CN109963591B - B7H3 antibody-drug conjugate and medical application thereof - Google Patents
B7H3 antibody-drug conjugate and medical application thereofDownload PDFInfo
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Abstract
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Description
Translated fromChinese技术领域Technical Field
本发明涉及B7H3抗体-药物偶联物及其在医药上的应用，进一步地，本发明涉及B7H3抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，以及包含前述偶联物或其药学上可接受的盐或溶剂化合物的药物组合物，以及其制备用于治疗B7H3介导的疾病或病症的药物中的用途；尤其在制备抗癌药物中的用途。The present invention relates to B7H3 antibody-drug conjugates and their use in medicine. Further, the present invention relates to B7H3 antibody-cytotoxic drug conjugates or pharmaceutically acceptable salts or solvent compounds thereof, and pharmaceutical compositions comprising the aforementioned conjugates or pharmaceutically acceptable salts or solvent compounds thereof, and their use in preparing drugs for treating B7H3-mediated diseases or conditions; in particular, their use in preparing anticancer drugs.
背景技术Background Art
T细胞介导的免疫反应在机体抗肿瘤过程中发挥着极其重要的作用，而T细胞的活化和增殖不仅需要TCR识别的抗原信号，还需要共刺激分子提供的第二信号。B7家族分子属于共刺激分子免疫球蛋白超家族，越来越多的研究表明，该家族分子在机体正常免疫功能和病理状态下均发挥了重要的调节作用。T cell-mediated immune response plays an extremely important role in the body's anti-tumor process, and the activation and proliferation of T cells require not only antigen signals recognized by TCR, but also second signals provided by co-stimulatory molecules. B7 family molecules belong to the immunoglobulin superfamily of co-stimulatory molecules. More and more studies have shown that this family of molecules plays an important regulatory role in both normal immune function and pathological conditions.
B7H3是B7家族的成员之一，属于I型跨膜蛋白，包含氨基端的一个信号肽，一个细胞外的免疫球蛋白样可变区(IgV)和恒定区(IgC)、一个跨膜区和一个含有45个氨基酸的胞质尾区(Tissue Antigens.2007 Aug；70(2)：96-104)。目前，B7H3主要存在2种剪切体，B7H3a和B7H3b。B7H3a胞外段由IgV-IgC 2个免疫球蛋白结构域组成，又称为2IgB7H3，而B7H3b胞外段由IgV-IgC-IgV-IgC 4个免疫球蛋白结构域组成，又称为4IgB7H3。B7H3 is a member of the B7 family and belongs to type I transmembrane protein. It contains a signal peptide at the amino terminal, an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region and a cytoplasmic tail region containing 45 amino acids (Tissue Antigens. 2007 Aug; 70(2): 96-104). At present, there are mainly two splice forms of B7H3, B7H3a and B7H3b. The extracellular segment of B7H3a is composed of two immunoglobulin domains, IgV-IgC, also known as 2IgB7H3, while the extracellular segment of B7H3b is composed of four immunoglobulin domains, IgV-IgC-IgV-IgC, also known as 4IgB7H3.
B7H3蛋白质在正常组织、细胞中不表达或极低表达，却高表达于多种肿瘤组织，并与肿瘤的进展、患者的生存及预后密切相关。临床上已经报道，B7H3在许多癌症类型中、特别是在非小细胞肺癌、肾癌、泌尿道上皮癌、结直肠癌、前列腺癌、多形性胶质母细胞瘤、卵巢癌和胰腺癌中过表达(Lung Cancer.2009 Nov；66(2)：245-249；Clin Cancer Res.2008Aug 15；14(16)：5150-5157)。此外，也有文献报道，在前列腺癌中，B7H3的表达强度与临床病理学恶性(诸如肿瘤体积、前列腺外侵袭或Gleason评分)正相关，且也与癌症进展相关(Cancer Res.2007 Aug 15；67(16)：7893-7900)。类似地，在多形性胶质母细胞瘤中，B7H3的表达与无事件存活负相关，且在胰腺癌中，B7H3的表达与淋巴结转移和病理学进展相关。因此，B7H3被认为是一种新的肿瘤标志物和潜在的治疗靶点B7H3 protein is not expressed or is expressed at very low levels in normal tissues and cells, but is highly expressed in a variety of tumor tissues and is closely related to tumor progression, patient survival and prognosis. It has been reported clinically that B7H3 is overexpressed in many types of cancer, especially in non-small cell lung cancer, renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer (Lung Cancer. 2009 Nov; 66 (2): 245-249; Clin Cancer Res. 2008 Aug 15; 14 (16): 5150-5157). In addition, there are also literature reports that in prostate cancer, the expression intensity of B7H3 is positively correlated with clinical pathological malignancy (such as tumor volume, extraprostatic invasion or Gleason score), and is also related to cancer progression (Cancer Res. 2007 Aug 15; 67 (16): 7893-7900). Similarly, in glioblastoma multiforme, B7H3 expression is negatively correlated with event-free survival, and in pancreatic cancer, B7H3 expression is associated with lymph node metastasis and pathological progression. Therefore, B7H3 is considered a new tumor marker and potential therapeutic target.
目前，已有针对B7H3靶点的治疗策略用于临床前研究，如靶向小鼠B7H3的抗体会增强瘤内的浸润性的CD8-阳性的T细胞和抑制肿瘤生长(Mod Pathol.2010 Aug；23(8)：1104-1112)。此外，WO 2008/066691专利显示，识别B7H3变体B7H3a的抗体会对腺癌表现出体内抗肿瘤作用。在临床研究中，一种鼠源的B7H3抗体与放射性I131的偶联药物可显著抑制患者成神经母细胞瘤的生长[J Neufooocol 97(3)：409-18(2010]。但目前在研的项目都是鼠源抗体经人源化改造的人源化抗体，而人源化抗体在免疫时存在免疫原性相对较高的问题，在人体应用时是一个不利的因素。At present, there are therapeutic strategies targeting B7H3 for preclinical research, such as antibodies targeting mouse B7H3, which can enhance the infiltration of CD8-positive T cells in tumors and inhibit tumor growth (Mod Pathol. 2010 Aug; 23(8): 1104-1112). In addition, WO 2008/066691 patent shows that antibodies that recognize the B7H3 variant B7H3a can exhibit in vivo anti-tumor effects on adenocarcinoma. In clinical studies, a conjugate drug of a mouse B7H3 antibody and radioactive I131 can significantly inhibit the growth of neuroblastoma in patients [J Neufooocol 97(3): 409-18 (2010). However, the projects currently under research are all humanized antibodies that are humanized by mouse antibodies, and humanized antibodies have the problem of relatively high immunogenicity during immunization, which is a disadvantageous factor when used in humans.
噬菌体展示技术(phage display technology)是将外源蛋白质或多肽与噬菌体外壳蛋白融合表达，从而将外源蛋白表达在噬菌体的表面。噬菌体抗体库是将噬菌体展示技术、PCR扩增技术、蛋白表达技术相结合的一项运用综合技术手段所建立起来的抗体库。Phage display technology is the process of fusing foreign proteins or peptides with phage coat proteins, thereby expressing foreign proteins on the surface of phages. Phage antibody library is an antibody library established by combining phage display technology, PCR amplification technology, and protein expression technology.
噬菌体抗体库最大的优点是不经体内免疫，模拟体内抗体生成的三个过程而制备出完全人源化抗体。除此之外，噬菌体抗体库还具有以下优势：①实现了基因型与表型的统一。此外，实验方法简单、快速，传统的通过杂交瘤技术抗体产生方法需历经数月，而抗体库技术只需短短几周的时间。②表达的是完全人源抗体，且分子量小，主要以活性片段Fab、scFV的形式表达，与完整抗体相比在组织穿透力方面都有明显优势。③筛选容量大，杂交瘤技术是在上千个克隆内筛选，抗体库技术可以对百万甚至亿万个分子选择。筛选到的抗体种类越多。④用途广泛，采用了原核表达系统，当大规模生产时优势更加明显(Curr OpinBiotechnol.2002 Dec；13(6)：598-602；Immunotechnology，2013，48(13)48(13)：63-73)。The biggest advantage of phage antibody library is that it can produce fully humanized antibodies without in vivo immunization, simulating the three processes of antibody production in vivo. In addition, phage antibody library has the following advantages: ① It realizes the unity of genotype and phenotype. In addition, the experimental method is simple and fast. The traditional method of producing antibodies through hybridoma technology takes several months, while antibody library technology only takes a few weeks. ② It expresses fully human antibodies with small molecular weight, mainly expressed in the form of active fragments Fab and scFV, which have obvious advantages in tissue penetration compared with complete antibodies. ③ It has a large screening capacity. Hybridoma technology screens in thousands of clones, while antibody library technology can select millions or even billions of molecules. The more types of antibodies are screened. ④ It has a wide range of uses. It adopts a prokaryotic expression system, and its advantages are more obvious when it is mass-produced (Curr Opin Biotechnol. 2002 Dec; 13(6): 598-602; Immunotechnology, 2013, 48(13): 63-73).
抗体-药物偶联物(antibody drug conjugate，ADC)把单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒性物质的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体-药物偶联物能更精准地结合肿瘤细胞并降低将对正常细胞的影响。Antibody-drug conjugates (ADCs) connect monoclonal antibodies or antibody fragments to biologically active cytotoxins through stable chemical linker compounds, making full use of the antibody's specificity for binding to surface antigens of normal cells and tumor cells and the high efficiency of cytotoxic substances, while avoiding the defects of the former's low efficacy and the latter's excessive toxic side effects. This means that compared with traditional chemotherapy drugs, antibody-drug conjugates can bind to tumor cells more accurately and reduce the impact on normal cells.
目前已有多种ADC药物被用于临床或临床研究，如Kadcyla，是靶向Her2的曲妥珠单抗与DM1形成的ADC药物。同时，也有靶向B7H3的抗体及ADC药物的专利报道，如WO2008100934、WO2012147713、WO2014061277、WO2015184203、WO2016044383。但仍没有B7H3靶点的ADC药物上市或用于临床治疗研究，因此，开发新的B7H3靶点的ADC药物具有广阔的前景。Currently, a variety of ADC drugs have been used in clinical practice or clinical research, such as Kadcyla, which is an ADC drug formed by trastuzumab targeting Her2 and DM1. At the same time, there are also patent reports on antibodies and ADC drugs targeting B7H3, such as WO2008100934, WO2012147713, WO2014061277, WO2015184203, and WO2016044383. However, there are still no ADC drugs targeting B7H3 on the market or used in clinical treatment research. Therefore, the development of new ADC drugs targeting B7H3 has broad prospects.
发明内容Summary of the invention
本发明的目的是提供与B7H3的胞外区的氨基酸序列或三维结构结合的单克隆抗体与细胞毒性物质偶联的ADC药物。通过筛选高活性和高稳定性的抗人B7H3全人抗体ADC药物，提供使用所述抗体ADC药物作为活性成分的治疗剂。The purpose of the present invention is to provide an ADC drug of a monoclonal antibody that binds to the amino acid sequence or three-dimensional structure of the extracellular region of B7H3 and a cytotoxic substance. By screening a highly active and highly stable anti-human B7H3 fully human antibody ADC drug, a therapeutic agent using the antibody ADC drug as an active ingredient is provided.
本发明提供一种通式(A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，The present invention provides an antibody-drug conjugate represented by general formula (A) or a pharmaceutically acceptable salt or solvent compound thereof,
Ab-(L2-L1-D)y (A)Ab-(L2 -L1 -D)y (A)
其中：in:
D是细胞毒性药物；D is a cytotoxic drug;
L1，L2是接头单元；L1 , L2 are linker units;
y为选自1-8的数，优选为选自2-4的数；y可以为小数或整数；y is a number selected from 1-8, preferably a number selected from 2-4; y can be a decimal or an integer;
Ab为B7H3抗体或其抗原结合片段，其与人B7H3结合，所述B7H3抗体或其抗原结合片段是选自下面(a)至(c)中任一种的单克隆抗体或其抗原结合片段：Ab is a B7H3 antibody or an antigen-binding fragment thereof, which binds to human B7H3, and the B7H3 antibody or the antigen-binding fragment thereof is a monoclonal antibody or the antigen-binding fragment thereof selected from any one of the following (a) to (c):
(a)单克隆抗体，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：(a) a monoclonal antibody comprising one or more CDR region sequences selected from the following or an amino acid sequence having at least 95% sequence identity thereto:
抗体重链可变区HCDR区序列：如SEQ ID NO：10、11和12氨基酸序列所示；和抗体轻链可变区LCDR区序列：如SEQ ID NO：13、14和15氨基酸序列所示；The antibody heavy chain variable region HCDR region sequence is as shown in SEQ ID NO: 10, 11 and 12 amino acid sequences; and the antibody light chain variable region LCDR region sequence is as shown in SEQ ID NO: 13, 14 and 15 amino acid sequences;
(b)单克隆抗体，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：(b) a monoclonal antibody comprising one or more CDR region sequences selected from the following or an amino acid sequence having at least 95% sequence identity thereto:
抗体重链可变区HCDR区序列：如SEQ ID NO：16、17和18氨基酸序列所示；和抗体轻链可变区LCDR区序列：如SEQ ID NO：19、20和21氨基酸序列所示；The antibody heavy chain variable region HCDR region sequence is as shown in SEQ ID NO: 16, 17 and 18 amino acid sequences; and the antibody light chain variable region LCDR region sequence is as shown in SEQ ID NO: 19, 20 and 21 amino acid sequences;
(c)单克隆抗体，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：(c) a monoclonal antibody comprising one or more CDR region sequences selected from the group consisting of:
抗体重链可变区HCDR区序列：如SEQ ID NO：30、31和32氨基酸序列所示；和抗体轻链可变区LCDR区序列：如SEQ ID NO：33、34和35氨基酸序列所示。The antibody heavy chain variable region HCDR region sequence is as shown in SEQ ID NO: 30, 31 and 32 amino acid sequences; and the antibody light chain variable region LCDR region sequence is as shown in SEQ ID NO: 33, 34 and 35 amino acid sequences.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述B7H3抗体或其抗原结合片段是选自下面(a)至(c)中任一种的单克隆抗体或其抗原结合片段：In a preferred embodiment, the antibody-drug conjugate as represented by general formula (A) or a pharmaceutically acceptable salt or solvent compound thereof, wherein the B7H3 antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof selected from any one of the following (a) to (c):
(a)单克隆抗体，其包含分别如SEQ ID NO：10、11和12氨基酸序列所示抗体重链可变区的HCDR1，HCDR2，HCDR3；和如SEQ ID NO：13、14和15氨基酸序列所示抗体轻链可变区的LCDR1，LCDR2，LCDR3；(a) a monoclonal antibody comprising HCDR1, HCDR2, HCDR3 of the antibody heavy chain variable region as shown in the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively; and LCDR1, LCDR2, LCDR3 of the antibody light chain variable region as shown in the amino acid sequences of SEQ ID NOs: 13, 14 and 15;
(b)单克隆抗体，其包含分别如SEQ ID NO：16、17和18氨基酸序列所示抗体重链可变区的HCDR1，HCDR2，HCDR3；和如SEQ ID NO：19、20和21氨基酸序列所示抗体轻链可变区的LCDR1，LCDR2，LCDR3；(b) a monoclonal antibody comprising HCDR1, HCDR2, HCDR3 of the antibody heavy chain variable region as shown in the amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively; and LCDR1, LCDR2, LCDR3 of the antibody light chain variable region as shown in the amino acid sequences of SEQ ID NOs: 19, 20 and 21;
(c)单克隆抗体，，其包含分别如SEQ ID NO：30、31和32氨基酸序列所示抗体重链可变区的HCDR1，HCDR2，HCDR3；和如SEQ ID NO：33、34和35氨基酸序列所示抗体轻链可变区的LCDR1，LCDR2，LCDR3。(c) a monoclonal antibody comprising HCDR1, HCDR2, HCDR3 of the antibody heavy chain variable region as shown in the amino acid sequences of SEQ ID NOs: 30, 31 and 32, respectively; and LCDR1, LCDR2, LCDR3 of the antibody light chain variable region as shown in the amino acid sequences of SEQ ID NOs: 33, 34 and 35, respectively.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，In a preferred embodiment, the antibody-drug conjugate as shown in general formula (A) or a pharmaceutically acceptable salt or solvent compound thereof,
Ab-(L2-L1-D)y (A)Ab-(L2 -L1 -D)y (A)
其中：in:
D是细胞毒性药物；D is a cytotoxic drug;
L1，L2是接头单元；L1 , L2 are linker units;
y为选自1-8的数，优选为选自2-4的数；y is a number selected from 1-8, preferably a number selected from 2-4;
Ab为与如上所定义的B7H3抗体或其抗原结合片段竞争结合人B7H3的单克隆抗体或其抗原结合片段。Ab is a monoclonal antibody or an antigen-binding fragment thereof that competes with the B7H3 antibody or antigen-binding fragment thereof as defined above for binding to human B7H3.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab是重组抗体。In a preferred embodiment, the antibody-drug conjugate as shown in general formula (A), wherein the Ab is a recombinant antibody.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab是人源的重组抗体或其抗原结合片段。In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (A), wherein the Ab is a human recombinant antibody or an antigen-binding fragment thereof.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab的轻链和重链可变区上的轻链和重链FR区序列分别来源于人种系轻链和重链序列或其突变序列。In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (A), wherein the light chain and heavy chain FR region sequences on the light chain and heavy chain variable regions of the Ab are derived from human germline light chain and heavy chain sequences or mutant sequences thereof, respectively.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab的恒定区包括来源于人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选人源IgG1重链恒定区；和来源于人源κ、λ链或其变体的轻链恒定区，优选人源κ链轻链恒定区。In a preferred embodiment, an antibody-drug conjugate as shown in general formula (A), wherein the constant region of the Ab includes a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably a human IgG1 heavy chain constant region; and a light chain constant region derived from human κ, λ chain or variants thereof, preferably a human κ chain light chain constant region.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab含有SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：36或SEQ ID NO：37所示的重链可变区或其变体；所述变体是在SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：36或SEQ ID NO：37所示的重链可变区序列上具有1-10个氨基酸替换的序列。In a preferred embodiment, the antibody-drug conjugate as shown in general formula (A), wherein the Ab contains a heavy chain variable region shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36 or SEQ ID NO: 37 or a variant thereof; the variant is a sequence having 1-10 amino acid substitutions in the heavy chain variable region sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36 or SEQ ID NO: 37.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab含有SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：38或SEQ ID NO：39所示的轻链可变区或其变体；所述变体是在SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：38或SEQ ID NO：39所示的轻链可变区序列上具有1-10个氨基酸替换的序列。In a preferred embodiment, the antibody-drug conjugate as shown in general formula (A), wherein the Ab contains a light chain variable region shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 38 or SEQ ID NO: 39 or a variant thereof; the variant is a sequence having 1-10 amino acid substitutions in the light chain variable region sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 38 or SEQ ID NO: 39.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab含有选自下面(1)至(7)中任一种的单克隆抗体或其抗原结合片段：In a preferred embodiment, the antibody-drug conjugate as shown in general formula (A), wherein the Ab comprises a monoclonal antibody or an antigen-binding fragment thereof selected from any one of the following (1) to (7):
(1)单克隆抗体，其包含SEQ ID NO：6所示的抗体重链可变区；和SEQ ID NO：7所示抗体轻链可变区；(1) A monoclonal antibody comprising an antibody heavy chain variable region as shown in SEQ ID NO: 6; and an antibody light chain variable region as shown in SEQ ID NO: 7;
(2)单克隆抗体，其包含SEQ ID NO：8所示的抗体重链可变区；和SEQ ID NO：9所示抗体轻链可变区；(2) a monoclonal antibody comprising an antibody heavy chain variable region as shown in SEQ ID NO: 8; and an antibody light chain variable region as shown in SEQ ID NO: 9;
(3)单克隆抗体，其包含SEQ ID NO：28所示的抗体重链可变区；和SEQ ID NO：29所示抗体轻链可变区；(3) a monoclonal antibody comprising an antibody heavy chain variable region as shown in SEQ ID NO: 28; and an antibody light chain variable region as shown in SEQ ID NO: 29;
(4)单克隆抗体，其包含SEQ ID NO：36所示的抗体重链可变区；和SEQ ID NO：38所示抗体轻链可变区；(4) a monoclonal antibody comprising an antibody heavy chain variable region as shown in SEQ ID NO: 36; and an antibody light chain variable region as shown in SEQ ID NO: 38;
(5)单克隆抗体，其包含SEQ ID NO：36所示的抗体重链可变区；和SEQ ID NO：39所示抗体轻链可变区；(5) a monoclonal antibody comprising an antibody heavy chain variable region as shown in SEQ ID NO: 36; and an antibody light chain variable region as shown in SEQ ID NO: 39;
(6)单克隆抗体，其包含SEQ ID NO：37所示的抗体重链可变区；和SEQ ID NO：38所示抗体轻链可变区；(6) a monoclonal antibody comprising an antibody heavy chain variable region as shown in SEQ ID NO: 37; and an antibody light chain variable region as shown in SEQ ID NO: 38;
(7)单克隆抗体，其包含SEQ ID NO：37所示的抗体重链可变区；和SEQ ID NO：39所示抗体轻链可变区。(7) A monoclonal antibody comprising the antibody heavy chain variable region shown in SEQ ID NO: 37; and the antibody light chain variable region shown in SEQ ID NO: 39.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab为全长抗体，其进一步包括人抗体恒定区；其中所述的全长抗体选自：In a preferred embodiment, the antibody-drug conjugate as shown in general formula (A), wherein the Ab is a full-length antibody, which further includes a human antibody constant region; wherein the full-length antibody is selected from:
h1702抗体，其是由SEQ ID NO：22所示的重链序列和SEQ ID NO：23所示的轻链序列组成的全长抗体，h1702 antibody, which is a full-length antibody consisting of the heavy chain sequence shown in SEQ ID NO: 22 and the light chain sequence shown in SEQ ID NO: 23,
h1703抗体，其是由SEQ ID NO：24所示的重链序列和SEQ ID NO：25所示的轻链序列组成的全长抗体，h1703 antibody, which is a full-length antibody consisting of the heavy chain sequence shown in SEQ ID NO: 24 and the light chain sequence shown in SEQ ID NO: 25,
h1702-DS抗体，其是由SEQ ID NO：22所示的重链序列和SEQ ID NO：26所示的轻链序列组成的全长抗体，h1702-DS antibody, which is a full-length antibody consisting of the heavy chain sequence shown in SEQ ID NO: 22 and the light chain sequence shown in SEQ ID NO: 26,
或者h1704-3抗体，其是由SEQ ID NO：40所示的重链序列和SEQ ID NO：41所示的轻链序列组成的全长抗体。Or the h1704-3 antibody, which is a full-length antibody consisting of the heavy chain sequence shown in SEQ ID NO:40 and the light chain sequence shown in SEQ ID NO:41.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述抗原结合片段选自Fab、Fab′、F(ab′)2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。In a preferred embodiment, the antibody-drug conjugate is as shown in the general formula (A), wherein the antigen-binding fragment is selected from Fab, Fab′, F(ab′)2, single-chain antibody (scFv), dimerized V region (diabody), disulfide bond-stabilized V region (dsFv) and an antigen-binding fragment of a peptide comprising CDR.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中细胞毒性药物选自毒素、化疗药物、抗生素、放射性同位素和核溶酶。In a preferred embodiment, the antibody-drug conjugate as represented by general formula (A), wherein the cytotoxic drug is selected from toxins, chemotherapeutic drugs, antibiotics, radioactive isotopes and nucleolytic enzymes.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中细胞毒性药物选自DM1、DM3、DM4、MMAF和MMAE。In a preferred embodiment, the antibody-drug conjugate as represented by general formula (A), wherein the cytotoxic drug is selected from DM1, DM3, DM4, MMAF and MMAE.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中细胞毒性药物选自：In a preferred embodiment, the antibody-drug conjugate as shown in general formula (A), wherein the cytotoxic drug is selected from:
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其为式I所示化合物或其药学上可接受的盐或溶剂化合物，In a preferred embodiment, the antibody-drug conjugate as shown in general formula (A) is a compound shown in formula I or a pharmaceutically acceptable salt or solvent compound thereof,
其中：in:
L1，L2是接头单元；L1 , L2 are linker units;
y为选自1-8的数，优选为选自2-4的数；y is a number selected from 1-8, preferably a number selected from 2-4;
Ab为如上所定义的B7H3抗体或其抗原结合片段、或与如上所定义的B7H3抗体或其抗原结合片段竞争结合人B7H3的单克隆抗体或其抗原结合片段。Ab is a B7H3 antibody or an antigen-binding fragment thereof as defined above, or a monoclonal antibody or an antigen-binding fragment thereof that competes with the B7H3 antibody or an antigen-binding fragment thereof as defined above for binding to human B7H3.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中L2如以下通式L2所示：In a preferred embodiment, the antibody-drug conjugate as shown in the general formula (A), whereinL2 is shown in the following general formulaL2 :
其中in
X1选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；X1 is selected from hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl;
X2选自烷基、环烷基和杂环基；X2 is selected from alkyl, cycloalkyl and heterocyclyl;
m为选自0-5的整数，优选为1、2或3；S为硫原子。m is an integer selected from 0 to 5, preferably 1, 2 or 3; S is a sulfur atom.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中L1如以下通式(L1)所示：In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (A), wherein L1 is represented by the following general formula (L1 ):
其中in
X3为烷基，任选所述的烷基进一步被选自卤素、羟基和氰基的取代基所取代；X3 is an alkyl group, optionally further substituted by a substituent selected from halogen, hydroxyl and cyano;
n为选自0-5的整数，优选为1、2或3。n is an integer selected from 0-5, preferably 1, 2 or 3.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中细胞毒性药物经与连接物L1连接后，得到化合物：In a preferred embodiment, in the antibody-drug conjugate as shown in general formula (A), the cytotoxic drug is connected to the linker L1 to obtain a compound:
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其为通式(II)所示的抗体-药物偶联物：In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A) is an antibody-drug conjugate represented by the general formula (II):
其中，L2是接头单元；Wherein, L2 is a linker unit;
y为选自1-8的数，优选为选自2-4的数；y is a number selected from 1-8, preferably a number selected from 2-4;
Ab为如上所定义的B7H3抗体或其抗原结合片段、或与如上所定义的B7H3抗体或其抗原结合片段竞争结合人B7H3的单克隆抗体或其抗原结合片段。Ab is a B7H3 antibody or an antigen-binding fragment thereof as defined above, or a monoclonal antibody or an antigen-binding fragment thereof that competes with the B7H3 antibody or an antigen-binding fragment thereof as defined above for binding to human B7H3.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其为通式(III)所示的抗体-药物偶联物：In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A) is an antibody-drug conjugate represented by the general formula (III):
其中，L1是接头单元；Wherein, L1 is a linker unit;
y为选自1-8的数，优选为选自2-4的数；y is a number selected from 1-8, preferably a number selected from 2-4;
Ab为如上所定义的B7H3抗体或其抗原结合片段、或与如上所定义的B7H3抗体或其抗原结合片段竞争结合人B7H3的单克隆抗体或其抗原结合片段。Ab is a B7H3 antibody or an antigen-binding fragment thereof as defined above, or a monoclonal antibody or an antigen-binding fragment thereof that competes with the B7H3 antibody or an antigen-binding fragment thereof as defined above for binding to human B7H3.
