and the relative tumor proliferation rates on D20 were all ⁇ 30%, and the tumor inhibition effect was significantly better than that of the control group 131I-IgG ( Figure 7). It shows that 131I-hu4G4 has the potential to treat glioma.

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Abstract

提出一种抗人B7-H3分子的人源化单克隆抗体，所述抗人B7-H3单克隆抗体为hu4G4，包括重链和轻链，所述hu4G4的重链可变区(VH)的氨基酸序列与SEQ ID NO.1相同；hu4G4的轻链可变区(VL)的氨基酸序列与SEQ ID NO.2相同；该人源化抗体在小鼠荷瘤模型中能特异识别肿瘤细胞，偶联碘131能有效抑制胶质瘤荷瘤小鼠的肿瘤生长，为靶向B7-H3分子的肿瘤免疫治疗提供治疗性抗体候选分子。

Description

靶向人B7-H3分子的人源化单克隆抗体及其应用 技术领域
本发明属于生物医学技术领域，具体涉及一种靶向人B7-H3分子的人源化单克隆抗体及其应用。
背景技术
近年来，诸多临床研究表明B7-H3蛋白在正常组织中几乎不表达，却高表达在多种肿瘤组织，包括胶质瘤、肺癌、肠癌、肾癌以及前列腺癌组织中。加之上世纪80年代一株识别肿瘤相关抗原的单抗(克隆号：376.96)被鉴定出其识别的分子就是B7-H3，因此B7-H3被认为是一个广泛表达在肿瘤细胞表面的标志，被称为“肿瘤相关抗原”。已有大量研究表明，B7-H3的异常表达与多种肿瘤的不良预后相关，包括与病人生存率、疾病进展和病理分级等，并促进肿瘤的生长、转移和复发。
干扰肿瘤细胞中B7-H3的表达后发现B7-H3分子参与肿瘤细胞的生物学行为、糖酵解能力以及与肿瘤血管生成。尽管由于B7-H3的受体或相互作用的分子尚未明确，肿瘤细胞表达B7-H3的作用和机制也有待进一步研究，但肿瘤细胞表达的B7-H3可以直接介导肿瘤细胞生物学行为和代谢的调节。B7-H3已成为肿瘤免疫治疗中的最受关注的免疫检查点分子，也为肿瘤靶向治疗提供极具临床价值的靶点。
发明内容
本发明的目的是提供种一种靶向人B7-H3分子的人源化单克隆抗体及其应用，所提供的单克隆抗体可特异性识别人B7-H3分子，动物实验证实该单克隆抗体偶联碘131能有效抑制小鼠肿瘤的生长，从而为肿瘤细胞免疫治疗提供新治疗性抗体候选分子。
本发明所提供的特异性靶向人B7-H3的人源化单克隆抗体hu4G4，其包含有重链和轻链，其中重链CDR区包含有CDR-H1、CDR-H2和CDR-H3，其序列信息如下：
CDR-H1：DYYIN(SEQ ID NO：1)、
CDR-H2：FIRNRVNGYTTEYTASVKG(SEQ ID NO：2)、
CDR-H3：DRGPGKHAIDY(SEQ ID NO：3)；
轻链CDR区包含有CDR-L1、CDR-L2和CDR-L3，其序列信息如下：
CDR-L1：RAAQDLEYYLD(SEQ ID NO：4)、
CDR-L2：HSARMDS(SEQ ID NO：5)、
CDR-L3：QNGYILPYT(SEQ ID NO：6)；
更进一步的，所述的人源化单克隆抗体hu4G4，其重链可变区(VH)的氨基酸序列如下：
Figure PCTCN2023070469-appb-000001
所述的人源化单克隆抗体hu4G4，其轻链可变区(VL)氨基酸序列如下：
Figure PCTCN2023070469-appb-000002
本发明所提供的单克隆抗体hu4G4可用于制备检测B7-H3蛋白的制品；
另一个方面，本发明所提供的单克隆抗体hu4G4还可用于制备用于阻断B7-H3蛋白的制品；
本发明还提供了一种药物组合物，其中包含有本发明的单克隆抗体hu4G4。
进一步，所述药物组合物还包括药学上可接受的载体和/或赋形剂。
本发明所提供的药物组合物在制备预防和/或治疗肿瘤的药物中的应用。
