WO2019100194A1

WO2019100194A1 - Anti-dr5 antibody and preparation method therefor and use thereof

Info

Publication number: WO2019100194A1
Application number: PCT/CN2017/112073
Authority: WO
Inventors: 万晓春; 陈倩; 夏蒙; 李俊鑫; 刘绿艳; 张青梅
Original assignee: 深圳先进技术研究院
Priority date: 2017-11-21
Filing date: 2017-11-21
Publication date: 2019-05-31

Abstract

Disclosed are an anti-DR5 antibody and a preparation method therefor and the use thereof. The antibody comprises a constant region and a variable region, wherein the heavy chain variable region comprises CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 and CDR3 as shown in SEQ ID NO: 3; the light chain variable region comprises CDR1' as shown in SEQ ID NO: 4, CDR2' as shown in SEQ ID NO: 5 and CDR3' as shown in SEQ ID NO: 6. The antibody can specifically bind to the antigen DR5 but has no cross reaction with DR4, has a very high affinity and biological activity, can induce apoptosis without the addition of crosslinking agents, and can synergistically induce apoptosis in combination with TRAIL.

Description

抗DR5的抗体及其制备方法和应用Anti-DR5 antibody and preparation method and application thereof

本发明属于生物医药技术领域，具体涉及一种抗DR5的抗体及其制备方法和应用。The invention belongs to the technical field of biomedicine, and particularly relates to an antibody against DR5 and a preparation method and application thereof.

根据世界卫生组织的研究显示，自1990年到2013年，癌症死亡率从12%上升至15%，已成为仅次于心血管疾病的高死亡率疾病，预测2020年全球癌症新发病例将达到2000万，因癌症死亡达1200万人；因此，人类亟需开发新的癌症治疗药物。According to research by the World Health Organization, from 1990 to 2013, cancer mortality increased from 12% to 15%, which has become a high-mortality disease after cardiovascular disease. It is predicted that new global cancer cases will reach 2020. 20 million people have died of cancers of 12 million people; therefore, humans need to develop new cancer treatment drugs.