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，或其可药用盐，包括但不限于：In a preferred embodiment, the antibody-drug conjugate as shown in general formula (A), or a pharmaceutically acceptable salt thereof, includes but is not limited to:
本发明进一步涉及一种药物组合物，其包含如本发明通式(A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，和一种或多种可药用的赋形剂、稀释剂或载体。The present invention further relates to a pharmaceutical composition, which comprises an antibody-drug conjugate as represented by the general formula (A) of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers.
本发明进一步涉及通式(A)所示的抗体-药物偶联物，或其药学上可接受的盐或溶剂化合物，或包含其的药物组合物，在制备用于治疗与人B7H3阳性细胞相关的疾病的药物中的用途；优选的，其中所述的用途，其在于制备用于治疗B7H3高表达癌症的药物中的用途。The present invention further relates to the use of an antibody-drug conjugate represented by general formula (A), or a pharmaceutically acceptable salt or solvent compound thereof, or a pharmaceutical composition comprising the same, in the preparation of a drug for treating a disease associated with human B7H3-positive cells; preferably, the use is in the preparation of a drug for treating cancers with high B7H3 expression.
本发明进一步涉及通式(A)所示的抗体-药物偶联物，或其药学上可接受的盐或溶剂化合物，或包含其的药物组合物在制备用于治疗疾病的药物中的用途，所述疾病选自治疗胶质瘤(非限制性实施例为人脑星形胶质母细胞瘤)、人咽头癌、肾上腺肿瘤、AIDS-相关癌症、腺泡状软组织肉瘤、星形细胞瘤、膀胱癌、骨癌、脑和脊髓癌、转移性脑瘤、乳腺癌、颈动脉体瘤、宫颈癌、软骨肉瘤、脊索瘤、肾嫌色细胞癌、透明细胞癌、结肠癌、结肠直肠癌、促结缔组织增生性小圆细胞肿瘤、室管膜细胞瘤、尤文肿瘤、骨外黏液样软骨肉瘤、骨纤维发育不全、骨纤维性发育不良、胆囊或胆管癌、胃癌、妊娠滋养细胞病、生殖细胞瘤、头颈癌、肝细胞癌、胰岛细胞瘤、卡波西肉瘤、肾癌、白血病、脂肪肉瘤/恶性脂肪瘤性肿瘤、肝癌、淋巴瘤、肺癌、成神经管细胞瘤、黑色素瘤、脑膜瘤、多发性内分泌瘤病、多发性骨髓瘤、骨髓增生异常综合征、成神经细胞瘤、神经内分泌肿瘤、卵巢癌、胰腺癌、乳头状甲状腺癌、甲状旁腺瘤、小儿癌症、外周神经鞘瘤、嗜铬细胞瘤、垂体肿瘤、前列腺癌、后葡萄膜黑色素瘤、肾转移性癌、横纹肌样瘤、横纹肌肉瘤、肉瘤、皮肤癌、软组织肉瘤、鳞状细胞癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲状腺转移性癌和子宫癌。The present invention further relates to the use of an antibody-drug conjugate represented by general formula (A), or a pharmaceutically acceptable salt or solvent compound thereof, or a pharmaceutical composition comprising the same in the preparation of a drug for treating a disease, wherein the disease is selected from the group consisting of treating glioma (a non-limiting example is human brain astrocytoma), human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, biliary tract infection, fibrous dysplasia ... Cyst or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumors, head and neck cancer, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma/malignant lipomatous tumors, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumors, pediatric cancer, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, and uterine cancer.
本发明进一步涉及一种治疗疾病的方法，该方法包括向需要其的患者施用治疗有效剂量的该通式(A)所示的抗体-药物偶联物，或其药学上可接受的盐或溶剂化合物，或包含其的药物组合物，所述疾病选自人脑星形胶质母细胞瘤、人咽头癌、肾上腺肿瘤、AIDS-相关癌症、腺泡状软组织肉瘤、星形细胞瘤、膀胱癌、骨癌、脑和脊髓癌、转移性脑瘤、乳腺癌、颈动脉体瘤、宫颈癌、软骨肉瘤、脊索瘤、肾嫌色细胞癌、透明细胞癌、结肠癌、结肠直肠癌、促结缔组织增生性小圆细胞肿瘤、室管膜细胞瘤、尤文肿瘤、骨外黏液样软骨肉瘤、骨纤维发育不全、骨纤维性发育不良、胆囊或胆管癌、胃癌、妊娠滋养细胞病、生殖细胞瘤、头颈癌、肝细胞癌、胰岛细胞瘤、卡波西肉瘤、肾癌、白血病、脂肪肉瘤/恶性脂肪瘤性肿瘤、肝癌、淋巴瘤、肺癌、成神经管细胞瘤、黑色素瘤、脑膜瘤、多发性内分泌瘤病、多发性骨髓瘤、骨髓增生异常综合征、成神经细胞瘤、神经内分泌肿瘤、卵巢癌、胰腺癌、乳头状甲状腺癌、甲状旁腺瘤、小儿癌症、外周神经鞘瘤、嗜铬细胞瘤、垂体肿瘤、前列腺癌、后葡萄膜黑色素瘤、肾转移性癌、横纹肌样瘤、横纹肌肉瘤、肉瘤、皮肤癌、软组织肉瘤、鳞状细胞癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲状腺转移性癌和子宫癌。The present invention further relates to a method for treating a disease, which comprises administering to a patient in need thereof a therapeutically effective dose of the antibody-drug conjugate represented by the general formula (A), or a pharmaceutically acceptable salt or solvent compound thereof, or a pharmaceutical composition comprising the same, wherein the disease is selected from the group consisting of human brain astrocytoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, biliary Cyst or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumors, head and neck cancer, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma/malignant lipomatous tumors, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumors, pediatric cancer, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, and uterine cancer.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1：抗体与U87MG细胞的结合力；Figure 1: Binding ability of antibodies to U87MG cells;
图2：不同抗体在U87MG细胞上的内吞效果；Figure 2: Internalization effects of different antibodies on U87MG cells;
图3：本发明不同ADC h1702-3024，h1703-3024在U87MG细胞上的内吞效果；FIG3 shows the endocytic effects of different ADCs h1702-3024 and h1703-3024 of the present invention on U87MG cells;
图4：本发明不同ADC h1702-cys-3024，h1702DS-cys-3024和Figure 4: Different ADCs of the present invention h1702-cys-3024, h1702DS-cys-3024 and
h1704-3-cys-3024，及其相应抗体在U87MG细胞上的内吞效果；The internalization effects of h1704-3-cys-3024 and its corresponding antibodies on U87MG cells;
图5：h1702-3024对裸鼠U87MG移植瘤的疗效；Figure 5: Efficacy of h1702-3024 on U87MG xenografts in nude mice;
图6：h1702-3024对U87MG裸鼠体重的影响。Figure 6: Effect of h1702-3024 on body weight of U87MG nude mice.
图7：h1702DS-cys-3024和h1704-3-cys-3024的稳定性结果Figure 7: Stability results of h1702DS-cys-3024 and h1704-3-cys-3024
具体实施方式DETAILED DESCRIPTION
一.术语1. Terminology
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。In order to make the present invention more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).
本发明所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。The "antibody" described in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain.
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3), or conform to the numbering rules of Kabat and Chothia (HCDR1).
本发明的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人B7H3的单克隆抗体。制备时用B7H3抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源B7H3抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。The term "murine antibody" in the present invention refers to a monoclonal antibody against human B7H3 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with the B7H3 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the murine B7H3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or a variant thereof.
术语“重组抗体”包括“嵌合抗体”、“人源化抗体”和“完全人源抗体”。The term "recombinant antibody" includes "chimeric antibodies", "humanized antibodies" and "fully human antibodies".
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本发明一个优选的实施方案中，所述的B7H3嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的B7H3嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(如YTE突变或回复突变)的IgG1、IgG2或IgG4变体。The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies must first be established, and then the variable region gene must be cloned from the mouse hybridoma cells, and then the constant region gene of the human antibody must be cloned as needed, and the mouse variable region gene and the human constant region gene must be connected into a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present invention, the antibody light chain of the B7H3 chimeric antibody further comprises a light chain constant region of a human κ, λ chain or a variant thereof. The antibody heavy chain of the B7H3 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or an IgG1, IgG2 or IgG4 variant using an amino acid mutation (such as a YTE mutation or a back mutation).
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-graftedantibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences ofProteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。在本发明一个优选的实施方案中，所述的B7H3人源化抗体中鼠的CDR序列选自SEQ IDNO：8-13或14-19；人的抗体可变区框架经过设计选择，其中所述抗体重链可变区上的重链FR区序列，来源于人种系重链IGHV1-18*01和hjh4.1的组合序列或IGHV3-7*01和hjh4.1的组合序列；其中所述抗体轻链可变区上的轻链FR区序列，来源于人种系轻链IGKV1-33*01和hjk4.1的组合序列或IGKV1-39*01和hjk2.1的组合序列。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the mouse CDR sequence into the human antibody variable region framework, i.e., different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components can be overcome. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internetwww.mrccpe.com.ac.uk/vbase ), and in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies of the present invention also include humanized antibodies after further affinity maturation of CDR by phage display. In a preferred embodiment of the present invention, the mouse CDR sequence in the B7H3 humanized antibody is selected from SEQ ID NO: 8-13 or 14-19; the human antibody variable region framework is designed and selected, wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from the combination sequence of human germline heavy chain IGHV1-18*01 and hjh4.1 or the combination sequence of IGHV3-7*01 and hjh4.1; wherein the light chain FR region sequence on the antibody light chain variable region is derived from the combination sequence of human germline light chain IGKV1-33*01 and hjk4.1 or the combination sequence of IGKV1-39*01 and hjk2.1. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
CDR的移植可由于与抗原接触的构架残基而导致产生的B7H3抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人B7H3抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查鼠单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚型共有序列或具有高相似性百分数的鼠序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。The transplantation of CDRs may result in a reduction in the affinity of the resulting B7H3 antibody or its antigen-binding fragment for the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of somatic hypermutation. Therefore, it may still be necessary to transplant such donor framework amino acids to the framework of the humanized antibody. The amino acid residues from non-human B7H3 antibodies or their antigen-binding fragments that participate in antigen binding can be identified by examining the sequence and structure of the mouse monoclonal antibody variable region. Each residue in the CDR donor framework that is different from the germline can be considered to be related. If the closest germline cannot be determined, the sequence can be compared with the subtype consensus sequence or the consensus sequence of the mouse sequence with a high similarity percentage. Rare framework residues are believed to be the result of somatic hypermutation, thus playing an important role in binding.
术语“完全人源抗体”或“全人抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。全人源抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。本发明中的“完全人抗体”采用噬菌体显示技术获得抗体可变区，可与抗体恒定区进一步重组获得完整抗体。The term "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", means that both the variable region and the constant region of the antibody are human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology. The "fully human antibody" in the present invention uses phage display technology to obtain the antibody variable region, which can be further recombined with the antibody constant region to obtain a complete antibody.
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，B7H3)的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab′)2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人，(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242：423-426；和Huston等人(1988)Proe.Natl.Acad.Sci USA85：5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., B7H3). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region, (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proe. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
本发明的抗原结合片段包括Fab、F(ab′)2、Fab′、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)、包含CDR的肽等。The antigen-binding fragments of the present invention include Fab, F(ab')2, Fab', single-chain antibodies (scFv), dimerized V regions (diabodies), disulfide-bond-stabilized V regions (dsFv), peptides containing CDRs, and the like.
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.
本发明的Fab可以通过用木瓜蛋白酶处理本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。The Fab of the present invention can be produced by treating the monoclonal antibody of the present invention that specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with papain. In addition, the Fab can be produced by inserting the DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab.
F(ab′)2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below two disulfide bonds in the hinge region of IgG with the enzyme pepsin and comprising two Fab regions linked at the hinge position.
本发明的F(ab′)2可以通过用胃蛋白酶处理本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab′来生产所述F(ab′)2。The F(ab')2 of the present invention can be produced by treating the monoclonal antibody of the present invention that specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with pepsin. In addition, the F(ab')2 can be produced by connecting the Fab' described below with a thioether bond or a disulfide bond.
Fab′是通过切割上述F(ab′)2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。本发明的Fab′可以通过用还原剂例如二硫苏糖醇处理本发明的特异性识别B7H3并与胞外区的氨基酸序列或其三维结构结合的F(ab′)2来生产。Fab' is an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2. The Fab' of the present invention can be produced by treating the F(ab')2 of the present invention that specifically recognizes B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent such as dithiothreitol.
此外，可以通过将编码抗体的Fab′片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab′来生产所述Fab′。Furthermore, the Fab' fragment of an antibody can be produced by inserting a DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or a eukaryotic organism to express the Fab'.
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90：6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，EurJ.Immuno 1.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述。The term "single-chain antibody", "single-chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure: NH2- VL-linker-VH-COOH or NH2- VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example variants using 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
本发明的scFv可以通过以下步骤来生产：获得本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。The scFv of the present invention can be produced by the following steps: obtaining cDNA encoding VH and VL of the monoclonal antibody of the present invention that specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, constructing a DNA encoding scFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or a eukaryotic organism to express the scFv.
双抗体是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。Diabodies are antibody fragments in which scFv is dimerized, and are antibody fragments having bivalent antigen-binding activity. In the bivalent antigen-binding activity, the two antigens may be the same or different.
本发明的双抗体可以通过以下步骤来生产：获得本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。The diabody of the present invention can be produced by the following steps: obtaining cDNA encoding VH and VL of the monoclonal antibody of the present invention that specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, constructing DNA encoding scFv so that the amino acid sequence length of the peptide linker is 8 residues or less, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or a eukaryotic organism to express the diabody.
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(ProteinEngineering，7，697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。dsFv is obtained by linking a polypeptide in which one amino acid residue in each VH and VL is substituted by a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residue substituted by the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).
本发明的dsFv可以通过以下步骤来生产：获得获得本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。The dsFv of the present invention can be produced by the following steps: obtaining cDNA encoding VH and VL of the monoclonal antibody of the present invention that specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, constructing a DNA encoding dsFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or a eukaryotic organism to express the dsFv.
包含CDR的肽是通过包含VH或VL的CDR中的一个或多个区域而构成的。包含多个CDR的肽可以被直接相连或经由适合的肽接头相连。The peptide containing CDR is constituted by including one or more regions in the CDR of VH or VL. Peptides containing multiple CDRs can be directly linked or linked via a suitable peptide linker.
本发明的包含CDR的肽可以通过以下步骤来生产：构建本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的CDR的编码DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达所述肽。也可以通过化学合成方法例如Fmoc方法或tBoc方法来生产所述包含CDR的肽。The peptides containing CDRs of the present invention can be produced by the following steps: constructing the coding DNA of the CDRs of VH and VL of the monoclonal antibody of the present invention that specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic organism to express the peptide. The peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins ofimmunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的LCDR1、LCDR2和LCDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的HCDR2和HCDR3(CDR H2、CDR H3或H2、H3)。The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR applies only to LCDR1, LCDR2, and LCDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and HCDR2 and HCDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain.
本文中使用的术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。The term "antibody framework" as used herein refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. In essence, it is a variable domain without CDRs.
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，B7H3分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols inMethods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a B7H3 molecule). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-continuous amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10-7M，例如大约小于10-8M、10-9M或10-10M或更小的亲和力(KD)结合。The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of less than about10-7 M, such as less than about10-8 M,10-9 M or10-10 M or less.
术语″KD″或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本发明的抗体以小于大约10-7M，例如小于大约10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合B7H3，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。The term "KD" or "Kd" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the invention bind to B7H3 with a dissociation equilibrium constant (KD) of less than about10-7 M, e.g., less than about10-8 M,10-9 M, or10-10 M or less, e.g., as determined in a BIACORE instrument using surface plasmon resonance (SPR) technology.