进一步，所述肿瘤选自急性骨髓白血病、慢性骨髓白血病、急性淋巴细胞白血病、非霍金性淋巴瘤、多发性骨髓瘤、黑色素瘤、肺癌、结肠直肠癌、肾肿瘤、膀胱癌、胃肠道癌、前列腺癌、肝癌、卵巢癌、胰腺癌、子宫内膜癌、胃癌、前 列腺癌、肾癌、宫颈癌、甲状腺癌、子宫癌、神经内分泌癌、头颈癌、鼻咽癌、睾丸癌、基底细胞皮肤癌、鳞状细胞皮肤癌、皮肤纤维肉瘤突出症、梅克尔细胞癌、胶质母细胞瘤、神经胶质瘤、肉瘤、间皮瘤或骨髓发育不良综合征。
本发明提供一种抗人B7-H3分子的人源化单克隆抗体，所述抗人B7-H3单克隆抗体为hu4G4，包括重链和轻链，所述hu4G4的重链可变区(VH)的氨基酸序列与SEQ ID NO.1相同；hu4G4的轻链可变区(VL)的氨基酸序列与SEQ ID NO.2相同；该人源化抗体在小鼠荷瘤模型中能特异识别肿瘤细胞，偶联碘131能有效抑制胶质瘤荷瘤小鼠的肿瘤生长，为靶向B7-H3分子的肿瘤免疫治疗提供治疗性抗体候选分子。
附图说明
图1：抗人B7-H3单抗hu4G4蛋白电泳染色鉴定图，其中重链：H chain；轻链：L chain；
图2：人源化B7-H3单抗hu4G4竞争结合m4G4单抗的抗原位点图，其中黑线为梯度加入母本抗体m4G4阻断后，hu4G4与抗原分子的结合；红线为梯度加入母本抗体m4G4阻断后，对照抗人B7-H3抗体与抗原分子的结合。
图3：人源化B7-H3单抗hu4G4竞争结合m4G4单抗细胞表面的抗原位点图，其中虚线峰为鼠源母本抗体m4G4与B7-H3转基因细胞的结合；实线峰为加入10ug/ml人源化抗体hu4G4阻断后，m4G4与B7-H3转基因细胞的结合；灰色峰为对照。
图4：人源化B7-H3单抗hu4G4的亲和力检测图，其中ka结合常数，kd解离常数，KD亲和常数。
图5.hu4G4结合U87细胞表面B7-H3分子
图6.抗人B7-H3抗体hu4G4利用89Zr-免疫PET 72h扫描MIP图
图7治疗性核素131I标记B7-H3抗体明显抑制脑胶质瘤肿瘤的生长
具体实施方式
本发明具体实施例中所使用的主要试剂有：人源抗体B7-H3(hu4G4)、鼠源抗体B7-H3(m4G4)；PBS、1640培养基、胎牛血清、PE-羊抗人Fc二抗、PE-鼠抗人Fc二抗、B7-H3抗原蛋白、89Zr和131I均为市售产品，此外下述具体实施例中所采用的未特别提及的试剂、操作工具和仪器等均为本领域常规试剂、操作工具和仪器，不再赘述。
本发明方法的基本流程如下：
1.流式细胞术鉴定抗人B7-H3单抗
选用L929/B7-H3细胞株作为检测细胞，收集hu4G4纯化抗体作为一抗，以PE标记的羊抗人Fc段抗体作为二抗，利用流式细胞仪对其进行检测。
2.Protein G亲和层析柱分离纯化hu4G4和Lowy法定量
采用CHO真核细胞表达hu4G4单抗，起始细胞浓度调整至2×106/ml并培养7d后收集表达上清，经超滤浓缩系统浓缩，按Phamacia公司提供的方案，采用Protein G亲和层析柱分离纯化后经Lowy法定量，抗体过滤除菌后分装于-20℃保存。
3.蛋白电泳对hu4G4重链和轻链的鉴定和纯度的初步检测
纯化后的抗体经12％的SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析，考马斯亮蓝染色过夜，脱色液脱色后，成像摄片，设标准蛋白为分子量对照，比对分子量大小。
4.hu4G4抗原结合表位的鉴定
将L929/4G4细胞株和已制备的抗人B7-H3人源抗体(hu4G4)于4℃反应40min，充分洗涤后，再和母本鼠源抗体m4G4于4℃反应40min，充分洗涤后，加入PE-羊抗小鼠二抗，流式细胞术检测。
将B7-H3蛋白包板，加入不同浓度的母本鼠源抗体m4G4 4℃反应60min，再加入抗人B7-H3人源抗体(hu4G4)反应，再加入hrp-羊抗人二抗，Elisa检测。
5.hu4G4识别U87细胞表面的B7-H3分子
取对数生长期的高表达B7-H3的人脑胶质瘤U87细胞与hu4G4单抗及相应的同型对照IgG在4℃孵育40min，洗涤后再加入二抗4℃孵育20min，PBS缓冲液洗涤2次后，重悬细胞并经流式细胞仪检测细胞表面B7-H3的表达。
6.hu4G4体内靶向识别肿瘤细胞
构建胶质瘤荷瘤小鼠模型。抗体用89Zr进行标记，通过尾静脉注射到小鼠体内，24h、72h、168h动态PET显像，观察核素在动物脏器、骨骼和肿瘤部位的聚集情况。