诱导癌细胞凋亡是多种癌症治疗方法之一，肿瘤坏死因子相关凋亡诱导配体（Tumor necrosis factor related apoptosis inducing ligand，TRAIL）能与死亡受体（Death receptor, DR）结合诱导癌细胞凋亡而不会损伤正常组织，因此TRAIL-DR成为抗肿瘤抗体药物研发的热门靶点。TRAIL又称为Apo2L，是Wiley 1995年首次发现的，是继TNF、FasL之后的第三个TNF家族的凋亡分子。TRAIL是一种Ⅱ型跨膜蛋白，胞外区可以被金属蛋白酶切下形成可溶性TRAIL，TRAIL在正常人体的各种组织内有广泛的表达，通过与靶细胞表面的受体特异性结合而发挥作用。目前，在人类中已发现5种TRAIL受体：①2个死亡受体DR4（death receptor 4）和DR5（death receptor 5）；②2个诱骗受体DcR1（decoy receptor l）和DcR2（decoy receptor 2）；③1个可溶性受体OPG（osteoprotegerin）。其中，DR4和DR5的胞内区拥有完整的死亡结构域，能够诱导细胞凋亡，而DcR1却没有死亡结构域，DcR2也只有一个截短的死亡结构域，均不能传递凋亡信号，OPG是这五种受体中唯一的可溶性蛋白，它主要参与骨骼密度的调节。在37℃条件下，TRAIL与DR5结合的亲和力高于其它膜表达的TRAIL受体；因此，在生理状态下，TRAIL更倾向与DR5结合，尤其当内源性TRAIL有限时。DR5高表达于人类癌症，包括结肠癌、胃癌、胰腺癌、卵巢癌、乳腺癌、非小细胞肺癌，在正常细胞无或低表达。死亡受体DR4或DR5与TRAIL结合后，形成配体/受体三聚复合物，诱导死亡受体胞浆段死亡结构域（death domain，DD）与Fas相关死亡结构域蛋白（Fas-associated death domain protein，FADD）C端DD结合，而FADD以其N端死亡效应结构域（death effector domain，DED）与procaspase-8结合，形成DR4/DR5/FADD/procaspase-8死亡诱导信号复合物（death-inducing signaling complex，DISC），促使其中procaspase-8自身催化成有活性的caspase-8，caspase-8活化后，通过2条信号途径传递凋亡信号（非线粒体依赖途径和线粒体依赖途径）。Inducing apoptosis in cancer cells is one of the many cancer treatment methods, tumor necrosis factor-related apoptosis-inducing ligands (Tumor Necrosis factor related apoptosis inducing Ligand, TRAIL) and death receptor (Death receptor, DR) combines to induce apoptosis in cancer cells without damaging normal tissues, so TRAIL-DR has become a hot target for the development of anti-tumor antibody drugs. TRAIL, also known as Apo2L, is Wiley First discovered in 1995, it is the third TNF family of apoptosis molecules following TNF and FasL. TRAIL is a type II transmembrane protein. The extracellular domain can be excised by metalloproteinases to form soluble TRAIL. TRAIL is widely expressed in various tissues of normal human body and is specifically bound by receptors on the surface of target cells. effect. Currently, five TRAIL receptors have been discovered in humans: 12 death receptors DR4 (death) Receptor 4) and DR5 (death receptor 5); 22 decoy receptor DcR1 (decoy Receptor l) and DcR2 (decoy receptor) 2); 31 soluble receptor OPG (osteoprotegerin). Among them, the intracellular region of DR4 and DR5 possesses a complete death domain, which can induce apoptosis, while DcR1 has no death domain, and DcR2 has only a truncated death domain, which can not transmit apoptosis signals. OPG is The only soluble protein of these five receptors, which is primarily involved in the regulation of bone density. At 37 °C, TRAIL binds to DR5 with higher affinity than other membrane-expressed TRAIL receptors; therefore, under physiological conditions, TRAIL is more likely to bind to DR5, especially when endogenous TRAIL is limited. DR5 is highly expressed in human cancer, including colon cancer, gastric cancer, pancreatic cancer, ovarian cancer, breast cancer, non-small cell lung cancer, and has no or low expression in normal cells. The death receptor DR4 or DR5 binds to TRAIL to form a ligand/receptor trimeric complex that induces death cytoplasmic segment death domain (death Domain, DD) Fas-associated death domain Protein, FADD) C-terminal DD binding, while FADD uses its N-terminal death effector Domain,DED) binds to procaspase-8 to form a DR4/DR5/FADD/procaspase-8 death-inducing signaling complex (death-inducing) The signaling complex (DISC) promotes the activation of procaspase-8 itself into active caspase-8, which is activated by two signaling pathways (non-mitochondria-dependent pathway and mitochondria-dependent pathway).

TRAIL能选择性杀伤肿瘤细胞的特点，使得多个TRAIL受体拮抗剂进入临床开发，其主要分为2类：1）重组TRAIL蛋白；2）拮抗DR4或DR5的抗体。但是，目前这些药物没有一个使临床癌症病人获益。重组TRAIL蛋白的主要缺陷是药物稳定性和靶向性不好，另外一些形式的重组TRAIL蛋白在高剂量下显示出肝毒性。TRAIL受体拮抗剂临床试验失败的主要原因有：1）候选药物的拮抗活性不足；2）许多原代癌细胞对单一抗体治疗有抗性；3）缺乏合适的生物标记物来鉴别病人对TRAIL受体拮抗剂的易感性。目前已进入临床研究的靶向DR5的单克隆抗体有：Lexatumumab、Drozitumab、Conatumumab、Tigatuzumab、LBY135等，而许多DR5抗体（如Drozitumab和Tigatuzumab）需要另外添加交联剂（cross-linking agent）才能在体外达到最佳的活性。TRAIL selectively kills tumor cells, allowing multiple TRAIL receptor antagonists to enter clinical development. It is mainly divided into two categories: 1) recombinant TRAIL protein; 2) antibodies that antagonize DR4 or DR5. However, none of these drugs currently benefit patients with clinical cancer. The main drawback of recombinant TRAIL protein is poor drug stability and targeting, and other forms of recombinant TRAIL protein show hepatotoxicity at high doses. The main reasons for the failure of TRAIL receptor antagonist clinical trials are: 1) insufficient antagonism of candidate drugs; 2) many primary cancer cells are resistant to single antibody therapy; 3) lack of suitable biomarkers to identify patients to TRAIL Susceptibility to receptor antagonists. Monoclonal antibodies targeting DR5 that have entered clinical research are: Lexatumumab, Drozitumab, Conatumumab, Tigatuzumab, LBY135, etc., while many DR5 antibodies (such as Drozitumab and Tigatuzumab) require an additional cross-linking agent to The best activity is achieved in vitro.