术语“与B7H3抗体竞争结合人B7H3的单克隆抗体”是指与本发明的单克隆抗体竞争识别人B7H3的胞外区上的相同表位(也称为抗原决定簇)或相同表位的一部分并与所述表位结合的抗体。与本发明的B7H3抗体结合相同表位的抗体是指识别并结合于本发明的B7H3抗体识别的人B7H3的氨基酸序列的抗体。The term "monoclonal antibody that competes with the B7H3 antibody for binding to human B7H3" refers to an antibody that competes with the monoclonal antibody of the present invention for recognizing the same epitope (also called antigenic determinant) or a portion of the same epitope on the extracellular region of human B7H3 and binds to the epitope. An antibody that binds to the same epitope as the B7H3 antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human B7H3 recognized by the B7H3 antibody of the present invention.
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. In another embodiment, the vector is a viral vector, in which other DNA segments can be connected to the viral genome. The vector disclosed herein can be autonomously replicated in the host cell introduced therein (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., non-additional mammalian vectors).
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人B7H3或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http：//imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold Spring Harbor Manual of Antibody Laboratory Techniques, Chapters 5-8 and 15. For example, mice can be immunized with human B7H3 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described in the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by aligning the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351.
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line) and NS0 cells.
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人B7H3特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human B7H3. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product should be immediately frozen, such as -70°C, or lyophilized.
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。"Administering" and "treating" as applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administering" and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering" and "treating" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic, a combination composition, or by another cell. "Treatment" as applied to humans, veterinary medicine, or research subjects refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
“治疗”意指给予患者内用或外用治疗剂，例如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本发明的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。"Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present invention, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent that is effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although an embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating every symptom of the target disease, it should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology ofthe Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。"Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity.
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。An "effective amount" includes an amount sufficient to improve or prevent a symptom or condition of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。"Exogenous" refers to substances produced outside the body of an organism, cell, or human body, as the case may be. "Endogenous" refers to substances produced inside the body of a cell, organism, or human body, as the case may be.
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, this is clear from the context.
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring HarborSymp.Ouant.Biol.51：263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press，N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a trace amount of a specific portion of a nucleic acid, RNA and/or DNA, is amplified as described, for example, in U.S. Pat. No. 4,683,195. In general, it is necessary to obtain sequence information from the ends of the target region or beyond so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotides of the two primers can be consistent with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, etc. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR used herein is considered as one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which method includes using a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific portion of a nucleic acid.
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。"Optional" or "optionally" means that the event or environment described later can but need not occur, and the description includes occasions where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence can but does not have to be present.
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。"Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.
此外，本发明涉及用于免疫检测或测定B7H3的方法、用于免疫检测或测定B7H3的试剂、用于免疫检测或测定表达B7H3的细胞的方法和用于诊断与B7H3阳性细胞相关的疾病的诊断剂，其包含本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体或抗体片段作为活性成分。In addition, the present invention relates to a method for immunodetection or determination of B7H3, a reagent for immunodetection or determination of B7H3, a method for immunodetection or determination of cells expressing B7H3, and a diagnostic agent for diagnosing a disease associated with B7H3-positive cells, which comprises the monoclonal antibody or antibody fragment of the present invention that specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or the three-dimensional structure thereof as an active ingredient.
在本发明中，用于检测或测定B7H3的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。In the present invention, the method for detecting or measuring the amount of B7H3 may be any known method. For example, it includes immunodetection or measurement methods.
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。Immunoassay or determination method is a method for detecting or determining the amount of antibody or antigen using labeled antigen or antibody. Examples of immunoassay or determination methods include radioactive material labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods, etc.
上述与B7H3阳性细胞相关的疾病可以通过用本发明的单克隆抗体或抗体片段检测或测定表达B7H3的细胞来诊断。The above-mentioned diseases associated with B7H3-positive cells can be diagnosed by detecting or measuring cells expressing B7H3 using the monoclonal antibody or antibody fragment of the present invention.
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(AppliedBiosystem)的荧光抗体染色法等。In order to detect cells expressing the polypeptide, known immunodetection methods can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistostaining, etc. In addition, fluorescent antibody staining using FMAT8100 HTS system (Applied Biosystem) can be used.
在本发明中，对用于检测或测定B7H3的活体样品没有特别限制，只要它具有包含表达B7H3的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。In the present invention, the living body sample used for detecting or measuring B7H3 is not particularly limited as long as it has the possibility of containing cells expressing B7H3, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.
根据所需的诊断方法，含有本发明的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。Depending on the desired diagnostic method, the diagnostic agent containing the monoclonal antibody or its antibody fragment of the present invention may also contain reagents for performing antigen-antibody reactions or reagents for detecting reactions. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include reagents commonly used in immunoassays or assays, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment, or its conjugate, and a substrate corresponding to the label, etc.
术语“细胞毒性药物”是指在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语意在包括放射性同位素(例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32和Lu的放射性同位素)，化疗药物，毒素如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，包括其片段和/或变体。The term "cytotoxic drug" refers to a chemical molecule that has a strong ability to destroy the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations, but due to lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, leading to serious side effects. The term is intended to include radioisotopes (e.g., At211 , I131 , I125 , Y90 , Re186 , Re188 , Sm153 , Bi212 , P32 and radioisotopes of Lu), chemotherapeutic drugs, toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
术语“毒素”指来自细菌、真菌、植物或动物的小分子毒素及其衍生物，包括美登木素生物碱及其衍生物(CN101573384)如DM1、DM3、DM4，auristatin F(AF)及其衍生物，如MMAF、MMAE、3024(WO 2016/127790 A1，化合物7)，白喉毒素、外毒素、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、modeccin、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)局限曲霉素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(trichothecenes)。The term "toxin" refers to small molecule toxins and derivatives thereof from bacteria, fungi, plants or animals, including maytansinoids and their derivatives (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivatives, such as MMAF, MMAE, 3024 (WO 2016/127790 A1, compound 7), diphtheria toxin, exotoxin, ricin A chain, abrin A chain, modeccin, α-sarcin, Aleurites fordii toxin, dianthin toxin, Phytolaca americana toxin (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.
MMAF、MMAE为澳瑞他汀衍生物，参见US2005/0238649及Doronina等(2006)Bioconiugate Chem.17：114-124，结构式如下：MMAF and MMAE are auristatin derivatives, see US2005/0238649 and Doronina et al. (2006) Bioconiugate Chem. 17: 114-124, and the structural formula is as follows:
具体而言，澳瑞他汀/多拉司他汀药物模块诸如MMAF及其衍生物可以使用US2005-0238649A1及Doronina等(2006)Bioconjugate Chem.17：114-124中记载的方法来制备。澳瑞他汀/多拉司他汀药物模块诸如MMAE及其衍生物可以使用Doronina等(2003)Nat.Biotech.21：778-784中记载的方法来制备。可以通过常规方法方便地合成药物-接头模块MC-MMAF、MC-MMAE、MC-vc-PAB-MMAF和MC-vc-PAB-MMAE，例如Doronina等(2003)Nat.Biotech.21：778-784及美国专利申请公开号US2005/0238649A1中所记载的，然后将它们偶联至感兴趣的抗体。Specifically, auristatin/dolastatin drug moieties such as MMAF and its derivatives can be prepared using the methods described in US2005-0238649A1 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. Auristatin/dolastatin drug moieties such as MMAE and its derivatives can be prepared using the methods described in Doronina et al. (2003) Nat. Biotech. 21: 778-784. Drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF and MC-vc-PAB-MMAE can be conveniently synthesized by conventional methods, such as those described in Doronina et al. (2003) Nat. Biotech. 21: 778-784 and US Patent Application Publication No. US2005/0238649A1, and then coupled to the antibody of interest.
本发明中所述药物2852，参见WO 2016/127790 A1(化合物实施例7)，其结构式如下：The drug 2852 described in the present invention is described in WO 2016/127790 A1 (Compound Example 7), and its structural formula is as follows:
通过该发明中所述方法(化合物实施例8)或所属领域人员所能推定的方法，合成药物-接头模块(本发明中所述的3024)后，其结构式如下：After synthesizing the drug-linker module (3024 described in the present invention) by the method described in the invention (Compound Example 8) or a method that can be deduced by a person skilled in the art, its structural formula is as follows:
术语“化疗药物”是在肿瘤治疗中使用的化学化合物。化疗药物实例包括烷化剂，如噻替哌(thiotepa)；环磷酰胺(cyclosphamide)(CYTOXANTM)；烷基磺酸脂如白消安(busulfan)，英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶(aziridine)如苯并多巴(benaodopa)，卡波醌(carboquone)，美妥替哌(meturedopa)和尿烷亚胺(uredopa)；氮丙啶和methylamelamine包括六甲蜜胺(altretamine)，三亚胺嗪(triethylenemelamine)，三亚乙基磷酰胺，三亚乙基硫代磷酰胺和三羟甲基蜜胺(trimethylolomelamine)；氮芥(nitrogen mustards)如苯丁酸氮芥，萘氮芥，胆磷酰胺(cholophosphamide)，雌氮芥(estramustine)，异环磷酰胺(ifosfamide)，氮芥(mechlorethamine)，盐酸氧氮芥；左旋苯丙氨酸氮芥(melphalan)，新氮芥(novembichin)，胆甾醇苯乙酸氮芥，松龙苯芥(prednimustine)，曲磷胺(trofosfamide)，尿嘧啶氮芥；亚硝基脲(nitrosureas)如亚硝基脲氮芥(carmustine)，氯脲菌素(chlorozotocin)，福莫司汀(fotemustine)，洛莫司汀(lomustine)，尼莫司汀(nimustine)，雷莫司汀(ranimustine)；抗生素如阿克拉霉素，放线菌素，authramycin，重氮丝氨酸，博来霉素，放线菌素C(cactinomycin)，加利车霉素(calicheamicin)，carabicin，洋红霉素(chromomycin)，嗜癌素(carzinophilin)，色霉素，放线菌素D，柔红菌素(daunorubicin)，地托比星(detorubicin)，6-重氮-5-氧-L-正亮氨酸，阿霉素(doxorubicin)，表阿霉素(epirubicin)，依索比星(esorubicin)，伊达比星(idarubicin)，发波霉素(marcellomycin)，丝裂霉素，霉酚酸，诺加霉素(nogalamycin)，橄榄霉素(olivomycin)，培洛霉素(peplomycin)，potfiromycin，嘌呤霉素，三铁阿霉素(quelamycin)，罗多比星(rodorubicin)，链黑菌素；链脲霉素(streptozocin)，杀结核菌素，乌苯美司(ubenimex)，净司他丁(zinostatin)，佐柔比星(zorubicin)；抗代谢药如氨甲蝶吟，5-氟尿嘧啶(5-FU)；叶酸类似物如二甲叶酸(denopterin)，氨甲蝶呤，蝶罗呤，三甲曲沙(trimetrexate)；喋吟类似物氟达拉滨(fludarabine)，6-巯基蝶呤，硫咪蝶呤，硫鸟蝶呤；嘧啶类似物如安西他滨(ancitabine)，阿扎胞苷(azacitidine)，6-氮尿苷，卡莫氟(carmofur)，阿糖胞苷，双脱氧尿苷，去氟氧尿苷(doxitluridine)，依诺他滨(enocitabine)，氟尿苷，5-FU；雄激素类如二甲睾酮(calusterone)，丙酸甲雄烷酮(dromostanolong propionate)，环硫雄醇(epitiostanol)，美雄氨(mepitiostane)，睾内酯(testolactone)；抗肾上腺类如氨鲁米特(aminoglutethimide)，米托坦(mitotane)，曲洛司坦(trilostane)；叶酸补充剂如frolinic acid；醋葡内脂；醛磷酰胺糖苷(aldophosphamideglycoside)；氨基乙酰丙酸(aminolevulinic acid)；安吖啶(amsacrine)；bestrabucil；比生群(biasntrene)；依达曲沙(edatraxate)；defofamine；秋水仙胺；地吖醌(diaziquone)；elfomithine；依利醋铵(elliptinium acetate)；依托格鲁(etoglucid)；硝酸镓；羟基脲；香菇多糖(lentinan)；氯尼达明(lonidamine)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫哌达醇(mopidamol)；硝呋旦(nitracrine)；喷司他丁(pintostatin)；phenamet；吡柔比星(pirarubicin)；鬼臼树酸(podophyllinic acid)；2-乙基酰肼；丙卡巴肼(procarbazine)；
术语“接头单元”在本发明中为L1和L2，指一端与抗体共价连接而另一端与细胞毒性药物相连的化学结构片段或键。The term "linker unit" in the present invention refers to L1 and L2, and refers to a chemical structure fragment or bond that is covalently linked to an antibody at one end and is linked to a cytotoxic drug at the other end.
术语“载药量”是指分子中每个配体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个配体(Pc)连接1-8个细胞毒性药物，在本发明的实施方式中，载药量表示为y，可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物品均数量。The term "drug loading" refers to the average amount of cytotoxic drugs loaded on each ligand in the molecule, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading can range from 1 to 8 cytotoxic drugs connected to each ligand (Pc). In an embodiment of the present invention, the drug loading is expressed as y, and conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC characterization can be used to identify the average amount of drug per ADC molecule after the coupling reaction.
在本发明中，y可能受连接位点数量的限制。本发明的一个实施方式中，细胞毒性药物通过接头单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。In the present invention, y may be limited by the number of attachment sites. In one embodiment of the present invention, the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the ε-amino group of the lysine residue via a linker unit. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.
可以用以下非限制性方法控制抗体细胞毒性药物偶联物的载量，包括：The loading capacity of the antibody cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:
(1)控制连接试剂和单抗的摩尔比，(1) Control the molar ratio of the linking reagent and the monoclonal antibody,
(2)控制反应时间和温度，(2) Control the reaction time and temperature,
(3)选择不同的反应试剂。(3) Select different reaction reagents.
术语“载体”用于本发明的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。The term "carrier" is used for the drug of the present invention and refers to a system that can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug and deliver the drug to the target organ. The drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, high molecular surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, and preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be used as excellent drug carriers.
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的黏合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。The term "excipient" refers to the additives in pharmaceutical preparations other than the main drug, which can also be called excipients. For example, the binders, fillers, disintegrants, and lubricants in tablets; the matrix part in semi-solid preparations such as ointments and creams; the preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients.
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。The term "diluent" is also called filler, and its main purpose is to increase the weight and volume of the tablet. The addition of diluent not only ensures a certain volume, but also reduces the dosage deviation of the main ingredients, improves the compression molding of the drug, etc. When the drug in the tablet contains oily components, an absorbent needs to be added to absorb the oily substance and keep it in a "dry" state to facilitate tableting.
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本发明化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的实例是Deltec CADD-PLUS.TM.5400型静脉注射泵。The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. The injection or microemulsion may be injected into the patient's bloodstream by local mass injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound of the invention. To maintain such a constant concentration, a continuous intravenous drug delivery device may be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous injection pump.
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。The pharmaceutical composition can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration. The suspension can be prepared by known techniques using suitable dispersants or wetting agents and suspending agents as described above. Sterile injection preparations can also be sterile injection solutions or suspensions prepared in nontoxic parenteral acceptable diluents or solvents. In addition, sterile fixed oils can be conveniently used as solvents or suspension media.
本发明还涉及治疗人B7H3阳性细胞相关的疾病的方法，特别是在治疗癌症和炎症中的用途。The present invention also relates to a method for treating diseases associated with human B7H3-positive cells, in particular, to a method for treating cancer and inflammation.
二.实施例与测试例2. Implementation examples and test cases
以下结合实施例进一步描述本发明，但这些实施例及测试例并非限制着本发明的范围。本发明实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。The present invention is further described below in conjunction with the examples, but these examples and test examples do not limit the scope of the present invention. The experimental methods in the examples or test examples of the present invention that do not specify specific conditions are usually carried out according to conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or product manufacturer. Reagents that do not specify the specific source are conventional reagents purchased on the market.
实施例1.B7H3抗原及检测用蛋白的制备Example 1. Preparation of B7H3 antigen and detection protein
以SEQ ID NO：1所示人B7H3作为本发明B7H3的模板，设计本发明涉及的抗原及检测用蛋白的氨基酸序列。以下B7H3抗原未特殊说明的均指人B7H3。The human B7H3 shown in SEQ ID NO: 1 was used as a template for the B7H3 of the present invention to design the amino acid sequence of the antigen and the detection protein involved in the present invention. The following B7H3 antigens, unless otherwise specified, refer to human B7H3.
1.1人B7H3全长氨基酸序列：B7H3(SEQ ID NO：1)：1.1 Human B7H3 full-length amino acid sequence: B7H3 (SEQ ID NO: 1):
注释：Notes:
双横线部分为信号肽(Signal peptide：1-28)；The double horizontal line part is the signal peptide (Signal peptide: 1-28);
划横线部分为B7H3胞外区(Extracellular domain：29-466)，其中29-139为Ig-样V-型1结构域，145-238为Ig-样C2-型1结构域；243-357为Ig-样V-型2结构域，363-456为Ig-样C2-型2结构域；The underlined part is the B7H3 extracellular domain (Extracellular domain: 29-466), of which 29-139 is the Ig-like V-
点划线部分为跨膜区部分(Transmembrane domain：467-487)；The dotted line part is the transmembrane domain (Transmembrane domain: 467-487);
斜体部分为胞内区(Cytoplasmic domain：488-534)。The italicized part is the intracellular domain (Cytoplasmic domain: 488-534).
1.2鼠B7H3全长氨基酸序列(SEQ ID NO：2)1.2 Full-length amino acid sequence of mouse B7H3 (SEQ ID NO: 2)
注释：Notes:
双横线部分为信号肽(Signal peptide：1-28)；The double horizontal line part is the signal peptide (Signal peptide: 1-28);
划横线部分为B7H3胞外区(Extracellular domain：29-248)，其中29-139为Ig-样V-型结构域，145-238为Ig-样C2-型结构域；The underlined part is the B7H3 extracellular domain (Extracellular domain: 29-248), of which 29-139 is the Ig-like V-type domain, and 145-238 is the Ig-like C2-type domain;
点划线部分为跨膜区部分(Transmembrane domain：249-269)；The dotted line part is the transmembrane domain (Transmembrane domain: 249-269);
斜体部分为胞内区(Cytoplasmic domain：270-316)。The italicized part is the intracellular domain (Cytoplasmic domain: 270-316).
1.3筛选及检测用人B7H3抗原(SEQ ID NO：3)1.3 Screening and detection of human B7H3 antigen (SEQ ID NO: 3)
为商业化产品(R&D cat#1949-B3-050/CF，简称2Ig-B7H3)，序列如下：It is a commercial product (R&D cat#1949-B3-050/CF, referred to as 2Ig-B7H3), and its sequence is as follows:
注释：划横线部分为B7H3胞外区；斜体部分为His-tag标记。Note: The underlined part is the B7H3 extracellular region; the italic part is the His-tag marker.
1.4检测用人B7H3抗原(SEQ ID NO：4)1.4 Detection of human B7H3 antigen (SEQ ID NO: 4)
为商业化产品(SinoBiological cat#11188-H08H，简称4Ig-B7H3)，序列如下：It is a commercial product (SinoBiological cat#11188-H08H, referred to as 4Ig-B7H3), and the sequence is as follows:
注释：划横线部分为B7H3胞外区；斜体部分为His-tag标记。Note: The underlined part is the B7H3 extracellular region; the italic part is the His-tag marker.
1.5筛选及检测用鼠B7H3抗原(SEQ ID NO：5)1.5 Mouse B7H3 antigen for screening and detection (SEQ ID NO: 5)
为商业化产品(R&D cat#1397-B3-050/CF)，序列如下：It is a commercial product (R&D cat#1397-B3-050/CF), and the sequence is as follows:
注释：划横线部分为B7H3胞外区；斜体部分为His-tag标记。Note: The underlined part is the B7H3 extracellular region; the italic part is the His-tag marker.