7.分析抗人B7-H3单抗偶联碘131对肿瘤生长的影响
构建胶质瘤荷瘤小鼠模型。抗体用治疗性核素碘131标记，通过尾静脉注射到小鼠体内，监测肿瘤大小和生长曲线。
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。
实施例1：酶联免疫吸附测定法(ELISA)流式细胞术鉴定人源化B7-H3单抗与母本鼠源抗体竞争识别抗原表位
纯化后的hu4G4SDS-PAGE电泳显示人B7-H3抗体重链和轻链位置和大小如图1所示。
测序确定人B7-H3抗体重链CDR区包含有CDR-H1、CDR-H2和CDR-H3，其序列信息如下：
CDR-H1：DYYIN(SEQ ID NO：1)、
CDR-H2：FIRNRVNGYTTEYTASVKG(SEQ ID NO：2)、
CDR-H3：DRGPGKHAIDY(SEQ ID NO：3)；
轻链CDR区包含有CDR-L1、CDR-L2和CDR-L3，其序列信息如下：
CDR-L1：RAAQDLEYYLD(SEQ ID NO：4)、
CDR-L2：HSARMDS(SEQ ID NO：5)、
CDR-L3：QNGYILPYT(SEQ ID NO：6)；
其重链可变区(VH)的氨基酸序列如下：
Figure PCTCN2023070469-appb-000003
所述的人源化单克隆抗体hu4G4，其轻链可变区(VL)的氨基酸序列如下：
Figure PCTCN2023070469-appb-000004
为了鉴定人源B7-H3单抗hu4G4与母本鼠源B7-H3单抗m4G4识别抗原位点的差异，用10μg/ml的B7-H3蛋白铺板，分别加入0.625、1.25、2.5、5、10、20和40μg/ml的鼠源母本抗体反应1h，再加入人源化抗体hu4G4抗体进行ELISA检测，结果显示，0.625μg/ml的m4G4就可以阻断hu4G4与抗原分子的结合，提示，hu4G4和m4G4识别同一个位点(图2)。
收集L929/B7-H3转基因细胞，PBS洗涤后重悬调整细胞浓度为5×105/管，采用免疫荧光标记竞争实验，加入10ug/ml的人源B7-H3单抗hu4G4，4℃作用40min并用PBS洗涤两遍后，加入鼠源B7-H3单抗m4G4(1.0μg/管)4℃作用40min，PBS洗涤两遍，PE标记羊抗鼠IgG抗体作为二抗4℃反应20min，PBS洗涤两遍后流式细胞仪分析。
结果显示，人源B7-H3单抗hu4G4可以有效地阻断鼠源B7-H3 m4G4与转基因细胞上CD40分子的特异性结合，提示，hu4G4和m4G4识别同一个位点(图3)。
实施例2：人源化B7-H3单抗与抗原结合能力及亲和力检测
将B7-H3抗原蛋白配成100nM，50nM，25nM，12.5nM，6.25nM，3.13nM和1.56nM梯度稀释的7个浓度，96孔板上样，选择人Protein G探针，固定相为抗原，流动相为人源B7-H3单抗hu4G4，根据实验方案将结合时间设定为180s，解离时间为300s。
检测人源单抗的结合及解离常数，计算亲和力，结果表明hu4G4亲和常数(KD)为2.625E-10(图4)。
实施例3：抗B7-H3单抗hu4G4识别B7-H3分子
取对数生长期的U87细胞和与hu4G4及相应的同型对照IgG在4℃孵育40min，洗涤后再加入相应的二抗4℃孵育20min，用PBS缓冲液洗涤2次后，重悬细胞并经流式细胞仪检测细胞表面B7-H3的表达。流式分析结果显示本发明筛选的hu4G4能识别、结合U87细胞表面B7-H3分子(图5)。进一步鉴定了hu4G4在胶质瘤荷瘤小鼠模型体内对肿瘤细胞的结合，发现hu4G4抗体在肿瘤部位有特异性浓聚(图6)。
实施例4：抗人B7-H3人源化抗体偶联碘131抑制肿瘤生长
利用治疗性核素131I对hu4G4抗体进行核素标记，并在脑胶质瘤模型中研究其抗肿瘤药效。研究结果显示，给予131I-hu4G4治疗7天后(放射性剂量400-500μCi/只)，瘤体积明显低于PBs和131I-IgG组对照组(p<0.05)；131I-hu4G4抑瘤效果明显，D23天抑瘤率均＞80％，D20天相对肿瘤增殖率均<30％，抑瘤效果明显优于对照组131I-IgG(图7)。显示131I-hu4G4具备治疗脑胶质瘤的潜能。