实施例2.完全人源抗体的制备Example 2. Preparation of fully human antibodies
2.1阳性序列的筛选2.1 Screening of positive sequences
利用人PBMC、脾脏、淋巴结组织分离B细胞，并提取RNA，构建天然单链噬菌体抗体库(库容32×1010)。将构建的天然单链噬菌体文库经过包装形成噬菌体颗粒后，采用液相法进行淘筛，噬菌体与生物素化的B7H3液相结合，再采用链霉亲和素磁珠分离。为了获得可分别与人B7H3(R&D cat#1949-B3-050/CF)和鼠B7H3(R&D cat#1397-B3-050/CF)交叉结合的阳性序列，分别采用生物素化的人B7H3和生物素化的鼠B7H3进行交替淘筛，首轮采用2μg/ml生物素化的人B7H3进行淘筛，第二轮采用2μg/ml生物素化的鼠B7H3进行淘筛，第三轮采用0.5μg/ml生物素化的人B7H3进行淘筛，经三轮淘筛后，挑取500个单克隆包装成噬菌体，用于噬菌体ELISA测试。分别测试单克隆噬菌体与人B7H3(R&D cat#1949-B3-050/CF)和鼠B7H3(R&D cat#1397-B3-050/CF)的结合活性：ELISA板上分别包被1μg/ml人B7H3或鼠B7H3以及1％BSA，加入1∶1封闭缓冲液稀释的噬菌体上清，最后用anti-M13 HRP检测；将ELISA测试到的OD450值大于0.5，以及结合人和鼠B7H3的ELISA OD450值除以结合1％BSA的ELISAOD450值的两个比值均大于2.0的克隆进行测序，得到9个特异性序列。B cells were isolated from human PBMC, spleen, and lymph node tissues, and RNA was extracted to construct a natural single-chain phage antibody library (library capacity 32×1010 ). The constructed natural single-chain phage library was packaged to form phage particles, which were then screened using a liquid phase method. The phages were combined with biotinylated B7H3 liquid phase and then separated using streptavidin magnetic beads. In order to obtain positive sequences that can cross-bind to human B7H3 (R&D cat#1949-B3-050/CF) and mouse B7H3 (R&D cat#1397-B3-050/CF), biotinylated human B7H3 and biotinylated mouse B7H3 were used for alternating panning. 2 μg/ml biotinylated human B7H3 was used for panning in the first round, 2 μg/ml biotinylated mouse B7H3 was used for panning in the second round, and 0.5 μg/ml biotinylated human B7H3 was used for panning in the third round. After three rounds of panning, 500 monoclones were picked and packaged into phages for phage ELISA testing. The binding activity of monoclonal phage to human B7H3 (R&D cat#1949-B3-050/CF) and mouse B7H3 (R&D cat#1397-B3-050/CF) was tested respectively: 1 μg/ml human B7H3 or mouse B7H3 and 1% BSA were coated on ELISA plates, phage supernatant diluted with 1:1 blocking buffer was added, and finally detected with anti-M13 HRP; clones with ELISA OD450 values greater than 0.5 and the ratio of the ELISA OD450 values binding to human and mouse B7H3 divided by the ELISA OD450 value binding to 1% BSA greater than 2.0 were sequenced to obtain 9 specific sequences.
2.2完整单克隆抗体的构建2.2 Construction of complete monoclonal antibodies
噬菌体库筛选得到的9个特异性序列构建完整抗体后通过ELISA结合实验确定其中2个抗体结合力强，分别是h1702和h1703。对其完整单克隆抗体构建的过程如下：After constructing complete antibodies from the 9 specific sequences screened from the phage library, ELISA binding experiments determined that two of them had strong binding ability, namely h1702 and h1703. The process of constructing their complete monoclonal antibodies is as follows:
根据测序得到的单链抗体序列，设计引物PCR搭建各单链抗体序列的VH/VK/VL基因片段。获得h1702和h1703的重轻链可变区。According to the sequenced single-chain antibody sequences, primers were designed for PCR to construct the VH/VK/VL gene fragments of each single-chain antibody sequence, and the heavy and light chain variable regions of h1702 and h1703 were obtained.
＞h1702重链可变区序列＞h1702 heavy chain variable region sequence
＞h1702轻链可变区序列＞h1702 light chain variable region sequence
＞h1703重链可变区序列＞h1703 heavy chain variable region sequence
＞h1703轻链可变区序列＞h1703 light chain variable region sequence
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The italics in the sequence are FR sequences, and the underlined ones are CDR sequences.
其中各抗体轻重链中CDR序列如表1所示。The CDR sequences in the light and heavy chains of each antibody are shown in Table 1.
表1各重链及轻链CDR区序列Table 1 Sequences of the heavy and light chain CDR regions
抗体可变区再与恒定区基因(CH1-FC/CL)片段进行同源重组，构建完整抗体VH-CH1-FC/VK-CL/VL-CL。The antibody variable region is then homologously recombined with the constant region gene (CH1-FC/CL) fragment to construct the complete antibody VH-CH1-FC/VK-CL/VL-CL.
构建的完整全长抗体h1702、h1703序列如下：The sequences of the constructed complete full-length antibodies h1702 and h1703 are as follows:
h1702：h1702:
h1702重链(IgG1)氨基酸序列：(SEQ ID NO：22)h1702 heavy chain (IgG1) amino acid sequence: (SEQ ID NO: 22)
h1702轻链氨基酸序列：Lamada(SEQ ID NO：23)h1702 light chain amino acid sequence: Lamada (SEQ ID NO: 23)
h1703：h1703:
h1703重链(IgG1)氨基酸序列：(SEQ ID NO：24)h1703 heavy chain (IgG1) amino acid sequence: (SEQ ID NO: 24)
h1703轻链氨基酸序列：Kappa(SEQ ID NO：25)h1703 light chain amino acid sequence: Kappa (SEQ ID NO: 25)
为进一步提高抗体的稳定性，对h1702的轻链序列进行氨基酸突变，具体突变为轻链(SEQ ID NO：23)N端第一个氨基酸残基Q替代为D，缺失突变C端第一个氨基酸残基S，以获得更加稳定和均一的单克隆抗体h1702-DS。To further improve the stability of the antibody, amino acid mutations were performed on the light chain sequence of h1702, specifically, the first amino acid residue Q at the N-terminus of the light chain (SEQ ID NO: 23) was replaced by D, and the first amino acid residue S at the C-terminus was deleted and mutated to obtain a more stable and uniform monoclonal antibody h1702-DS.
突变修饰后的h1702-DS的重链序列为SEQ ID NO：22，轻链氨基酸序列如下：(SEQID NO：26)。The heavy chain sequence of the mutated h1702-DS is SEQ ID NO: 22, and the light chain amino acid sequence is as follows: (SEQ ID NO: 26).
2.3全人抗体的表达与纯化2.3 Expression and purification of fully human antibodies
分别表达抗体轻重链的质粒以1.5∶1的比例转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。Plasmids expressing the light and heavy chains of the antibody were transfected into HEK293E cells at a ratio of 1.5:1. After 6 days, the expression supernatant was collected, impurities were removed by high-speed centrifugation, and purified using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, it was further purified using a gel chromatography Superdex200 (GE) equilibrated with PBS to remove aggregates, collect the monomer peak, and aliquot for standby use.
B7H3抗体-药物偶联物制备实施例Example of Preparation of B7H3 Antibody-Drug Conjugate
实施例3、鼠源抗体的制备Example 3. Preparation of mouse antibodies
3.1抗原的制备3.1 Preparation of Antigen
编码带huFc标签的人B7-H3(h-B7H3-Fc)序列由Integrated DNA Technology(IDT)公司合成(以上B7-H3重组蛋白均由本发明设计模版序列)，分别克隆到pTT5载体上(Biovector)。重组的B7-H3蛋白在293T细胞表达后，通过所属领域的常规技术进行纯化。纯化的蛋白可用于小鼠免疫获得抗体。The sequence encoding human B7-H3 (h-B7H3-Fc) with huFc tag was synthesized by Integrated DNA Technology (IDT) (the above B7-H3 recombinant proteins were all designed by the present invention) and cloned into pTT5 vector (Biovector). After the recombinant B7-H3 protein was expressed in 293T cells, it was purified by conventional techniques in the field. The purified protein can be used to immunize mice to obtain antibodies.
h-B7H3-Fc的序列：Sequence of h-B7H3-Fc:
3.2抗体的制备3.2 Antibody Preparation
抗人B7H3单克隆抗体通过免疫小鼠产生。实验用Swiss Webster白小鼠，雌性，6周龄(Charles River公司)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。免疫抗原为带Fc标签的人B7H3重组蛋白(huB7H3-Fc)。用Titermax(sigma Lot Num：T2684)为佐剂。抗原与佐剂(titermax)比例为1∶1，抗原乳化后进行接种，时间为第0、21、35、49、63天。第0天腹膜内(IP)注射15μg+爪垫(footpad)25/只的乳化后抗原。21，35，49，63天腹膜内(IP)注射15μg+爪垫(footpad)15/只的乳化后抗原，在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射15μg+爪垫(footpad)15/只的生理盐水配制的抗原溶液。于第42，56，70天进行血检，用ELISA及FACS方法检测小鼠血清，确定小鼠血清中的抗体滴度。在第5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合，采用优化的电融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
融合后的杂交瘤细胞培养7-14天后，取培养基上清，使用B7-H3重组蛋白，ELISA实验对杂交瘤上清进行抗体筛选，得到的阳性抗体株进一步使用稳转表达B7-H3的CHO-S细胞，对比空白CHO-S细胞以排除非特异性结合抗体杂交瘤株，用流式分选方法进行筛选，从而选定两株结合重组蛋白且也结合细胞表达抗原的杂交瘤。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen，15596-018)提取RNA并反转录(PrimeScriptTM ReverseTranscriptase，Takara#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen，TB326Rev.B 0503)进行PCR扩增后测序，最终得到鼠源抗体m1704的序列。After the fused hybridoma cells were cultured for 7-14 days, the culture supernatant was taken, and the hybridoma supernatant was screened for antibodies using B7-H3 recombinant protein and ELISA experiments. The positive antibody strains were further used to stably express B7-H3 CHO-S cells, and compared with blank CHO-S cells to exclude non-specific binding antibody hybridoma strains, and screened by flow sorting method, thereby selecting two hybridomas that bind to recombinant proteins and cell-expressed antigens. Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted and reverse transcribed (PrimeScriptTM Reverse Transcriptase, Takara #2680A) using Trizol (Invitrogen, 15596-018). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sequenced, and the sequence of mouse antibody m1704 was finally obtained.
鼠单抗m1704的重链和轻链可变区序列如下：The heavy chain and light chain variable region sequences of mouse monoclonal antibody m1704 are as follows:
m1704重链m1704 heavy chain
m1704轻链m1704 light chain
为了提高抗体的稳定性，对上述鼠抗的CDR序列进行优化设计。优化后的CDR区具有如下序列：In order to improve the stability of the antibody, the CDR sequence of the above mouse antibody was optimized and designed. The optimized CDR region has the following sequence:
将鼠抗的重链和轻链可变区分别克隆进入含人IgG1重链恒定区和κ轻链恒定区的pTT载体质粒(Biovector)，然后瞬转转染入HEK293细胞，得到了抗B7-H3的嵌合抗体，按常规技术方法纯化后备用。The heavy chain and light chain variable regions of the mouse antibody were cloned into the pTT vector plasmid (Biovector) containing the human IgG1 heavy chain constant region and the kappa light chain constant region, respectively, and then transiently transfected into HEK293 cells to obtain anti-B7-H3 chimeric antibodies, which were purified according to conventional technical methods and set aside.
3.3小鼠抗体的人源化3.3 Humanization of mouse antibodies
鼠源抗人B7-H3单克隆抗体人源化如本领域许多文献公示的方法进行。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种抗体序列，本发明将抗体m1704进行人源化。The humanization of the mouse anti-human B7-H3 monoclonal antibody was carried out as disclosed in many documents in the art. In short, the parent (mouse antibody) constant domain was replaced with a human constant domain, and the human antibody sequence was selected based on the homology between the mouse antibody and the human antibody. The antibody m1704 was humanized in the present invention.
在所获得的鼠源抗体VH/VL CDR典型结构的基础上，将重、轻链可变区序列与人源抗体种系数据库比较，获得同源性高的人种系模板。其中人类种系轻链框架区来自人κ轻链基因人种系轻链模板IGkV1-33，人类种系重链框架区来自人重链模版IGHV3-23Based on the typical structure of mouse antibody VH/VL CDR, the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain human germline templates with high homology. The human germline light chain framework region is derived from the human κ light chain gene human germline light chain template IGkV1-33, and the human germline heavy chain framework region is derived from the human heavy chain template IGHV3-23
将鼠源抗体m1704的CDR区移植到选择好的相应人源化模板上，替换人源化可变区，再与IgG恒定区(优选重链为IgG1，轻链为κ)重组。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并对CDR区化学不稳定氨基酸残基优化，设计并检测了由如下人源化轻重链可变区序列组合而成的抗体。The CDR region of the mouse antibody m1704 was transplanted to the selected corresponding humanized template, replacing the humanized variable region, and then recombined with the IgG constant region (preferably IgG1 for the heavy chain and κ for the light chain). Then, based on the three-dimensional structure of the mouse antibody, the buried residues, the residues that directly interact with the CDR region, and the residues that have an important influence on the conformation of VL and VH were back-mutated, and the chemically unstable amino acid residues in the CDR region were optimized. The antibody composed of the following humanized light and heavy chain variable region sequences was designed and tested.
h1704VH1(SEQ ID NO：36)：h1704VH1 (SEQ ID NO: 36):
h1704VH2(SEQ ID NO：37)：h1704VH2 (SEQ ID NO: 37):
h1704VL1(SEQ ID NO：38)：h1704VL1 (SEQ ID NO: 38):
h1704VL2(SEQ ID NO：39)：h1704VL2 (SEQ ID NO: 39):
经表达测试和回复突变数量对比，选择出最终的人源化h1704-3抗体分子(使用VH1重链可变区和VL2轻链可变区)，其重链和轻链序列如SEQ ID NO：40和41所示。After expression testing and comparison of the number of back mutations, the final humanized h1704-3 antibody molecule (using the VH1 heavy chain variable region and the VL2 light chain variable region) was selected, and its heavy chain and light chain sequences are shown in SEQ ID NOs: 40 and 41.
h1704-3抗体重链(IgG1)序列：h1704-3 antibody heavy chain (IgG1) sequence:
h1704-3抗体轻链(Kappa)序列：h1704-3 antibody light chain (Kappa) sequence:
根据以上各人源化抗体轻链和重链的基因序列合成cDNA片段，插入到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences，Inc.Cat.No.23966)以1∶2的比例转染HEK293细胞(Life TechnologiesCat.No.11625019)，并置于CO2孵育箱中孵育4-5天。表达的抗体通过离心回收后，进行抗体纯化，得到本发明的人源化抗体蛋白。According to the gene sequences of the above humanized antibody light chain and heavy chain, cDNA fragments were synthesized and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and placed in aCO2 incubator for 4-5 days. After the expressed antibody was recovered by centrifugation, the antibody was purified to obtain the humanized antibody protein of the present invention.
B7H3抗体-药物偶联物制备实施例Example of Preparation of B7H3 Antibody-Drug Conjugate
以下实施例4至实施例11为本发明相关ADC的制备过程。其中实施例4至实施例7中抗体(h1702、h1703)通过在其赖氨酸上偶联带有连接单元的药物(MMAF或3024)反应，制备ADC；实施例8至实施例11通过抗体(h1702、h1704-3、h1702DS)半胱氨酸上的巯基，与带有连接单元的药物(3024)反应制备ADC。The following Examples 4 to 11 are the preparation processes of the ADCs of the present invention. In Examples 4 to 7, the antibodies (h1702, h1703) are prepared by reacting with a drug (MMAF or 3024) with a linker on their lysines; and in Examples 8 to 11, the ADCs are prepared by reacting the sulfhydryl groups on the cysteine of the antibodies (h1702, h1704-3, h1702DS) with a drug (3024) with a linker.
实施例4：B7H3-h1702-L2-MC-MMAF(h1702-MMAF)的制备Example 4: Preparation of B7H3-h1702-L2-MC-MMAF (h1702-MMAF)
将硫代乙酸S-(3-羰基丙基)酯(0.10mg，0.75μmol)，溶解于02mL乙腈溶液，备用；向抗体B7H3-h1702，PH＝4.3的乙酸/乙酸钠缓冲液(2.5mg/ml，2.0mL，0.034umol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加0.1mL的氰基硼氢化钠(3.4mg，53μmol)的水溶液，于25℃下振荡反应2小时。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，除去未反应的硫代乙酸S-(3-羰基丙基)酯以及氰基硼氢化钠，得到标题产物1b的PBS缓冲溶液(约5.5mL)，超滤离心浓缩至约2.5ml进行下一步反应。S-(3-carbonylpropyl)thioacetate (0.10 mg, 0.75 μmol) was dissolved in 0.2 mL of acetonitrile solution for later use; the above-prepared acetonitrile solution of S-(3-carbonylpropyl)thioacetate was added to antibody B7H3-h1702, acetic acid/sodium acetate buffer (2.5 mg/ml, 2.0 mL, 0.034 μmol) at pH = 4.3, and then 0.1 mL of an aqueous solution of sodium cyanoborohydride (3.4 mg, 53 μmol) was added dropwise, and the reaction was shaken at 25° C. for 2 hours. The reaction solution was purified by desalting with Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to remove unreacted S-(3-carbonylpropyl)thioacetate and sodium cyanoborohydride to obtain a PBS buffer solution (about 5.5 mL) of the title product 1b, which was concentrated to about 2.5 ml by ultrafiltration and centrifugation for the next reaction.
第二步
向1b的PBS缓冲溶液(2.5mL)中加入0.07mL的2.0M盐酸羟胺溶液，加毕，置于水浴振荡器，于25℃下振荡反应30分钟，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，得到标题产物B7H3-h1702单抗-丙硫醇1c的PBS缓冲溶液(浓度0.84mg/ml，5.0mL)。Add 0.07 mL of 2.0 M hydroxylamine hydrochloride solution to the PBS buffer solution (2.5 mL) of 1b, place in a water bath shaker, shake at 25°C for 30 minutes to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product B7H3-h1702 monoclonal antibody-propanethiol 1c in a PBS buffer solution (concentration 0.84 mg/ml, 5.0 mL).
第三步
将化合物(S)-2-((2R，3R)-3-((1S，3S，5S)-2-((3R，4S，5S)-4-((S)-2-((S)-2-(6-(2，5-二羰基-2，5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N，3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸MC-MMAF(0.32mg，0.34μmol)溶解于0.5mL乙腈中，加入B7H3-h1702单抗-丙硫醇PBS缓冲溶液1c(0.84mg/mL，5.0mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。Compound (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamide)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo [3.1.0] Hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-phenylpropanoic acid MC-MMAF (0.32 mg, 0.34 μmol) was dissolved in 0.5 mL of acetonitrile and added to B7H3-h1702 mAb-propanethiol PBS buffer solution 1c (0.84 mg/mL, 5.0 mL), placed in a water bath shaker, and shaken at 25°C for 4 hours before stopping the reaction.
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物h1702-MMAF的PBS缓冲液(0.21mg/mL，14.5mL)，于4℃冷冻储存。The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product h1702-MMAF in PBS buffer (0.21 mg/mL, 14.5 mL), which was stored frozen at 4°C.
Q-TOF LC/MS计算平均值：y＝1.76。Q-TOF LC/MS calculated average value: y=1.76.
实施例5：B7H3-h1703-L2-MC-MMAF(h1703-MMAF)的制备Example 5: Preparation of B7H3-h1703-L2-MC-MMAF (h1703-MMAF)
将硫代乙酸S-(3-羰基丙基)酯(0.11mg，0.82μmol)，溶解于02mL乙腈溶液，备用；向抗体B7H3-h1703，PH＝4.3的乙酸/乙酸钠缓冲液(2.5mg/ml，2.0mL，0.034umol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加0.1mL的氰基硼氢化钠(3.4mg，53μmol)的水溶液，于25℃下振荡反应2小时。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，除去未反应的硫代乙酸S-(3-羰基丙基)酯以及氰基硼氢化钠，得到标题产物2b的PBS缓冲溶液(约5.5mL)，超滤离心浓缩至约2.5ml进行下一步反应。S-(3-carbonylpropyl)thioacetate (0.11 mg, 0.82 μmol) was dissolved in 0.2 mL of acetonitrile solution for later use; the above-prepared acetonitrile solution of S-(3-carbonylpropyl)thioacetate was added to antibody B7H3-h1703, PH=4.3 acetic acid/sodium acetate buffer (2.5 mg/ml, 2.0 mL, 0.034 μmol), and then 0.1 mL of an aqueous solution of sodium cyanoborohydride (3.4 mg, 53 μmol) was added dropwise, and the reaction was shaken at 25°C for 2 hours. The reaction solution was purified by desalting with Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to remove unreacted S-(3-carbonylpropyl)thioacetate and sodium cyanoborohydride to obtain a PBS buffer solution (about 5.5 mL) of the title product 2b, which was concentrated to about 2.5 ml by ultrafiltration and centrifugation for the next reaction.
第二步
向2b的PBS缓冲溶液(2.5mL)中加入0.07mL的2.0M盐酸羟胺溶液，加毕，置于水浴振荡器，于25℃下振荡反应30分钟，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，得到标题产物B7H3-h1703单抗-丙硫醇2c的PBS缓冲溶液(浓度0.82mg/ml，5.0mL)。0.07 mL of 2.0 M hydroxylamine hydrochloride solution was added to the PBS buffer solution (2.5 mL) of 2b, and after the addition, the mixture was placed in a water bath shaker and shaken at 25° C. for 30 minutes to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (concentration 0.82 mg/ml, 5.0 mL) of the title product B7H3-h1703 monoclonal antibody-propanethiol 2c.
第三步
将化合物(S)-2-((2R，3R)-3-((1S，3S，5S)-2-((3R，4S，5S)-4-((S)-2-((S)-2-(6-(2，5-二羰基-2，5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N，3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸MC-MMAF(0.32mg，0.34μmol)溶解于0.5mL乙腈中，加入B7H3-h1703单抗-丙硫醇PBS缓冲溶液2c(0.82mg/mL，5.0mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。Compound (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamide)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo [3.1.0] Hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-phenylpropanoic acid MC-MMAF (0.32 mg, 0.34 μmol) was dissolved in 0.5 mL of acetonitrile and added to B7H3-h1703 mAb-propanethiol PBS buffer solution 2c (0.82 mg/mL, 5.0 mL), placed in a water bath shaker, and shaken at 25°C for 4 hours before stopping the reaction.
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物h1703-MMAF的PBS缓冲液(0.20mg/mL，13.0mL)，于4℃冷冻储存。The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product h1703-MMAF in PBS buffer (0.20 mg/mL, 13.0 mL), which was stored frozen at 4°C.
Q-TOF LC/MS计算平均值：y＝2.29。Q-TOF LC/MS calculated average value: y=2.29.
实施例6：B7H3-h1702-L2-3024(h1702-3024)的制备Example 6: Preparation of B7H3-h1702-L2-3024 (h1702-3024)
将硫代乙酸S-(3-羰基丙基)酯(0.45mg，3.38μmol)，溶解于0.8mL乙腈溶液，备用；向抗体B7H3-h1702，pH＝4.3的乙酸/乙酸钠缓冲液(5.0mg/ml，8.0mL，0.270μmol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加0.1mL的氰基硼氢化钠(12.8mg，0.2mmol)的水溶液，于25℃下振荡反应2小时。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，除去未反应的硫代乙酸S-(3-羰基丙基)酯以及氰基硼氢化钠，得到标题产物3b的PBS缓冲溶液(约14.5mL)，超滤离心浓缩至约7.5ml进行下一步反应。S-(3-carbonylpropyl)thioacetate (0.45 mg, 3.38 μmol) was dissolved in 0.8 mL of acetonitrile solution for later use; the above-prepared acetonitrile solution of S-(3-carbonylpropyl)thioacetate was added to the antibody B7H3-h1702, pH = 4.3 acetic acid/sodium acetate buffer (5.0 mg/ml, 8.0 mL, 0.270 μmol), and then 0.1 mL of an aqueous solution of sodium cyanoborohydride (12.8 mg, 0.2 mmol) was added dropwise, and the reaction was shaken at 25°C for 2 hours. The reaction solution was purified by desalting with Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to remove unreacted S-(3-carbonylpropyl)thioacetate and sodium cyanoborohydride to obtain a PBS buffer solution (about 14.5 mL) of the title product 3b, which was concentrated to about 7.5 ml by ultrafiltration and centrifugation for the next reaction.
第二步
向3b的PBS缓冲溶液(7.5mL)中加入0.20mL的2.0M盐酸羟胺溶液，加毕，置于水浴振荡器，于25℃下振荡反应30分钟，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，得到标题产物B7H3-h1702单抗-丙硫醇3c的PBS缓冲溶液(浓度2.84mg/ml，14.0mL)。0.20 mL of 2.0 M hydroxylamine hydrochloride solution was added to the PBS buffer solution (7.5 mL) of 3b, and after the addition, the mixture was placed in a water bath shaker and oscillated at 25° C. for 30 minutes to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (concentration 2.84 mg/ml, 14.0 mL) of the title product B7H3-h1702 monoclonal antibody-propanethiol 3c.
第三步
将化合物(S)-2-((2R，3R)-3-((1S，3S，5S)-2-((3R，4S，5S)-4-((S)-2-((S)-2-(6-(2，5-二羰基-2，5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N，3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸化合物3024(2.6mg，2.7μmol)溶解于1.4mL乙腈中，加入B7H3-h1702单抗-丙硫醇PBS缓冲溶液3c(2.84mg/mL，14.0mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。Compound (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamide)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3 .1.0]hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid compound 3024 (2.6 mg, 2.7 μmol) was dissolved in 1.4 mL of acetonitrile and added to B7H3-h1702 monoclonal antibody-propanethiol PBS buffer solution 3c (2.84 mg/mL, 14.0 mL), placed in a water bath shaker, and shaken at 25°C for 4 hours before stopping the reaction.
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物h1702-3024的PBS缓冲液(1.35mg/mL，27.5mL)，于4℃冷冻储存。The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product h1702-3024 in PBS buffer (1.35 mg/mL, 27.5 mL), which was stored frozen at 4°C.
Q-TOF LC/MS计算平均值：y＝2.05。Q-TOF LC/MS calculated average value: y=2.05.
实施例7：B7H3-h1703-L2-3024(h1703-3024)的制备Example 7: Preparation of B7H3-h1703-L2-3024 (h1703-3024)
将硫代乙酸S-(3-羰基丙基)酯(0.50mg，3.78μmol)，溶解于0.8mL乙腈溶液，备用；向抗体B7H3-h1703，pH＝4.3的乙酸/乙酸钠缓冲液(5.0mg/ml，8.0mL，0.270umol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加0.1mL的氰基硼氢化钠(12.8mg，0.2mmol)的水溶液，于25℃下振荡反应2小时。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，除去未反应的硫代乙酸S-(3-羰基丙基)酯以及氰基硼氢化钠，得到标题产物4b的PBS缓冲溶液(约14.0mL)，超滤离心浓缩至约7.5ml进行下一步反应。S-(3-carbonylpropyl)thioacetate (0.50 mg, 3.78 μmol) was dissolved in 0.8 mL of acetonitrile solution for later use; the above-prepared acetonitrile solution of S-(3-carbonylpropyl)thioacetate was added to the antibody B7H3-h1703, pH = 4.3 acetic acid/sodium acetate buffer (5.0 mg/ml, 8.0 mL, 0.270 umol), and then 0.1 mL of an aqueous solution of sodium cyanoborohydride (12.8 mg, 0.2 mmol) was added dropwise, and the reaction was shaken at 25°C for 2 hours. The reaction solution was purified by desalting with Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to remove unreacted S-(3-carbonylpropyl)thioacetate and sodium cyanoborohydride to obtain a PBS buffer solution (about 14.0 mL) of the title product 4b, which was concentrated to about 7.5 ml by ultrafiltration and centrifugation for the next reaction.
第二步
向4b的PBS缓冲溶液(7.5mL)中加入0.20mL的2.0M盐酸羟胺溶液，加毕，置于水浴振荡器，于25℃下振荡反应30分钟，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，得到标题产物B7H3-h1703单抗-丙硫醇4c的PBS缓冲溶液(浓度2.64mg/ml，14.5mL)。0.20 mL of 2.0 M hydroxylamine hydrochloride solution was added to the PBS buffer solution (7.5 mL) of 4b, and after the addition, the mixture was placed in a water bath shaker and oscillated at 25° C. for 30 minutes to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (concentration 2.64 mg/ml, 14.5 mL) of the title product B7H3-h1703 monoclonal antibody-propanethiol 4c.
第三步
将化合物(S)-2-((2R，3R)-3-((1S，3S，5S)-2-((3R，4S，5S)-4-((S)-2-((S)-2-(6-(2，5-二羰基-2，5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N，3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸化合物3024(2.6mg，2.7μmol)溶解于1.45mL乙腈中，加入B7H3-h1703单抗-丙硫醇PBS缓冲溶液6c(2.64mg/mL，14.5mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。The compound (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamide)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3. The 1.0]hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid compound 3024 (2.6 mg, 2.7 μmol) was dissolved in 1.45 mL of acetonitrile, added to B7H3-h1703 mAb-propanethiol PBS buffer solution 6c (2.64 mg/mL, 14.5 mL), placed in a water bath shaker, and shaken at 25°C for 4 hours before stopping the reaction.
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物h1703-3024的PBS缓冲液(1.20mg/mL，28.0mL)，于4℃冷冻储存。The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product h1703-3024 in PBS buffer (1.20 mg/mL, 28.0 mL), which was stored frozen at 4°C.
Q-TOF LC/MS计算平均值：y＝1.62。Q-TOF LC/MS calculated average value: y=1.62.
实施例8.B7H3-h1702-cys-3024(h1702-cys-3024)的制备Example 8. Preparation of B7H3-h1702-cys-3024 (h1702-cys-3024)
在37℃条件下，向抗体h1702，pH＝6.5的0.05M的PBS缓冲水溶液(10.0mg/ml，5.0mL，0.333umol)加入配置好的0.01mM的三(2-羧乙基)膦(TCEP)的10mM水溶液0.07mL(0.7umol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应；At 37°C, add 0.07 mL (0.7 umol) of 10 mM aqueous solution of 0.01 mM tris(2-carboxyethyl)phosphine (TCEP) to 0.05 M PBS buffer solution (10.0 mg/ml, 5.0 mL, 0.333 umol) of antibody h1702 at pH = 6.5, place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction;
将反应液用水浴降温至25℃，再将化合物(S)-2-((2R，3R)-3-((1S，3S，5S)-2-((3R，4S，5S)-4-((S)-2-((S)-2-(6-(2，5-二羰基-2，5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N，3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸SHR3024(2.6mg，2.7μmol)溶解于0.5mL乙腈中，加入到降温至25℃的抗体h1702(含有TCEP)中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应；The reaction solution was cooled to 25°C in a water bath, and then compound (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamide)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy -5-methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid SHR3024 (2.6 mg, 2.7 μmol) was dissolved in 0.5 mL of acetonitrile, added to antibody h1702 (containing TCEP) cooled to 25°C, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped;
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物的PBS缓冲液(6.85mg/mL，7.5mL)，于4℃冷冻储存。The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product in PBS buffer (6.85 mg/mL, 7.5 mL), which was stored frozen at 4°C.
HIC-HPLC计算平均值：y＝3.5。The average value calculated by HIC-HPLC was: y=3.5.
实施例9.B7H3-h1704-3-cys-3024(h1704-3-cys-3024)的制备Example 9. Preparation of B7H3-h1704-3-cys-3024 (h1704-3-cys-3024)
在37℃条件下，向抗体B7H3-h1704-3，PH＝6.5的0.05M的PBS缓冲水溶液(10.0mg/ml，5.0mL，0.333umol)加入配置好的0.01mM的三(2-羧乙基)膦(TCEP)的10mM水溶液0.075mL(0.75umol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应；At 37°C, add 0.075 mL (0.75 umol) of 10 mM aqueous solution of 0.01 mM tris(2-carboxyethyl)phosphine (TCEP) to 0.05 M PBS buffer solution (10.0 mg/ml, 5.0 mL, 0.333 umol) of antibody B7H3-h1704-3 at pH = 6.5, place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction;
将反应液用水浴降温至25℃，再将化合物(S)-2-((2R，3R)-3-((1S，3S，5S)-2-((3R，4S，5S)-4-((S)-2-((S)-2-(6-(2，5-二羰基-2，5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N，3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸化合物3024(2.6mg，2.7μmol)溶解于0.5mL乙腈中，加入到降温至25℃的抗体B7H3-h1704-3(含有TCEP)中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应；The reaction solution was cooled to 25°C in a water bath, and then compound (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamide)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methyl Compound 3024 (2.6 mg, 2.7 μmol) was dissolved in 0.5 mL of acetonitrile, added to antibody B7H3-h1704-3 (containing TCEP) cooled to 25°C, placed in a water bath shaker, and shaken at 25°C for 3 hours to stop the reaction;
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物的PBS缓冲液(6.75mg/mL，7.8mL)，于4℃冷冻储存。The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product in PBS buffer (6.75 mg/mL, 7.8 mL), which was stored frozen at 4°C.
HIC-HPLC计算平均值：y＝3.4。The average value calculated by HIC-HPLC was: y=3.4.
实施例10.B7H3-h1702DS-cys-3024(h1702DS-cys-3024)的制备Example 10. Preparation of B7H3-h1702DS-cys-3024 (h1702DS-cys-3024)
在37℃条件下，向抗体H1702-DS，pH＝6.5的0.05M的PBS缓冲水溶液(10.0mg/ml，2.0mL，0.133umol)加入配置好的0.01mM的三(2-羧乙基)膦(TCEP)的10mM水溶液0.028mL(0.28umol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应；At 37°C, add 0.028 mL (0.28 umol) of 10 mM aqueous solution of 0.01 mM tris(2-carboxyethyl)phosphine (TCEP) to 0.05 M PBS buffer solution (10.0 mg/ml, 2.0 mL, 0.133 umol) of antibody H1702-DS, pH=6.5, and place in a water bath shaker, shake at 37°C for 3 hours to stop the reaction;
将反应液用水浴降温至25℃，再将化合物(S)-2-((2R，3R)-3-((1S，3S，5S)-2-((3R，4S，5S)-4-((S)-2-((S)-2-(6-(2，5-二羰基-2，5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N-3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸化合物3024(1.02mg，1.1μmol)溶解于0.2mL乙腈中，加入到降温至25℃的抗体h1702-DS(含有TCEP)中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应；The reaction solution was cooled to 25°C in a water bath, and then compound (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamide)-3-methylbutanamide)-N-3-dimethylbutanamide)-3-methoxy-5-nitropropane -methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl)propionic acid compound 3024 (1.02 mg, 1.1 μmol) was dissolved in 0.2 mL of acetonitrile, added to antibody h1702-DS (containing TCEP) cooled to 25°C, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped;
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物的PBS缓冲液(6.65mg/mL，2.7mL)，于4℃冷冻储存。The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product in PBS buffer (6.65 mg/mL, 2.7 mL), which was stored frozen at 4°C.
HIC-HPLC计算平均值：y＝3.55。The average value calculated by HIC-HPLC was: y=3.55.
以下用测试方法验证本发明抗体性能及有益效果。The following test methods are used to verify the performance and beneficial effects of the antibodies of the present invention.
测试例1.ELISA结合实验Test Example 1. ELISA binding assay
为检测筛选到的B7H3抗体，及相应的不同B7H3-ADC对于人不同形式B7H3的体外结合能力，人2Ig-B7H3(Cat.#1949-B3-050/CF，R&D)和人4Ig-B7H3(Cat.#11188-H08H，SinoBiological)被用于进行体外结合检测。To detect the in vitro binding ability of the screened B7H3 antibodies and the corresponding different B7H3-ADCs to different forms of human B7H3, human 2Ig-B7H3 (Cat.#1949-B3-050/CF, R&D) and human 4Ig-B7H3 (Cat.#11188-H08H, SinoBiological) were used for in vitro binding assays.
用pH7.4的PBS(Sigma，P4417-100TAB)缓冲液将人B7H3蛋白(2Ig/4Ig)稀释至1μg/ml浓度，以100μl/孔的体积加入96孔酶标板(Corning，CLS3590-100EA)中，于4℃放置过夜16-20小时。弃去液体后，加入用PBST缓冲液(PH7.4PBS含0.05％Tween-20)稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液120μl/孔，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板4次后，加入100μl/孔初始浓度为1μM的相应B7H3抗体(或B7H3-ADC)，用PBST缓冲液倍比稀释8个梯度，置于37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板4次，加入100μl/孔用PBST稀释(1∶4000)的HRP标记的羊抗人IgG(山羊抗人IgG)Fcγ片段特异性的二抗(Jackson Immuno Research，109-005-008)，37℃孵育1小时。用PBST洗板4次后，加入100μl/孔TMB显色底物(KPL，52-00-03)，于室温孵育3-5min，加入100μl/孔1M H2SO4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算抗体对抗原的结合EC50值，结果见表2。The human B7H3 protein (2Ig/4Ig) was diluted to a concentration of 1 μg/ml with PBS (Sigma, P4417-100TAB) buffer at pH 7.4, and added to a 96-well ELISA plate (Corning, CLS3590-100EA) at a volume of 100 μl/well, and placed at 4°C overnight for 16-20 hours. After discarding the liquid, 120 μl/well of 5% skim milk (Bright skim milk powder) blocking solution diluted with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20) was added, and the plate was incubated in a 37°C incubator for 2 hours for blocking. After the blocking was completed, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer, and then 100 μl/well of the corresponding B7H3 antibody (or B7H3-ADC) with an initial concentration of 1 μM was added, and 8 gradients were diluted with PBST buffer, and the plate was incubated in a 37°C incubator for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded, the plate was washed 4 times with PBST, and 100 μl/well of HRP-labeled goat anti-human IgG (goat anti-human IgG) Fcγ fragment-specific secondary antibody (Jackson Immuno Research, 109-005-008) diluted with PBST (1:4000) was added, and incubated at 37°C for 1 hour. After washing the
表2.不同抗体及抗体ADC与人2Ig-B7H3及4Ig-B7H3抗原的结合力Table 2. Binding affinity of different antibodies and antibody ADCs to human 2Ig-B7H3 and 4Ig-B7H3 antigens
结论：针对人2Ig-B7H3及人4Ig-B7H3，ADC与裸抗在ELISA实验上具有类似的结合力，即ADC标记后未降低结合力。Conclusion: For human 2Ig-B7H3 and human 4Ig-B7H3, ADC and naked antibody have similar binding affinity in ELISA experiments, that is, the binding affinity is not reduced after ADC labeling.
测试例2.与不同种属B7H3的交叉结合实验Test Example 2. Cross-binding experiment with B7H3 of different species
为检测筛选到的B7H3抗体，及相应的不同B7H3-ADC，对于不同种属来源的B7H3的体外结合能力，小鼠B7H3(Cat.#1397-B3-050/CF，R&D)被用于进行体外结合检测。To detect the in vitro binding ability of the screened B7H3 antibodies and corresponding different B7H3-ADCs to B7H3 from different species, mouse B7H3 (Cat.#1397-B3-050/CF, R&D) was used for in vitro binding assays.
用pH7.4的PBS(Sigma，P4417-100TAB)缓冲液将不同种属B7H3蛋白(mouse B7H3)稀释至1μg/ml浓度，以100μl/孔的体积加入96孔酶标板中，于4℃放置过夜16-20小时。弃去液体后，加入用PBST缓冲液(PH7.4PBS含0.05％Tween-20)稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液120μl/孔，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板4次后，加入100μl/孔初始浓度为1μM的相应B7H3抗体(或B7H3-ADC)，用PBST缓冲液倍比稀释8个梯度，置于37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板4次，加入100μl/孔用PBST稀释(1∶4000)的HRP标记的山羊抗人IgG，Fcγ片段特异性的二抗(Jackson Immuno Research，109-005-008)，37℃孵育1小时。用PBST洗板4次后，加入100μl/孔TMB显色底物(KPL，52-00-03)，于室温孵育3-5min，加入100μl/孔1M H2SO4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算抗体对抗原的结合EC50值(结果见表3)。Different species of B7H3 proteins (mouse B7H3) were diluted to a concentration of 1 μg/ml with PBS (Sigma, P4417-100TAB) buffer at pH 7.4, added to a 96-well ELISA plate at a volume of 100 μl/well, and placed at 4°C overnight for 16-20 hours. After discarding the liquid, 120 μl/well of 5% skim milk (Bright skim milk powder) blocking solution diluted with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20) was added, and the plate was incubated in a 37°C incubator for 2 hours for blocking. After the blocking was completed, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer, and then 100 μl/well of the corresponding B7H3 antibody (or B7H3-ADC) with an initial concentration of 1 μM was added, and 8 gradients were diluted with PBST buffer, and the plate was incubated in a 37°C incubator for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded, the plate was washed 4 times with PBST, 100 μl/well of HRP-labeled goat anti-human IgG, Fcγ fragment-specific secondary antibody (Jackson Immuno Research, 109-005-008) diluted with PBST (1:4000) was added, and incubated at 37°C for 1 hour. After washing the
表3.不同抗体与鼠B7H3抗原的结合力Table 3. Binding ability of different antibodies to mouse B7H3 antigen
结果显示，h1702和h1703，及不同ADC与鼠B7H3结合力相对较弱，显示出两个单克隆抗体具有良好的人B7H3结合特异性。The results showed that h1702 and h1703, as well as different ADCs, had relatively weak binding to mouse B7H3, indicating that the two monoclonal antibodies had good human B7H3 binding specificity.
测试例3.Biacore抗体亲和力实验Test Example 3. Biacore Antibody Affinity Experiment
用Biacore，GE仪器测定抗B7H3抗体，及抗B7H3-ADC和人2Ig-B7H3抗原，人4Ig-B7H3抗原各种抗原之间的反应亲和力。The reaction affinity between anti-B7H3 antibodies and anti-B7H3-ADC and human 2Ig-B7H3 antigen and human 4Ig-B7H3 antigen was determined using Biacore, GE instrument.
用生物传感芯片Protein A(Cat.#29127556，GE)亲和捕获一定量的待测抗体/待测ADC，然后于芯片表面流经一系列浓度梯度下的人2Ig-B7H3抗原(Cat.#1949-B3-050/CF，R&D)、人4Ig-B7H3抗原(Cat.#11188-H08H，Sino Biological)、利用Biacore仪器(BiacoreT200，GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用甘氨酸-盐酸再生溶液(pH 1.5)(Cat#BR-1003-54，GE)将生物芯片洗净再生。实验中用到的缓冲液为HBS-EP缓冲溶液(pH 7.4)(Cat.#BR-1001-88，GE)。A certain amount of the antibody/ADC to be tested was captured by affinity using the biosensor chip Protein A (Cat.#29127556, GE), and then a series of concentration gradients of human 2Ig-B7H3 antigen (Cat.#1949-B3-050/CF, R&D) and human 4Ig-B7H3 antigen (Cat.#11188-H08H, Sino Biological) flowed over the chip surface, and the reaction signal was detected in real time using a Biacore instrument (BiacoreT200, GE) to obtain the binding and dissociation curves. After each cycle of dissociation was completed, the biochip was washed and regenerated with a glycine-hydrochloric acid regeneration solution (pH 1.5) (Cat#BR-1003-54, GE). The buffer used in the experiment was HBS-EP buffer solution (pH 7.4) (Cat.#BR-1001-88, GE).
实验得到的数据用BIAevaluation version 4.1，GE软件以(1∶1)Langmuir模型进行拟合，从而得出亲和力数值。实验结果见表4。The data obtained from the experiment were fitted with the (1:1) Langmuir model using BIAevaluation version 4.1, GE software, to obtain the affinity value. The experimental results are shown in Table 4.
表4.不同抗体/ADC和各种抗原之间的反应亲和力(单位：M)Table 4. Reaction affinity between different antibodies/ADCs and various antigens (unit: M)
结论：针对与人2Ig-B7H3，人4Ig-B7H3在Biacore实验上的亲和力测试表明，ADC与裸抗的亲和力相似。Conclusion: The affinity test of human 2Ig-B7H3 and human 4Ig-B7H3 in Biacore experiment showed that the affinity of ADC was similar to that of naked antibody.