Claims (9)

  1. 一种靶向人B7-H3的人源化单克隆抗体，其特征在于，所述的人源化单克隆抗体包含有重链和轻链，其中重链CDR区包含有CDR-H1、CDR-H2和CDR-H3，其序列分别为SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3；
    所述的轻链CDR区包含有CDR-L1、CDR-L2和CDR-L3，其序列分别为SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6。
  2. 如权利要求1所述的单克隆抗体，其特征在于，所述的单克隆抗体的重链可变区的氨基酸序列为SEQ ID NO：7。
  3. 如权利要求1所述的单克隆抗体，其特征在于，所述的单克隆抗体的轻链可变区的氨基酸序列为SEQ ID NO：8。
  4. 权利要求1-3任一项所述的单克隆抗体在制备用于检测B7-H3蛋白的制品中的应用。
  5. 权利要求1-3任一项所述的单克隆抗体在制备用于阻断B7-H3蛋白的制品中的应用。
  6. 一种药物组合物，其特征在于，所述的药物组合物中包含有权利要求1-3任一项所述的单克隆抗体。
  7. 如权利要求6所述的药物组合物，其特征在于，所述的药物组合物中包含有药学上可接受的载体和/或赋形剂。
  8. 权利要求6或7所述的药物组合物在制备预防和/或治疗肿瘤的药物中的应用。
  9. 如权利要求8所述的应用，其特征在于，所述的肿瘤为急性骨髓白血病、慢性骨髓白血病、急性淋巴细胞白血病、非霍金性淋巴瘤、多发性骨髓瘤、黑色素瘤、肺癌、结肠直肠癌、肾肿瘤、膀胱癌、胃肠道癌、前列腺癌、肝癌、卵巢癌、胰腺癌、子宫内膜癌、胃癌、前列腺癌、肾癌、宫颈癌、甲状腺癌、子宫癌、神经内分泌癌、头颈癌、鼻咽癌、睾丸癌、基底细胞皮肤癌、鳞状细胞皮肤癌、皮肤纤维肉瘤突出症、梅克尔细胞癌、胶质母细胞瘤、神经胶质瘤、肉瘤、间皮 瘤或骨髓发育不良综合征。
PCT/CN2023/070469 2022-01-07 2023-01-04 靶向人b7-h3分子的人源化单克隆抗体及其应用 WO2023131193A1 (zh)