测试例4.体外细胞结合实验Test Example 4. In vitro cell binding assay
本实验通过检测细胞表面抗体的荧光信号，根据荧光信号强弱来评价抗体的结合。将10μg一抗与2×105个U87MG细胞在冰上孵育30分钟后洗掉多余的抗体。将细胞与APC抗人IgG Fc(Biolegend，409306)在室温孵育30分钟，洗掉多余抗体后使用BD Verse读取细胞表面的荧光信号(结果见图1)。In this experiment, the fluorescence signal of the cell surface antibody was detected, and the binding of the antibody was evaluated according to the strength of the fluorescence signal. 10 μg of primary antibody was incubated with 2×105 U87MG cells on ice for 30 minutes, and then the excess antibody was washed off. The cells were incubated with APC anti-human IgG Fc (Biolegend, 409306) at room temperature for 30 minutes, and the fluorescence signal on the cell surface was read using BD Verse after washing off the excess antibody (see Figure 1 for the results).
结果表明：h1702与细胞表面的B7H3抗原具有很强的结合能力。The results showed that h1702 had a strong binding ability with B7H3 antigen on the cell surface.
测试例5.体外细胞内吞实验Test Example 5. In vitro cell endocytosis experiment
本实验通过检测细胞内抗体或不同ADC的荧光信号，根据荧光信号强弱来评价抗体的内吞效果。将B7H3抗体/ADC和APC抗人IgG Fc(Biolegend，409306)以1∶2的摩尔比例混合在冰上孵育15分钟。将抗体混合物与2×105个U87MG细胞在冰上孵育30分钟后洗掉多余的抗体，然后将细胞转入预温37℃的培养基中，在37℃分别孵育0，15，30，60和120分钟。离心细胞并将细胞重悬在抗体洗脱液中室温孵育7分钟，洗掉抗体洗脱液，使用BD Verse读取细胞内荧光信号，见图2，图3，图4。结果可见ADC在U87MG细胞上的内吞效果与裸抗相当。This experiment evaluates the antibody internalization effect by detecting the fluorescence signal of intracellular antibodies or different ADCs according to the strength of the fluorescence signal. B7H3 antibody/ADC and APC anti-human IgG Fc (Biolegend, 409306) were mixed at a molar ratio of 1:2 and incubated on ice for 15 minutes. The antibody mixture was incubated with 2×105 U87MG cells on ice for 30 minutes, and then the excess antibody was washed off. The cells were then transferred to a pre-warmed 37°C culture medium and incubated at 37°C for 0, 15, 30, 60 and 120 minutes. The cells were centrifuged and resuspended in the antibody eluate and incubated at room temperature for 7 minutes. The antibody eluate was washed off and the intracellular fluorescence signal was read using BD Verse, as shown in Figures 2, 3, and 4. The results show that the internalization effect of ADC on U87MG cells is equivalent to that of naked antibodies.
测试例6.SD大鼠T1/2评价Test Example 6. Evaluation of T1/2 in SD rats
SD大鼠4只，雌雄各半，12/12小时光/暗调节，温度24±3℃恒温，湿度50-60％，自由进食饮水。购自杰思捷实验动物有限公司。实验当天对SD大鼠分别尾静脉注射受试药物B7H3抗体/ADC，给药剂量为3mg/kg，注射体积5ml/kg。Four SD rats, half male and half female, were placed under 12/12 hour light/dark regulation, constant temperature of 24±3℃, humidity of 50-60%, and free access to food and water. They were purchased from JST Experimental Animal Co., Ltd. On the day of the experiment, the SD rats were injected with the test drug B7H3 antibody/ADC through the tail vein, with a dose of 3 mg/kg and an injection volume of 5 ml/kg.
取血时间点为：第1天给药后5min、8h、24h(第2天)，第3天，第5天，第8天，第11天，第15天，于大鼠眼底静脉取血，每次200μL(相当于取血清100μL)；收集的血样在室温下置放半小时至凝集，然后4℃下10000×g离心10分钟。收集上清，立即放置-80℃贮存。Blood was collected from the rats at 5 minutes, 8 hours, 24 hours (day 2) after administration on
用ELISA检测血清中的B7H3抗体浓度，进行PK分析，结果见表5。The B7H3 antibody concentration in serum was detected by ELISA and PK analysis was performed. The results are shown in Table 5.
表5.B7H3抗体在SD大鼠中的T1/2Table 5. T1/2 of B7H3 antibody in SD rats
结果表明，本发明h1702在大鼠体内的半衰期约为185h(7.7天)。The results showed that the half-life of h1702 of the present invention in rats was about 185h (7.7 days).
用ELISA检测血清中的B7H3抗体ADC的浓度，进行PK分析，结果见表6The concentration of B7H3 antibody ADC in serum was detected by ELISA, and PK analysis was performed. The results are shown in Table 6
表6.B7H3抗体偶联物在SD大鼠中的T1/2Table 6. T1/2 of B7H3 antibody conjugates in SD rats
结果表明，大鼠静脉给予3mg/kg受试抗体偶联物h1702-3024，h1702DS-cys-3024，h1704-3-cys-3024后，总ADC(total ADC)在大鼠体内的半衰期约为97h(4.04天)，98h(4.08天)，65h(2.71天)。The results showed that after rats were intravenously administered 3 mg/kg of the test antibody conjugates h1702-3024, h1702DS-cys-3024, and h1704-3-cys-3024, the half-life of the total ADC in rats was approximately 97h (4.04 days), 98h (4.08 days), and 65h (2.71 days).
测试例7.B7H3抗体的物理稳定性Test Example 7. Physical stability of B7H3 antibody
利用DSC检测不同抗体的热稳定性，比较了不同的缓冲体系不同pH条件下的热稳定性情况，不同pH对应的示例性缓冲体系如10mM PB(pH7)，10mM Acetate(pH5.2)。将样品置换到对应缓冲液中，控制样品浓度在1mg/ml左右，利用MicroCal*VP-Capillary DSC(Malvern)进行检测。检测前，将各个样品及空白缓冲液用真空脱气器脱气1～2min。样品板每个孔加入400μl样品或空白缓冲液(仪器上样量为300μl)。最后两对孔板分别加入14％Decon 90和ddH2O，以备清洗用，样品板加样完毕后，套上塑料软盖板。扫描温度从25℃开始到100℃结束，扫描速率60℃/h。具体结果如表7所示，在几个测试体系中h1702、h1703均表现了较好的热稳定性。DSC was used to detect the thermal stability of different antibodies, and the thermal stability of different buffer systems under different pH conditions was compared. Exemplary buffer systems corresponding to different pH values are 10mM PB (pH7) and 10mM Acetate (pH5.2). The samples were replaced in the corresponding buffer, the sample concentration was controlled at about 1mg/ml, and the detection was performed using MicroCal*VP-Capillary DSC (Malvern). Before the detection, each sample and blank buffer were degassed with a vacuum degasser for 1-2min. 400μl of sample or blank buffer was added to each well of the sample plate (the instrument loading volume was 300μl). Finally, 14
表7.不同抗体的DSC实验结果Table 7. DSC experimental results of different antibodies
通过SEC-HPLC监测样品纯度考察一定浓度条件下周期性稳定性，示例性的条件比如将样品浓度控制在约40-50mg/ml，在PBS(pH7.4)体系及pH5.2醋酸/蔗糖体系中比较不同抗体在比如-80℃反复冻融及4℃、30℃、40℃保存一个月的稳定性情况。利用Xbridgeprotein BEH SEC 200A(Waters)HPLC柱子检测抗体纯度，通过一个月的考察，h1702、h1703均表现了良好的稳定性，结果如表8所示。The sample purity was monitored by SEC-HPLC to investigate the periodic stability under certain concentration conditions. For example, the sample concentration was controlled at about 40-50 mg/ml, and the stability of different antibodies was compared in a PBS (pH 7.4) system and a pH 5.2 acetic acid/sucrose system at, for example, -80°C repeated freeze-thaw and 4°C, 30°C, and 40°C for one month. The antibody purity was detected using an Xbridgeprotein BEH SEC 200A (Waters) HPLC column. After one month of investigation, h1702 and h1703 both showed good stability, as shown in Table 8.
表8.不同抗体的周期稳定性结果Table 8. Cycle stability results of different antibodies
结果显示，h1702和h1703在醋酸和PBS缓冲液中均显示出良好的周期稳定性。The results showed that h1702 and h1703 exhibited good cycle stability in both acetic acid and PBS buffers.
测试例8.B7H3抗体的化学稳定性Test Example 8. Chemical stability of B7H3 antibody
抗体制备后化学修饰是导致产品稳定性的常见问题之一，尤其是CDR区域的部分氨基酸高度脱酰胺、氧化或者异构化修饰一般选择尽量避免或者突变降低。取500μg待测抗体溶于500μl pH 7.4的PBS中，40℃水浴；分别于0、10、20天取样，用于酶解实验。将100μg不同时间点取样的样品溶于100μl 0.2M His-HCl，8M Gμa-HCl，pH 6.0溶液中，加3μl 0.1g/mL DTT，50℃水浴1小时，后用0.02M His-HCl，pH 6.0的溶液超滤两次，加入3μL 0.25mg/mL的胰蛋白酶(trypsin)，37℃水浴酶解过夜。Agilent 6530 Q-TOF进行LC-MS检测分析，对潜在的修饰位点进行质谱分析(结果见表9)，结果显示本发明中涉及的h1702、h1703均无明显的脱酰胺、氧化或者异构化加剧趋势，提示分子良好的理化稳定性。Chemical modification after antibody preparation is one of the common problems leading to product stability, especially the highly deamidated, oxidized or isomerized modifications of some amino acids in the CDR region are generally avoided as much as possible or reduced by mutation. Take 500μg of the antibody to be tested and dissolve it in 500μl PBS at pH 7.4, and water bath at 40℃; take samples at 0, 10, and 20 days for enzymatic hydrolysis experiments. Dissolve 100μg of samples taken at different time points in 100μl 0.2M His-HCl, 8M Gμa-HCl, pH 6.0 solution, add 3μl 0.1g/mL DTT, and water bath at 50℃ for 1 hour, then ultrafilter twice with 0.02M His-HCl, pH 6.0 solution, add 3μL 0.25mg/mL trypsin, and enzymolysis overnight at 37℃ water bath. Agilent 6530 Q-TOF was used for LC-MS detection and analysis, and mass spectrometry analysis was performed on potential modification sites (results are shown in Table 9). The results showed that h1702 and h1703 involved in the present invention had no obvious deamidation, oxidation or isomerization aggravation trend, indicating good physical and chemical stability of the molecules.
表9.不同抗体的化学稳定性Table 9. Chemical stability of different antibodies
测试例9：细胞增殖实验Test Example 9: Cell Proliferation Experiment
本实验通过检测细胞内ATP含量，根据IC50大小评价B7H3-ADC对U87MG细胞和ZR-75-1细胞增殖的抑制效果。In this experiment, the intracellular ATP content was detected and the inhibitory effect of B7H3-ADC on the proliferation of U87MG cells and ZR-75-1 cells was evaluated according to the IC50 size.
1、对U87MG细胞增殖的抑制效果1. Inhibitory effect on U87MG cell proliferation
U87MG细胞(中科院细胞库，Catalog#TCHu138)培养在含10％FBS的EMEM培养基中，一周传代2～3次，传代比列1∶2或1∶5。传代时，吸掉培养基，用5ml 0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞。在96孔细胞培养板中加入90μL的细胞悬液，密度为4×104细胞/ml，培养基为10％FBS的DMEM，96孔板外围只加入100μl 10％FBS的DMEM培养基。将培养板在培养箱培养24小时(37℃，5％CO2)。U87MG cells (Cell Bank of Chinese Academy of Sciences, Catalog #TCHu138) were cultured in EMEM medium containing 10% FBS and passaged 2-3 times a week at a passage ratio of 1:2 or 1:5. During passage, the medium was aspirated, the cell layer was rinsed with 5 ml of 0.25% trypsin, then the trypsin was aspirated, the cells were placed in an incubator for digestion for 3-5 minutes, and fresh medium was added to resuspend the cells. 90 μL of cell suspension was added to a 96-well cell culture plate, with a density of 4×104 cells/ml, and the culture medium was DMEM with 10% FBS. Only 100 μL of DMEM with 10% FBS was added to the periphery of the 96-well plate. The culture plate was cultured in an incubator for 24 hours (37°C, 5% CO2 ).
将待测样品用PBS或DMSO稀释成50mM，并以3倍依次稀释成10个浓度，设置成空白和对照的孔。取10μl配制成梯度浓度的化合物溶液加入到90μl新鲜培养基中。再向培养板中加入10μl上述含药物的培养基溶液。将培养板在培养箱孵育3天(37℃，5％CO2)。在96孔细胞培养板中，每孔加入100μl CellTiter-Glo试剂，室温避光放置5-10min，在Victor3中读取化学发光信号值，数据使用GraphPad软件处理。测得的IC50值见表10。The sample to be tested was diluted to 50 mM with PBS or DMSO, and diluted 3 times in sequence to 10 concentrations, and set as blank and control wells. Take 10 μl of the compound solution prepared into gradient concentrations and add it to 90 μl of fresh culture medium. Then add 10 μl of the above-mentioned drug-containing culture medium solution to the culture plate. Incubate the culture plate in an incubator for 3 days (37°C, 5% CO2 ). In a 96-well cell culture plate, add 100 μl of CellTiter-Glo reagent to each well, place it at room temperature away from light for 5-10 minutes, read the chemiluminescent signal value in Victor3, and process the data using GraphPad software. The measured IC50 values are shown in Table 10.
表10.不同ADC在U87MG细胞上的增殖实验结果Table 10. Proliferation test results of different ADCs on U87MG cells
结论：裸抗偶联毒素得到的ADC在U87MG细胞上具有很好的杀伤作用。Conclusion: The ADC obtained by naked antibody-conjugated toxin has a good killing effect on U87MG cells.
2、对ZR-75-1细胞增殖的抑制效果2. Inhibitory effect on ZR-75-1 cell proliferation
ZR-75-1细胞(ATCC，Catalog#CRL-1500)培养在含10％FBS的RPMI-1640培养基中，一周传代2～3次，传代比列1∶4或1∶6。传代时，吸掉培养基，用5mL0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞。在96孔细胞培养板中加入90μL的细胞悬液，密度为2.8×104细胞/mL，96孔板外围只加入100μL10％FBS的RPMI-1640培养基。将培养板在培养箱培养24小时(37℃，5％CO2)。ZR-75-1 cells (ATCC, Catalog #CRL-1500) were cultured in RPMI-1640 medium containing 10% FBS and passaged 2-3 times a week at a passage ratio of 1:4 or 1:6. During passage, the medium was aspirated, the cell layer was rinsed with 5 mL of 0.25% trypsin, and then the trypsin was aspirated. The cells were placed in an incubator for digestion for 3-5 minutes, and fresh medium was added to resuspend the cells. 90 μL of cell suspension was added to a 96-well cell culture plate at a density of 2.8×104 cells/mL, and only 100 μL of RPMI-1640 medium containing 10% FBS was added to the periphery of the 96-well plate. The culture plate was cultured in an incubator for 24 hours (37°C, 5% CO2 ).
将待测样品用PBS或DMSO稀释成50mM，并以3倍依次稀释成10个浓度，设置成空白和对照的孔。取10μl配制成梯度浓度的化合物溶液加入到90μl新鲜培养基中。再向培养板中加入10μl上述含药物的培养基溶液。将培养板在培养箱孵育6天(37℃，5％CO2)。在96孔细胞培养板中，每孔加入100μL CellTiter-Glo试剂，室温避光放置5-10min，在Victor3中读取化学发光信号值，数据使用GraphPad软件处理。测得的IC50值见表11。The sample to be tested was diluted to 50 mM with PBS or DMSO, and diluted 3 times in sequence to 10 concentrations, and set as blank and control wells. Take 10 μl of the compound solution prepared into gradient concentrations and add it to 90 μl of fresh culture medium. Then add 10 μl of the above-mentioned drug-containing culture medium solution to the culture plate. Incubate the culture plate in an incubator for 6 days (37°C, 5% CO2 ). In a 96-well cell culture plate, add 100 μL of CellTiter-Glo reagent to each well, place it at room temperature away from light for 5-10 minutes, read the chemiluminescent signal value in Victor3, and process the data using GraphPad software. The measured IC50 values are shown in Table 11.
表11.不同ADC在ZR-75-1细胞上的增殖实验结果Table 11. Proliferation test results of different ADCs on ZR-75-1 cells
结论：裸抗偶联毒素得到的ADC在ZR-75-1细胞上具有很好的杀伤作用。Conclusion: The ADC obtained by naked antibody-conjugated toxin has a good killing effect on ZR-75-1 cells.
3、对Detroit 562细胞增殖的抑制效果3. Inhibitory effect on Detroit 562 cell proliferation
Detroit 562细胞(ATCC，Catalog#CCL-138)培养在含10％FBS的EMEM培养基中，一周传代2～3次，传代比列1∶4或1∶6。传代时，吸掉培养基，用5mL 0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞。在96孔细胞培养板中加入90μL的细胞悬液，密度为22×104细胞/mL，96孔板外围只加入100μL 10％FBS的EMEM培养基。将培养板在培养箱培养24小时(37℃，5％CO2)。Detroit 562 cells (ATCC, Catalog # CCL-138) were cultured in EMEM medium containing 10% FBS and passaged 2-3 times a week at a passage ratio of 1:4 or 1:6. During passage, the medium was aspirated, the cell layer was rinsed with 5 mL of 0.25% trypsin, and then the trypsin was aspirated. The cells were placed in an incubator for digestion for 3-5 minutes, and fresh medium was added to resuspend the cells. 90 μL of cell suspension was added to a 96-well cell culture plate at a density of 22×104 cells/mL, and only 100 μL of 10% FBS EMEM medium was added to the periphery of the 96-well plate. The culture plate was cultured in an incubator for 24 hours (37°C, 5% CO2 ).
将待测样品用PBS或DMSO稀释成50mM，并以3倍依次稀释成10个浓度，设置成空白和对照的孔。取10μl配制成梯度浓度的化合物溶液加入到90μl新鲜培养基中。再向培养板中加入10μl上述含药物的培养基溶液。将培养板在培养箱孵育6天(37℃，5％CO2)。在96孔细胞培养板中，每孔加入100μL CellTiter-Glo试剂，室温避光放置5-10min，在Victor3中读取化学发光信号值，数据使用GraphPad软件处理。测得的IC50值见表12。The sample to be tested was diluted to 50 mM with PBS or DMSO, and diluted 3 times in sequence to 10 concentrations, and set as blank and control wells. Take 10 μl of the compound solution prepared into gradient concentrations and add it to 90 μl of fresh culture medium. Then add 10 μl of the above-mentioned drug-containing culture medium solution to the culture plate. Incubate the culture plate in an incubator for 6 days (37°C, 5% CO2 ). In a 96-well cell culture plate, add 100 μL of CellTiter-Glo reagent to each well, place it at room temperature away from light for 5-10 minutes, read the chemiluminescent signal value in Victor3, and process the data using GraphPad software. The measured IC50 values are shown in Table 12.
表12.不同ADC在Detroit 562细胞上的增殖实验结果Table 12. Proliferation test results of different ADCs on Detroit 562 cells
结论：裸抗偶联毒素得到的ADC在Detroit 562细胞上具有很好的杀伤作用。Conclusion: The ADC obtained by naked antibody-conjugated toxin has a good killing effect on Detroit 562 cells.
4、以上述方法对h1702DS，h1704-3抗体，及其相应的ADC：h1702-cys-3024，h1702DS-cys-3024，h1704-3-cys-3024测试对U87MG ZR-75-1及Detroit 562细胞的增殖抑制效果，结果如下表13：4. The above method was used to test the h1702DS, h1704-3 antibodies, and their corresponding ADCs: h1702-cys-3024, h1702DS-cys-3024, and h1704-3-cys-3024 for their proliferation inhibition effects on U87MG ZR-75-1 and Detroit 562 cells. The results are shown in Table 13 below:
结论：裸抗偶联毒素得到的ADC在U87MG，ZR-75-1和Detroit562细胞上具有很好的杀伤作用。Conclusion: The ADC obtained by naked antibody-conjugated toxin has a good killing effect on U87MG, ZR-75-1 and Detroit562 cells.
体内活性生物学评价In vivo biological evaluation
测试例10：ADC对人脑星形胶质母细胞瘤U87MG裸小鼠移植瘤的疗效评价Test Example 10: Evaluation of the efficacy of ADC on human brain glioblastoma U87MG nude mouse transplanted tumors
一、测试方法1. Test Method
实验用BALB/cA-nude裸小鼠，雌性，6-7周，购自上海西普尔·必凯实验动物有限责任公司(合格证号：SCXK(沪)2008-0016)。饲养环境：SPF级。裸小鼠皮下接种人脑星形胶质母细胞瘤U87MG细胞(中科院，货号TCHu138)，接种细胞后第十天(肿瘤平均体积122mm3)，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，每周测2-3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：The BALB/cA-nude nude mice used in the experiment, female, 6-7 weeks old, were purchased from Shanghai Xipu·Bikai Experimental Animal Co., Ltd. (certificate number: SCXK (Shanghai) 2008-0016). Housing environment: SPF grade. Nude mice were subcutaneously inoculated with human brain astroglioma U87MG cells (Chinese Academy of Sciences, item number TCHu138). On the tenth day after cell inoculation (average tumor volume 122mm3 ), the animals were randomly divided into groups (D0), 8 in each group, and intraperitoneal injection was started once a week for a total of 3 times. The tumor volume and body weight were measured 2-3 times a week and the data were recorded. The tumor volume (V) was calculated as follows:
V＝1/2×a×b2V＝1/2×a×b2
其中a、b分别表示长、宽。Where a and b represent length and width respectively.
相对体积(RTV)＝VT/V0Relative volume (RTV) = VT/V0
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)Tumor inhibition rate (%) = (CRTV-TRTV)/CRTV (%)
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。CRTV、TRTV分别为实验结束时的对照组(空白)及实验组的相对肿瘤体积。V0 and VT are the tumor volumes at the beginning and end of the experiment, respectively. CRTV and TRTV are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment, respectively.
二、测试对象2. Test Objects
h1702-3024 ADC(1mpk，3mpk，10mpk)；h1702-3024 ADC(1mpk, 3mpk, 10mpk);
h1702DS-cys-3024 ADC(1mpk，3mpk)；h1702DS-cys-3024 ADC(1mpk, 3mpk);
h1704-3-cys-3024 ADC(1mpk，3mpk)；h1704-3-cys-3024 ADC(1mpk, 3mpk);
空白组(空白)：PH7.4的PBS缓冲液Blank group (blank): PBS buffer at pH 7.4
三、抗体ADC的抑瘤效果3. Anti-tumor effect of antibody ADC
1)观察至给药开始后第26天(D26)时，受试抗体h1702-3024ADC(1mpk，3mpk，10mpk)的抑瘤率分别为33.68％，99.13％，100％；见图5，与对照组相比均有显著差异。给药第21天时，对照组由于肿瘤过大，个别鼠体重下降明显，并于给药第26天时，对照组有两只鼠因肿瘤太大死亡。其他各组均体重正常，见图6，表14，给药过程中未出现小鼠死亡，提示h1702-3024各给药剂量没有明显毒副作用。1) The tumor inhibition rates of the tested antibody h1702-3024ADC (1 mpk, 3 mpk, 10 mpk) were observed to be 33.68%, 99.13%, and 100% on the 26th day after the start of administration (D26), respectively; see Figure 5, which are significantly different from the control group. On the 21st day of administration, the weight of some mice in the control group decreased significantly due to the large tumor, and on the 26th day of administration, two mice in the control group died due to the large tumor. The weights of the other groups were normal, see Figure 6, Table 14, and no mice died during the administration, indicating that the h1702-3024 doses had no obvious toxic side effects.
h1702-3024各剂量间有一定的剂量依赖关系，在3mpk的时候已经达到95％以上的抑瘤率，10mpk达到100％的抑瘤率。There is a certain dose-dependency relationship between the different doses of h1702-3024. At 3 mpk, the tumor inhibition rate reached more than 95%, and at 10 mpk, the tumor inhibition rate reached 100%.
表14.给药抗体h1702-3024对荷瘤裸鼠U87MG移植瘤的疗效Table 14. Efficacy of antibody h1702-3024 on U87MG xenografts in nude mice
vs空白：*p＜0.05，**p＜0.01，***p＜0.001vs blank: *p<0.05, **p<0.01, ***p<0.001
2)对受试抗体h1702DS-cys-3024ADC，h1704-3-cys-3024ADC抑瘤效果实验结果见表15。结果显示，腹腔注射给药1次/周，共给药3次，观察至D26时，受试ADC h1702DS-cys-3024(1mpk)的抑瘤率达到22.99％；h1702DS-cys-3024(3mpk)的抑瘤率达到98.16％；h1704-3-cys-3024(1mpk)的抑瘤率达到31.05％；h1704-3-cys-3024(3mpk)的抑瘤率达到94.83％；1mpk的3个受试抗体组与空白组相比都无显著差异(P＞0.05)，3mpk的3个受试抗体组与空白组相比均有极显著差异(P＜0.001)。2) The experimental results of the anti-tumor effects of the tested antibodies h1702DS-cys-3024ADC and h1704-3-cys-3024ADC are shown in Table 15. The results showed that after intraperitoneal injection once a week for a total of 3 times, the tumor inhibition rate of the tested ADC h1702DS-cys-3024 (1 mpk) reached 22.99% on D26; the tumor inhibition rate of h1702DS-cys-3024 (3 mpk) reached 98.16%; the tumor inhibition rate of h1704-3-cys-3024 (1 mpk) reached 31.05%; the tumor inhibition rate of h1704-3-cys-3024 (3 mpk) reached 94.83%; there was no significant difference between the three tested antibody groups at 1 mpk and the blank group (P>0.05), and there were extremely significant differences between the three tested antibody groups at 3 mpk and the blank group (P<0.001).
给药过程中各组动物体重正常，提示ADC无明显毒副作用。The body weights of animals in each group were normal during the administration process, indicating that ADC had no obvious toxic side effects.
空白组和1mpk各组在26天时肿瘤体积较大，因此D26时将空白和1mpk各组处死，其余组观察至D36时，h1702DS-cys-3024(3mpk)组停药后肿瘤生长仍然较慢，抑瘤效果最好。h1702DS-cys-3024(3mpk)和h1704-3-cys-3024(3mpk)在D26和D36时的抑瘤率组间比较无明显差异(P＞0.05)。The tumor volume of the blank group and the 1mpk group was large at 26 days, so the blank group and the 1mpk group were killed at D26, and the other groups were observed until D36. The tumor growth of the h1702DS-cys-3024 (3mpk) group was still slow after drug withdrawal, and the tumor inhibition effect was the best. There was no significant difference in the tumor inhibition rate between the h1702DS-cys-3024 (3mpk) and h1704-3-cys-3024 (3mpk) groups at D26 and D36 (P>0.05).
表15.给药抗体对荷瘤裸鼠U87MG移植瘤的疗效(D26)Table 15. Efficacy of the administered antibodies on U87MG xenografts in nude mice (D26)
vs空白：*p＜0.05，**p＜0.01，***p＜0.001vs blank: *p<0.05, **p<0.01, ***p<0.001
测试例11：ADC对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效评价Test Example 11: Evaluation of the efficacy of ADC on nude mouse transplanted tumors of Detroit 562 cells of human pharyngeal carcinoma pleural effusion metastatic cells
一、测试方法1. Test Method
实验用BALB/cA-nude裸小鼠，雌性，6-7周，皮下接种人咽头癌胸水转移细胞Detroit 562细胞。接种细胞后第十天，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，每周测2-3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：The experiment used BALB/cA-nude nude mice, female, 6-7 weeks old, subcutaneously inoculated with Detroit 562 cells, human pharyngeal carcinoma pleural effusion metastatic cells. On the tenth day after cell inoculation, the animals were randomly divided into groups (D0), 8 in each group, and intraperitoneal injection was started once a week for a total of 3 times. The tumor volume and body weight were measured 2-3 times a week and the data were recorded. The tumor volume (V) was calculated as follows:
V＝1/2×a×b2V＝1/2×a×b2
其中a、b分别表示长、宽。Where a and b represent length and width respectively.
相对体积(RTV)＝VT/V0Relative volume (RTV) = VT/V0
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)Tumor inhibition rate (%) = (CRTV-TRTV)/CRTV (%)
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。CRTV、TRTV分别为实验结束时的对照组(空白)及实验组的相对肿瘤体积。V0 and VT are the tumor volumes at the beginning and end of the experiment, respectively. CRTV and TRTV are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment, respectively.
二、测试对象2. Test Objects
h1702DS-cys-3024 ADC(1mpk，3mpk)；h1702DS-cys-3024 ADC(1mpk, 3mpk);
空白组(空白)：PH7.4的PBS缓冲液Blank group (blank): PBS buffer at pH 7.4
三、抗体ADC的抑瘤效果3. Anti-tumor effect of antibody ADC
观察至D35时，受试抗体ADC抑瘤率分别是：h1702DS-cys-3024 ADC(1mpk)的抑瘤率达到39.22％(P＜0.05)；h1702DS-cys-3024 ADC(3mpk)的抑瘤率达到70.50％(P＜0.001)(见表16)。药过程中各组动物体重正常。At D35, the tumor inhibition rates of the tested antibody ADCs were: h1702DS-cys-3024 ADC (1 mpk) reached 39.22% (P < 0.05); h1702DS-cys-3024 ADC (3 mpk) reached 70.50% (P < 0.001) (see Table 16). The body weights of the animals in each group were normal during the medication process.
表16.给药抗体对荷瘤裸鼠Detroit 562移植瘤的疗效Table 16. Efficacy of the administered antibodies on Detroit 562 xenografts in nude mice
vs空白：*p＜0.05，***p＜0.001vs blank: *p<0.05, ***p<0.001
稳定性评价Stability evaluation
测试例12：B7H3 ADC的物理稳定性Test Example 12: Physical Stability of B7H3 ADC
利用DSC检测不同抗体ADC的热稳定性，比较了不同的缓冲体系不同pH条件下的热稳定性情况，不同pH对应的示例性缓冲体系如10mM PBS(pH7.4)，10mM Acetate(pH5.5)。将样品置换到对应缓冲液中，控制样品浓度在1mg/ml左右，利用MicroCal*VP-Capillary DSC(Malvern)进行检测。检测前，将各个样品及空白缓冲液用真空脱气器脱气1～2min。样品板每个孔加入400μl样品或空白缓冲液(仪器上样量为300μl)。最后两对孔板分别加入14％Decon 90和ddH2O，以备清洗用，样品板加样完毕后，套上塑料软盖板。扫描温度从25℃开始到100℃结束，扫描速率60℃/h。具体结果如表17所示，在几个测试体系中h1702-3024、h1703-3024均表现了较好的热稳定性。DSC was used to detect the thermal stability of different antibody ADCs, and the thermal stability of different buffer systems under different pH conditions was compared. Exemplary buffer systems corresponding to different pH values are 10mM PBS (pH7.4) and 10mM Acetate (pH5.5). The samples were replaced in the corresponding buffer, the sample concentration was controlled at about 1mg/ml, and the samples were detected using MicroCal*VP-Capillary DSC (Malvern). Before the test, each sample and blank buffer were degassed with a vacuum degasser for 1-2min. 400μl of sample or blank buffer was added to each well of the sample plate (the instrument loading volume was 300μl). Finally, 14
表17.不同ADC的DSC实验结果Table 17. DSC experimental results of different ADCs
测试例13：ADC的稳定性Test Example 13: ADC Stability
通过SEC-HPLC监测样品(h1702DS-cys-3024 ADC，h1704-3-cys-3024 ADC)纯度考察一定浓度条件下稳定性，示例性的条件比如将样品浓度控制在约50mg/ml，在PBS(pH7.4)体系及pH5.5醋酸/蔗糖体系(代称559体系)中比较不同抗体在40℃保存28天的稳定性情况。利用Xbridge protein BEH SEC 200A(Waters)HPLC柱子检测抗体纯度，通过28天的考察，h1702DS-cys-3024 ADC表现了良好的稳定性，结果如图7所示。The purity of samples (h1702DS-cys-3024 ADC, h1704-3-cys-3024 ADC) was monitored by SEC-HPLC to examine the stability under certain concentration conditions. For example, the sample concentration was controlled at about 50 mg/ml, and the stability of different antibodies stored at 40°C for 28 days was compared in PBS (pH 7.4) system and pH 5.5 acetic acid/sucrose system (referred to as 559 system). The antibody purity was detected using Xbridge protein BEH SEC 200A (Waters) HPLC column. After 28 days of investigation, h1702DS-cys-3024 ADC showed good stability, as shown in Figure 7.
结果显示，h1702DS-cys-3024在醋酸和PBS缓冲液中均显示出良好的稳定性。The results showed that h1702DS-cys-3024 exhibited good stability in both acetic acid and PBS buffers.
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本发明的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。Although the above invention has been described in detail with the aid of drawings and examples for clear understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.
序列表Sequence Listing
<110> 江苏恒瑞医药股份有限公司、上海恒瑞医药有限公司<110> Jiangsu Hengrui Medicine Co., Ltd., Shanghai Hengrui Medicine Co., Ltd.
<120> B7H3抗体-药物偶联物及其医药用途<120> B7H3 antibody-drug conjugates and their medical uses
<130> 780060CPCN<130> 780060CPCN
<160> 41<160> 41
<170> PatentIn version 3.3<170> PatentIn version 3.3
<210> 1<210> 1
<211> 534<211> 534
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 人B7H3全长氨基酸序列<223> Full-length amino acid sequence of human B7H3
<400> 1<400> 1
Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly AlaMet Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala
1 5 10 151 5 10 15
Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val GlnAla Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln
20 25 3020 25 30
Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr LeuVal Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu
35 40 4535 40 45
Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu AsnCys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn
50 55 6050 55 60
Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe AlaLeu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala
65 70 75 8065 70 75 80
Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu PheGlu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe
85 90 9585 90 95
Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg ValPro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val
100 105 110100 105 110
Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg AspArg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp
115 120 125115 120 125
Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser LysPhe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys
130 135 140130 135 140
Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp ThrPro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr
145 150 155 160145 150 155 160
Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu ValVal Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val
165 170 175165 170 175
Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr ThrPhe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr
180 185 190180 185 190
Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile LeuSer Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu
195 200 205195 200 205
Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg AsnArg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn
210 215 220210 215 220
Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro GlnPro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln
225 230 235 240225 230 235 240
Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro ValArg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val
245 250 255245 250 255
Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser ProVal Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro
260 265 270260 265 270
Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu ThrGlu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr
275 280 285275 280 285
Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln GlyAsp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly
290 295 300290 295 300
Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala GlnSer Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln
305 310 315 320305 310 315 320
Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu GlyGly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly
325 330 335325 330 335
Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala ValSer Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val
340 345 350340 345 350
Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu GluSer Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu
355 360 365355 360 365
Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys SerPro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser
370 375 380370 375 380
Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly GlnSer Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln
385 390 395 400385 390 395 400
Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn GluGly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu
405 410 415405 410 415
Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly AlaGln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala
420 425 430420 425 430
Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln AspAsn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp
435 440 445435 440 445
Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro ProAla His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro
450 455 460450 455 460
Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala LeuGlu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu
465 470 475 480465 470 475 480
Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser CysLeu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys
485 490 495485 490 495
Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu GlyGlu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly
500 505 510500 505 510
Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu AspSer Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp
515 520 525515 520 525
Asp Gly Gln Glu Ile AlaAsp Gly Gln Glu Ile Ala
530530
<210> 2<210> 2
<211> 316<211> 316
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鼠B7H3全长氨基酸序列<223> Full-length amino acid sequence of mouse B7H3
<400> 2<400> 2
Met Leu Arg Gly Trp Gly Gly Pro Ser Val Gly Val Cys Val Arg ThrMet Leu Arg Gly Trp Gly Gly Pro Ser Val Gly Val Cys Val Arg Thr
1 5 10 151 5 10 15
Ala Leu Gly Val Leu Cys Leu Cys Leu Thr Gly Ala Val Glu Val GlnAla Leu Gly Val Leu Cys Leu Cys Leu Thr Gly Ala Val Glu Val Gln
20 25 3020 25 30
Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr Asp Ala Thr LeuVal Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr Asp Ala Thr Leu
35 40 4535 40 45
Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu AsnArg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn
50 55 6050 55 60
Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe ThrLeu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr
65 70 75 8065 70 75 80
Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn Arg Thr Ala Leu PheGlu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn Arg Thr Ala Leu Phe
85 90 9585 90 95
Pro Asp Leu Leu Val Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg ValPro Asp Leu Leu Val Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val
100 105 110100 105 110
Arg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val Ser Ile Gln AspArg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val Ser Ile Gln Asp
115 120 125115 120 125
Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser LysPhe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys
130 135 140130 135 140
Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asn MetPro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asn Met
145 150 155 160145 150 155 160
Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu ValVal Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val
165 170 175165 170 175
Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr ThrPhe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr
180 185 190180 185 190
Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val His Ser Val LeuSer Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val His Ser Val Leu
195 200 205195 200 205
Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg AsnArg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn
210 215 220210 215 220
Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly GlnPro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln
225 230 235 240225 230 235 240
Pro Leu Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu SerPro Leu Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser
245 250 255245 250 255
Val Cys Leu Val Val Leu Leu Val Ala Leu Ala Phe Val Cys Trp ArgVal Cys Leu Val Val Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg
260 265 270260 265 270
Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp GlnLys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln
275 280 285275 280 285
Asp Gly Asp Gly Glu Gly Ser Lys Thr Ala Leu Arg Pro Leu Lys ProAsp Gly Asp Gly Glu Gly Ser Lys Thr Ala Leu Arg Pro Leu Lys Pro
290 295 300290 295 300
Ser Glu Asn Lys Glu Asp Asp Gly Gln Glu Ile AlaSer Glu Asn Lys Glu Asp Asp Gly Gln Glu Ile Ala
305 310 315305 310 315
<210> 3<210> 3
<211> 223<211> 223
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 人B7H3抗原：2Ig-B7H3<223> Human B7H3 antigen: 2Ig-B7H3
<400> 3<400> 3
Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly ThrLeu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr
1 5 10 151 5 10 15
Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser LeuAsp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu
20 25 3020 25 30
Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu ValAla Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val
35 40 4535 40 45
His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn ArgHis Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg
50 55 6050 55 60
Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu ArgThr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg
65 70 75 8065 70 75 80
Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe ValLeu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val
85 90 9585 90 95
Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala AlaSer Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala
100 105 110100 105 110
Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu ArgPro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg
115 120 125115 120 125
Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr ProPro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro
130 135 140130 135 140
Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr GlyGlu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly
145 150 155 160145 150 155 160
Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp ValAsn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val
165 170 175165 170 175
His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser CysHis Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys
180 185 190180 185 190
Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val ThrLeu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr
195 200 205195 200 205
Ile Thr Pro Gln Arg Ser Pro Thr Gly His His His His His HisIle Thr Pro Gln Arg Ser Pro Thr Gly His His His His His
210 215 220210 215 220
<210> 4<210> 4
<211> 439<211> 439
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 人B7H3抗原：4Ig-B7H3<223> Human B7H3 antigen: 4Ig-B7H3
<400> 4<400> 4
Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly ThrLeu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr
1 5 10 151 5 10 15
Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser LeuAsp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu
20 25 3020 25 30
Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu ValAla Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val
35 40 4535 40 45
His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn ArgHis Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg
50 55 6050 55 60
Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu ArgThr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg
65 70 75 8065 70 75 80
Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe ValLeu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val
85 90 9585 90 95
Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala AlaSer Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala
100 105 110100 105 110
Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu ArgPro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg
115 120 125115 120 125
Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr ProPro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro
130 135 140130 135 140
Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr GlyGlu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly
145 150 155 160145 150 155 160
Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp ValAsn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val
165 170 175165 170 175
His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser CysHis Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys
180 185 190180 185 190
Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val ThrLeu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr
195 200 205195 200 205
Ile Thr Pro Gln Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val ProIle Thr Pro Gln Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro
210 215 220210 215 220
Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg CysGlu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys
225 230 235 240225 230 235 240
Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu IleSer Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile
245 250 255245 250 255
Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu GlyTrp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly
260 265 270260 265 270
Arg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro AspArg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp
275 280 285275 280 285
Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg ValLeu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val
290 295 300290 295 300
Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe GlyAla Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly
305 310 315 320305 310 315 320
Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro SerSer Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser
325 330 335325 330 335
Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val ThrMet Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr
340 345 350340 345 350
Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe TrpIle Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp
355 360 365355 360 365
Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser GlnGln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln
370 375 380370 375 380
Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu Arg ValMet Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val
385 390 395 400385 390 395 400
Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro ValVal Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val
405 410 415405 410 415
Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro MetLeu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met
420 425 430420 425 430
Thr His His His His His HisThr His His His His His
435435
<210> 5<210> 5
<211> 222<211> 222
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鼠B7H3抗原<223> Mouse B7H3 antigen
<400> 5<400> 5
Val Glu Val Gln Val Ser Glu Asp Pro Val Val Ala Leu Val Asp ThrVal Glu Val Gln Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr
1 5 10 151 5 10 15
Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser LeuAsp Ala Thr Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu
20 25 3020 25 30
Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu ValAla Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val
35 40 4535 40 45
His Ser Phe Thr Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn ArgHis Ser Phe Thr Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn Arg
50 55 6050 55 60
Thr Ala Leu Phe Pro Asp Leu Leu Val Gln Gly Asn Ala Ser Leu ArgThr Ala Leu Phe Pro Asp Leu Leu Val Gln Gly Asn Ala Ser Leu Arg
65 70 75 8065 70 75 80
Leu Gln Arg Val Arg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe ValLeu Gln Arg Val Arg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val
85 90 9585 90 95
Ser Ile Gln Asp Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala AlaSer Ile Gln Asp Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala
100 105 110100 105 110
Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu ArgPro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg
115 120 125115 120 125
Pro Gly Asn Met Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr ProPro Gly Asn Met Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro
130 135 140130 135 140
Glu Ala Glu Val Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr GlyGlu Ala Glu Val Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly
145 150 155 160145 150 155 160
Asn Val Thr Thr Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp ValAsn Val Thr Thr Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val
165 170 175165 170 175
His Ser Val Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser CysHis Ser Val Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys
180 185 190180 185 190
Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Gly Ser Val ThrLeu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr
195 200 205195 200 205
Ile Thr Gly Gln Pro Leu Thr Phe His His His His His HisIle Thr Gly Gln Pro Leu Thr Phe His His His His His
210 215 220210 215 220
<210> 6<210> 6
<211> 119<211> 119
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702重链可变区<223> h1702 heavy chain variable region
<400> 6<400> 6
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly ThrGln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser SerSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser
20 25 3020 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValAla Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 4535 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser ValAla Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val
50 55 6050 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln GlyAla Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly
100 105 110100 105 110
Ala Leu Val Thr Val Ser SerAla Leu Val Thr Val Ser Ser
115115
<210> 7<210> 7
<211> 110<211> 110
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702轻链可变区<223> h1702 light chain variable region
<400> 7<400> 7
Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly GlyGln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly
1 5 10 151 5 10 15
Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr SerThr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser
20 25 3020 25 30
His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg MetHis Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met
35 40 4535 40 45
Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg PheLeu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe
50 55 6050 55 60
Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly AlaSer Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 8065 70 75 80
Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp ArgGln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg
85 90 9585 90 95
Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val LeuAsp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110100 105 110
<210> 8<210> 8
<211> 119<211> 119
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703重链可变区<223> h1703 heavy chain variable region
<400> 8<400> 8
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlyGln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 3020 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 4535 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser ValSer Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val
50 55 6050 55 60
Lys Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Lys Gly Val Gly Pro Val His Ala Leu Asp Val Trp Gly Gln GlyAla Lys Gly Val Gly Pro Val His Ala Leu Asp Val Trp Gly Gln Gly
100 105 110100 105 110
Thr Thr Val Thr Val Ser SerThr Thr Val Thr Val Ser Ser
115115
<210> 9<210> 9
<211> 108<211> 108
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703轻链可变区<223> h1703 light chain variable region
<400> 9<400> 9
Asp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr TyrAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr
20 25 3020 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu IleLeu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu Ile
35 40 4535 40 45
Asn Ala Val Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser GlyAsn Ala Val Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln ProSer Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro MetGlu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Met
85 90 9585 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile LysTrp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105100 105
<210> 10<210> 10
<211> 8<211> 8
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702 HCDR1<223> h1702 HCDR1