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CN114380910B (zh) * 2022-01-07 2023-04-28 苏州旭光科星抗体生物科技有限公司 靶向人b7-h3分子的人源化单克隆抗体及其应用
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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103687945A (zh) * 2011-04-25 2014-03-26 第一三共株式会社 抗-b7-h3抗体
US20170073416A1 (en) * 2014-05-29 2017-03-16 Spring Bioscience Corporation Anti-b7-h3 antibodies and diagnostic uses thereof
WO2018161872A1 (zh) * 2017-03-06 2018-09-13 江苏恒瑞医药股份有限公司 抗b7-h3抗体、其抗原结合片段及其医药用途
CN110950953A (zh) * 2018-09-26 2020-04-03 福建医科大学 抗b7-h3的单克隆抗体及其在细胞治疗中的应用
WO2020103100A1 (en) * 2018-11-22 2020-05-28 Suzhou Kanova Biopharmaceutical Co., Ltd. An anti-b7-h3 antibody
WO2020140094A1 (en) * 2018-12-27 2020-07-02 Gigagen, Inc. Anti-b7-h3 binding proteins and methods of use thereof
CN111454357A (zh) * 2019-08-14 2020-07-28 上海岺樾生物医药科技有限公司 一种含有抗体的肿瘤治疗剂的开发和应用
CN112189021A (zh) * 2018-05-24 2021-01-05 Abl生物公司 抗b7-h3抗体及其用途
WO2021027687A1 (zh) * 2019-08-14 2021-02-18 博源润生医药（杭州）有限公司 一种嵌合抗原受体以及表达该嵌合抗原受体的免疫效应细胞
WO2021099347A1 (en) * 2019-11-18 2021-05-27 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Antibodies targeting, and other modulators of, the cd276 antigen, and uses thereof
CN113330034A (zh) * 2018-11-16 2021-08-31 阿尔伯特爱因斯坦医学院 针对B7-H3的IgV结构域的单克隆抗体及其用途
WO2021244721A1 (en) * 2020-06-04 2021-12-09 Y-Mabs Therapeutics, Inc. Anti-b7h3 antibodies for the treatment of cancer
CN114380910A (zh) * 2022-01-07 2022-04-22 苏州旭光科星抗体生物科技有限公司 靶向人b7-h3分子的人源化单克隆抗体及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109851673B (zh) * 2019-01-22 2023-07-25 苏州旭光科星抗体生物科技有限公司 一种抗人b7-h3单克隆抗体的制备方法及其免疫组化检测方法及其应用及其试剂盒

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103687945A (zh) * 2011-04-25 2014-03-26 第一三共株式会社 抗-b7-h3抗体
US20170073416A1 (en) * 2014-05-29 2017-03-16 Spring Bioscience Corporation Anti-b7-h3 antibodies and diagnostic uses thereof
WO2018161872A1 (zh) * 2017-03-06 2018-09-13 江苏恒瑞医药股份有限公司 抗b7-h3抗体、其抗原结合片段及其医药用途
CN112189021A (zh) * 2018-05-24 2021-01-05 Abl生物公司 抗b7-h3抗体及其用途
CN110950953A (zh) * 2018-09-26 2020-04-03 福建医科大学 抗b7-h3的单克隆抗体及其在细胞治疗中的应用
CN113330034A (zh) * 2018-11-16 2021-08-31 阿尔伯特爱因斯坦医学院 针对B7-H3的IgV结构域的单克隆抗体及其用途
CN112533955A (zh) * 2018-11-22 2021-03-19 苏州鑫康合生物医药科技有限公司 抗b7-h3抗体
WO2020103100A1 (en) * 2018-11-22 2020-05-28 Suzhou Kanova Biopharmaceutical Co., Ltd. An anti-b7-h3 antibody
WO2020140094A1 (en) * 2018-12-27 2020-07-02 Gigagen, Inc. Anti-b7-h3 binding proteins and methods of use thereof
CN111454357A (zh) * 2019-08-14 2020-07-28 上海岺樾生物医药科技有限公司 一种含有抗体的肿瘤治疗剂的开发和应用
WO2021027687A1 (zh) * 2019-08-14 2021-02-18 博源润生医药（杭州）有限公司 一种嵌合抗原受体以及表达该嵌合抗原受体的免疫效应细胞
WO2021099347A1 (en) * 2019-11-18 2021-05-27 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Antibodies targeting, and other modulators of, the cd276 antigen, and uses thereof
WO2021244721A1 (en) * 2020-06-04 2021-12-09 Y-Mabs Therapeutics, Inc. Anti-b7h3 antibodies for the treatment of cancer
CN114380910A (zh) * 2022-01-07 2022-04-22 苏州旭光科星抗体生物科技有限公司 靶向人b7-h3分子的人源化单克隆抗体及其应用

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