<400> 10<400> 10
Gly Phe Ile Phe Ser Ser Ser AlaGly Phe Ile Phe Ser Ser Ser Ala
1 51 5
<210> 11<210> 11
<211> 8<211> 8
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702 HCDR2<223> h1702 HCDR2
<400> 11<400> 11
Ile Ser Tyr Asp Gly Ser Asn LysIle Ser Tyr Asp Gly Ser Asn Lys
1 51 5
<210> 12<210> 12
<211> 12<211> 12
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702 HCDR3<223> h1702 HCDR3
<400> 12<400> 12
Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp TyrAla Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr
1 5 101 5 10
<210> 13<210> 13
<211> 9<211> 9
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702 LCDR1<223> h1702 LCDR1
<400> 13<400> 13
Ser Gly Ser Val Ser Thr Ser His TyrSer Gly Ser Val Ser Thr Ser His Tyr
1 51 5
<210> 14<210> 14
<211> 3<211> 3
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702 LCDR2<223> h1702 LCDR2
<400> 14<400> 14
Asn Thr AsnAsn Thr Asn
11
<210> 15<210> 15
<211> 10<211> 10
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702 LCDR3<223> h1702 LCDR3
<400> 15<400> 15
Ala Ile His Val Asp Arg Asp Ile Trp ValAla Ile His Val Asp Arg Asp Ile Trp Val
1 5 101 5 10
<210> 16<210> 16
<211> 8<211> 8
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703 HCDR1<223> h1703 HCDR1
<400> 16<400> 16
Gly Phe Thr Phe Ser Ser Tyr AlaGly Phe Thr Phe Ser Ser Tyr Ala
1 51 5
<210> 17<210> 17
<211> 8<211> 8
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703 HCDR2<223> h1703 HCDR2
<400> 17<400> 17
Ile Ser Gly Ser Gly Gly Ser ThrIle Ser Gly Ser Gly Gly Ser Thr
1 51 5
<210> 18<210> 18
<211> 12<211> 12
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703 HCDR3<223> h1703 HCDR3
<400> 18<400> 18
Ala Lys Gly Val Gly Pro Val His Ala Leu Asp ValAla Lys Gly Val Gly Pro Val His Ala Leu Asp Val
1 5 101 5 10
<210> 19<210> 19
<211> 6<211> 6
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703 LCDR1<223> h1703 LCDR1
<400> 19<400> 19
Gln Ser Ile Ser Thr TyrGln Ser Ile Ser Thr Tyr
1 51 5
<210> 20<210> 20
<211> 3<211> 3
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703 LCDR2<223> h1703 LCDR2
<400> 20<400> 20
Ala Val SerAla Val Ser
11
<210> 21<210> 21
<211> 10<211> 10
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703 LCDR3<223> h1703 LCDR3
<400> 21<400> 21
Gln Gln Ser Tyr Ser Thr Pro Met Trp ThrGln Gln Ser Tyr Ser Thr Pro Met Trp Thr
1 5 101 5 10
<210> 22<210> 22
<211> 449<211> 449
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702 重链<223> h1702 heavy chain
<400> 22<400> 22
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly ThrGln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser SerSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser
20 25 3020 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValAla Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 4535 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser ValAla Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val
50 55 6050 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln GlyAla Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly
100 105 110100 105 110
Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val PheAla Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala LeuPro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser TrpGly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val LeuAsn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro SerGln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys ProSer Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp LysSer Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly ProThr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu AspArg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His AsnPro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg ValAla Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys GluVal Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu LysTyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr ThrThr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu ThrLeu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp GluCys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuSer Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp LysAsp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His GluSer Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyAla Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445435 440 445
LysLys
<210> 23<210> 23
<211> 216<211> 216
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702轻链<223> h1702 light chain
<400> 23<400> 23
Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly GlyGln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly
1 5 10 151 5 10 15
Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr SerThr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser
20 25 3020 25 30
His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg MetHis Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met
35 40 4535 40 45
Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg PheLeu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe
50 55 6050 55 60
Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly AlaSer Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 8065 70 75 80
Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp ArgGln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg
85 90 9585 90 95
Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly GlnAsp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110100 105 110
Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu GluPro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe TyrLeu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val LysPro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys
145 150 155 160145 150 155 160
Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys TyrAla Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser HisAla Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu LysArg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205195 200 205
Thr Val Ala Pro Thr Glu Cys SerThr Val Ala Pro Thr Glu Cys Ser
210 215210 215
<210> 24<210> 24
<211> 449<211> 449
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703 重链<223> h1703 heavy chain
<400> 24<400> 24
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlyGln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 3020 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 4535 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser ValSer Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val
50 55 6050 55 60
Lys Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Lys Gly Val Gly Pro Val His Ala Leu Asp Val Trp Gly Gln GlyAla Lys Gly Val Gly Pro Val His Ala Leu Asp Val Trp Gly Gln Gly
100 105 110100 105 110
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val PheThr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala LeuPro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser TrpGly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val LeuAsn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro SerGln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys ProSer Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp LysSer Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly ProThr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu AspArg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His AsnPro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg ValAla Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys GluVal Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu LysTyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr ThrThr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu ThrLeu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp GluCys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuSer Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp LysAsp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His GluSer Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyAla Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445435 440 445
LysLys
<210> 25<210> 25
<211> 215<211> 215
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1703轻链<223> h1703 light chain
<400> 25<400> 25
Asp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr TyrAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr
20 25 3020 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu IleLeu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu Ile
35 40 4535 40 45
Asn Ala Val Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser GlyAsn Ala Val Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln ProSer Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro MetGlu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Met
85 90 9585 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val AlaTrp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys SerAla Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg GluGly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerAla Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser LeuGln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys ValSer Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr LysTyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205195 200 205
Ser Phe Asn Arg Gly Glu CysSer Phe Asn Arg Gly Glu Cys
210 215210 215
<210> 26<210> 26
<211> 215<211> 215
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1702-DS 轻链序列<223> h1702-DS light chain sequence
<400> 26<400> 26
Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly GlyAsp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly
1 5 10 151 5 10 15
Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr SerThr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser
20 25 3020 25 30
His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg MetHis Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met
35 40 4535 40 45
Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg PheLeu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe
50 55 6050 55 60
Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly AlaSer Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 8065 70 75 80
Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp ArgGln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg
85 90 9585 90 95
Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly GlnAsp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110100 105 110
Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu GluPro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe TyrLeu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val LysPro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys
145 150 155 160145 150 155 160
Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys TyrAla Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser HisAla Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu LysArg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205195 200 205
Thr Val Ala Pro Thr Glu CysThr Val Ala Pro Thr Glu Cys
210 215210 215
<210> 27<210> 27
<211> 666<211> 666
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗原h-B7H3-FC<223> Antigen h-B7H3-FC
<400> 27<400> 27
Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly ThrLeu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr
1 5 10 151 5 10 15
Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser LeuAsp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu
20 25 3020 25 30
Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu ValAla Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val
35 40 4535 40 45
His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn ArgHis Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg
50 55 6050 55 60
Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu ArgThr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg
65 70 75 8065 70 75 80
Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe ValLeu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val
85 90 9585 90 95
Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala AlaSer Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala
100 105 110100 105 110
Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu ArgPro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg
115 120 125115 120 125
Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr ProPro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro
130 135 140130 135 140
Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr GlyGlu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly
145 150 155 160145 150 155 160
Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp ValAsn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val
165 170 175165 170 175
His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser CysHis Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys
180 185 190180 185 190
Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val ThrLeu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr
195 200 205195 200 205
Ile Thr Pro Gln Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val ProIle Thr Pro Gln Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro
210 215 220210 215 220
Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg CysGlu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys
225 230 235 240225 230 235 240
Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu IleSer Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile
245 250 255245 250 255
Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu GlyTrp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly
260 265 270260 265 270
Arg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro AspArg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp
275 280 285275 280 285
Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg ValLeu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val
290 295 300290 295 300
Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe GlyAla Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly
305 310 315 320305 310 315 320
Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro SerSer Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser
325 330 335325 330 335
Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val ThrMet Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr
340 345 350340 345 350
Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe TrpIle Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp
355 360 365355 360 365
Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser GlnGln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln
370 375 380370 375 380
Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu Arg ValMet Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val
385 390 395 400385 390 395 400
Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro ValVal Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val
405 410 415405 410 415
Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro MetLeu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met
420 425 430420 425 430
Thr Gly Ser Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro CysThr Gly Ser Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys
435 440 445435 440 445
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro ProPro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
450 455 460450 455 460
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr CysLys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
465 470 475 480465 470 475 480
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn TrpVal Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
485 490 495485 490 495
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg GluTyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
500 505 510500 505 510
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val LeuGlu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
515 520 525515 520 525
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser AsnHis Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
530 535 540530 535 540
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyLys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
545 550 555 560545 550 555 560
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp GluGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
565 570 575565 570 575
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe TyrLeu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
580 585 590580 585 590
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu AsnPro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
595 600 605595 600 605
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe PheAsn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
610 615 620610 615 620
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly AsnLeu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
625 630 635 640625 630 635 640
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr ThrVal Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
645 650 655645 650 655
Gln Lys Ser Leu Ser Leu Ser Pro Gly LysGln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665660 665
<210> 28<210> 28
<211> 118<211> 118
<212> PRT<212> PRT
<213> Murine adenovirus<213> Murine adenovirus
<400> 28<400> 28
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Ser Gly GlyGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Ser Gly Gly
1 5 10 151 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg TyrSer Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 3020 25 30
Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp ValGly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 4535 40 45
Ala Ala Ile Ser Ser Asn Gly Gly Ser Ile Tyr Tyr Pro Asp Thr ValAla Ala Ile Ser Ser Asn Gly Gly Ser Ile Tyr Tyr Pro Asp Thr Val
50 55 6050 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu TyrLys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Ile Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Phe CysLeu Gln Met Ile Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Phe Cys
85 90 9585 90 95
Thr Arg His Tyr Leu Leu Phe Glu Met Asp Tyr Trp Gly Arg Gly ThrThr Arg His Tyr Leu Leu Phe Glu Met Asp Tyr Trp Gly Arg Gly Thr
100 105 110100 105 110
Ser Val Thr Val Ser SerSer Val Thr Val Ser Ser
115115
<210> 29<210> 29
<211> 106<211> 106
<212> PRT<212> PRT
<213> Murine adenovirus<213> Murine adenovirus
<400> 29<400> 29
Asp Ile Val Met Thr Gln Ser Gln Glu Phe Met Ser Thr Thr Val GlyAsp Ile Val Met Thr Gln Ser Gln Glu Phe Met Ser Thr Thr Val Gly
1 5 10 151 5 10 15
Asp Arg Val Tyr Ile Thr Cys Lys Ala Ser Gln Asn Val Asn Thr AlaAsp Arg Val Tyr Ile Thr Cys Lys Ala Ser Gln Asn Val Asn Thr Ala
20 25 3020 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu IleVal Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 4535 40 45
Phe Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr GlyPhe Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln SerSer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 8065 70 75 80
Glu Asp Leu Ala Ala Tyr Phe Cys Gln Gln Tyr Ser Ser Ser Leu ThrGlu Asp Leu Ala Ala Tyr Phe Cys Gln Gln Tyr Ser Ser Ser Leu Thr
85 90 9585 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu LysPhe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105100 105
<210> 30<210> 30
<211> 5<211> 5
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 1704-HCDR1<223> 1704-HCDR1
<400> 30<400> 30
Arg Tyr Gly Met SerArg Tyr Gly Met Ser
1 51 5
<210> 31<210> 31
<211> 16<211> 16
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 1704-HCDR2<223> 1704-HCDR2
<400> 31<400> 31
Ile Ser Ser Gly Gly Gly Ser Ile Tyr Tyr Pro Asp Thr Val Lys GlyIle Ser Ser Gly Gly Gly Ser Ile Tyr Tyr Pro Asp Thr Val Lys Gly
1 5 10 151 5 10 15
<210> 32<210> 32
<211> 11<211> 11
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 1704-HCDR3<223> 1704-HCDR3
<400> 32<400> 32
Thr Arg His Tyr Leu Leu Phe Glu Met Asp TyrThr Arg His Tyr Leu Leu Phe Glu Met Asp Tyr
1 5 101 5 10
<210> 33<210> 33
<211> 11<211> 11
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 1704-LCDR1<223> 1704-LCDR1
<400> 33<400> 33
Lys Ala Ser Gln Asn Val Asn Thr Ala Val AlaLys Ala Ser Gln Asn Val Asn Thr Ala Val Ala
1 5 101 5 10
<210> 34<210> 34
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 1704-LCDR2<223> 1704-LCDR2
<400> 34<400> 34
Ser Ala Ser Asn Arg Tyr ThrSer Ala Ser Asn Arg Tyr Thr
1 51 5
<210> 35<210> 35
<211> 8<211> 8
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 1704-LCDR3<223> 1704-LCDR3
<400> 35<400> 35
Gln Gln Tyr Ser Ser Ser Leu ThrGln Gln Tyr Ser Ser Ser Leu Thr
1 51 5
<210> 36<210> 36
<211> 118<211> 118
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1704VH1<223> h1704VH1
<400> 36<400> 36
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly GlyGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 3020 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 4535 40 45
Ser Ala Ile Ser Ser Gly Gly Gly Ser Ile Tyr Tyr Pro Asp Thr ValSer Ala Ile Ser Ser Gly Gly Gly Ser Ile Tyr Tyr Pro Asp Thr Val
50 55 6050 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu TyrLys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 9585 90 95
Thr Arg His Tyr Leu Leu Phe Glu Met Asp Tyr Trp Gly Gln Gly ThrThr Arg His Tyr Leu Leu Phe Glu Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110100 105 110
Thr Val Thr Val Ser SerThr Val Thr Val Ser Ser
115115
<210> 37<210> 37
<211> 118<211> 118
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1704VH2<223> h1704VH2
<400> 37<400> 37
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlyGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 3020 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 4535 40 45
Ala Ala Ile Ser Ser Gly Gly Gly Ser Ile Tyr Tyr Pro Asp Thr ValAla Ala Ile Ser Ser Gly Gly Gly Ser Ile Tyr Tyr Pro Asp Thr Val
50 55 6050 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 9585 90 95
Thr Arg His Tyr Leu Leu Phe Glu Met Asp Tyr Trp Gly Gln Gly ThrThr Arg His Tyr Leu Leu Phe Glu Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110100 105 110
Thr Val Thr Val Ser SerThr Val Thr Val Ser Ser
115115
<210> 38<210> 38
<211> 106<211> 106
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1704VL1<223> h1704VL1
<400> 38<400> 38
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asn Thr AlaAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asn Thr Ala
20 25 3020 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleVal Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 4535 40 45
Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser GlyTyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Ser Ser Leu ThrGlu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Ser Ser Leu Thr
85 90 9585 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile LysPhe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105100 105
<210> 39<210> 39
<211> 106<211> 106
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1704VL2<223> h1704VL2
<400> 39<400> 39
Asp Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val GlyAsp Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asn Thr AlaAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asn Thr Ala
20 25 3020 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleVal Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 4535 40 45
Phe Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser GlyPhe Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Val Ala Ala Tyr Phe Cys Gln Gln Tyr Ser Ser Ser Leu ThrGlu Asp Val Ala Ala Tyr Phe Cys Gln Gln Tyr Ser Ser Ser Leu Thr
85 90 9585 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile LysPhe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105100 105
<210> 40<210> 40
<211> 448<211> 448
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1704-3抗体重链序列<223> h1704-3 antibody heavy chain sequence
<400> 40<400> 40
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly GlyGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 3020 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 4535 40 45
Ser Ala Ile Ser Ser Gly Gly Gly Ser Ile Tyr Tyr Pro Asp Thr ValSer Ala Ile Ser Ser Gly Gly Gly Ser Ile Tyr Tyr Pro Asp Thr Val
50 55 6050 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu TyrLys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 9585 90 95
Thr Arg His Tyr Leu Leu Phe Glu Met Asp Tyr Trp Gly Gln Gly ThrThr Arg His Tyr Leu Leu Phe Glu Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110100 105 110
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe ProThr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu GlyLeu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp AsnCys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu GlnSer Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser SerSer Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro SerSer Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys ThrAsn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro SerHis Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp ProThr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaGlu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val ValLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu TyrSer Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys ThrLys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr LeuIle Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr CysPro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu AspAsn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys SerSer Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu AlaArg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly LysLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445435 440 445
<210> 41<210> 41
<211> 213<211> 213
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> h1704-3抗体轻链序列<223> h1704-3 antibody light chain sequence
<400> 41<400> 41
Asp Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val GlyAsp Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asn Thr AlaAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asn Thr Ala
20 25 3020 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleVal Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 4535 40 45
Phe Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser GlyPhe Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Val Ala Ala Tyr Phe Cys Gln Gln Tyr Ser Ser Ser Leu ThrGlu Asp Val Ala Ala Tyr Phe Cys Gln Gln Tyr Ser Ser Ser Leu Thr
85 90 9585 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala ProPhe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly ThrSer Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala LysAla Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln GluVal Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser SerSer Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr AlaThr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser PheCys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205195 200 205
Asn Arg Gly Glu CysAsn Arg Gly Glu Cys
210210
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710660432 | 2017-08-04 | ||
| CN2017106604329 | 2017-08-04 | ||
| PCT/CN2018/098480WO2019024911A1 (en) | 2017-08-04 | 2018-08-03 | B7h3 antibody-drug conjugate and medical use thereof |
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| CN109963591A CN109963591A (en) | 2019-07-02 |
| CN109963591Btrue CN109963591B (en) | 2023-04-04 |
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| CN201880004425.6AActiveCN109963591B (en) | 2017-08-04 | 2018-08-03 | B7H3 antibody-drug conjugate and medical application thereof |
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| Country | Link |
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| CN (1) | CN109963591B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018161872A1 (en)* | 2017-03-06 | 2018-09-13 | 江苏恒瑞医药股份有限公司 | Anti-b7-h3 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof |
| KR20210068457A (en)* | 2018-09-30 | 2021-06-09 | 지앙수 헨그루이 메디슨 컴퍼니 리미티드 | Anti-B7H3 antibody-exatecan analog conjugates and pharmaceutical uses thereof |
| WO2021190480A1 (en)* | 2020-03-24 | 2021-09-30 | 上海翰森生物医药科技有限公司 | Antibody-drug conjugate and medical use thereof |
| TWI885105B (en)* | 2020-03-25 | 2025-06-01 | 大陸商江蘇恆瑞醫藥股份有限公司 | Pharmaceutical composition comprising an antibody drug conjugate, and use thereof |
| WO2021190586A1 (en)* | 2020-03-25 | 2021-09-30 | 江苏恒瑞医药股份有限公司 | B7h3 antibody-exatecan analogue conjugate and pharmaceutical use thereof |
| CN115279781B (en)* | 2020-03-26 | 2025-05-09 | 上海翰森生物医药科技有限公司 | Antibody drug conjugates and their medical uses |
| WO2022104692A1 (en)* | 2020-11-20 | 2022-05-27 | Bliss Biopharmaceutical (Hangzhou) Co., Ltd. | Engineered antibody, antibody-drug conjugate, and use thereof |
| CN117098548A (en)* | 2020-12-23 | 2023-11-21 | 信达生物制药(苏州)有限公司 | anti-B7-H3 antibodies and uses thereof |
| IL305847A (en) | 2021-03-26 | 2023-11-01 | Innate Pharma | Multispecific proteins comprising an nkp46-binding site, a cancer antgienge binding site fused to a cytokine for nk cell engaging |
| MX2023014647A (en) | 2021-06-09 | 2024-01-31 | Innate Pharma | MULTI-SPECIFIC NKP46 BINDING PROTEINS. |
| WO2022258691A1 (en) | 2021-06-09 | 2022-12-15 | Innate Pharma | Multispecific proteins binding to nkg2d, a cytokine receptor, a tumour antigen and cd16a |
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| TW201909926A (en) | 2019-03-16 |
| WO2019024911A1 (en) | 2019-02-07 |
| CN109963591A (en) | 2019-07-02 |
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