发明内容Summary of the invention

本发明的目的在于克服现有技术的上述不足，提供一种抗DR5的抗体及其制备方法和应用，旨在解决现有TRAIL受体拮抗剂类抗肿瘤药物的拮抗活性不足，需要添加交联剂才能诱导细胞凋亡的技术问题。The object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide an anti-DR5 antibody, a preparation method and application thereof, and aim to solve the problem that the antagonistic activity of the existing TRAIL receptor antagonist antitumor drugs is insufficient, and cross-linking needs to be added. The technical problem of the agent to induce apoptosis.

为实现上述发明目的，本发明采用的技术方案如下：In order to achieve the above object, the technical solution adopted by the present invention is as follows:

一方面，本发明提供一种抗体的可变区，包括重链可变区和轻链可变区，所述重链可变区包括以下3个互补决定区CDR：In one aspect, the invention provides a variable region of an antibody, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the following three complementarity determining region CDRs:

SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2，和SEQ ID NO:3所示的CDR3；CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3;

所述轻链可变区包括以下3个互补决定区CDR：The light chain variable region comprises the following three complementarity determining region CDRs:

SEQ ID NO:4所示的CDR1’，SEQ ID NO:5所示的CDR2’，和SEQ ID NO:6所示的CDR3’。CDR1' shown in SEQ ID NO: 4, CDR2' shown in SEQ ID NO: 5, and SEQ ID NO: CDR3' shown by 6.

进一步地，所述重链可变区含有如SEQ ID NO:7所示的氨基酸序列，或如SEQ ID NO:7所示的氨基酸序列经缺失、***或替换所获得的具有相同功能的氨基酸序列；和／或Further, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having the same function obtained by deletion, insertion or substitution of the amino acid sequence shown in SEQ ID NO: ;and / or

所述轻链可变区含有如SEQ ID NO:8所示的氨基酸序列，或如SEQ ID NO:8所示的氨基酸序列经缺失、***或替换所获得的具有相同功能的氨基酸序列。The light chain variable region contains an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having the same function obtained by deletion, insertion or substitution of the amino acid sequence shown in SEQ ID NO: 8.

另一方面，本发明还提供一种抗体，所述抗体包括恒定区以及上述可变区，所述恒定区包括重链恒定区和轻链恒定区。In another aspect, the invention provides an antibody comprising a constant region and a variable region as described above, the constant region comprising a heavy chain constant region and a light chain constant region.

另一方面，本发明还提供一种重组蛋白，所述的重组蛋白含有具有上述可变区的序列；和／或In another aspect, the present invention provides a recombinant protein, the recombinant protein comprising a sequence having the above variable region; and/or

含有上述抗体的序列，以及协助表达和/或纯化的标签序列。Sequences containing the above antibodies, as well as tag sequences that facilitate expression and/or purification.

另一方面，本发明还提供一种核酸分子，所述核酸分子编码上述可变区；和／或In another aspect, the invention provides a nucleic acid molecule encoding the variable region; and/or

编码上述抗体；和／或Encoding the above antibodies; and/or

编码上述重组蛋白。The above recombinant protein is encoded.

进一步地，编码所述重链可变区的核苷酸序列如SEQ ID NO:9所示，或如SEQ ID NO:9所示的核苷酸序列经缺失、***或替换所获得的具有相同功能的核苷酸序列；和/或Further, the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 9, or the nucleotide sequence shown in SEQ ID NO: 9 is obtained by deletion, insertion or substitution. Functional nucleotide sequence; and/or

编码所述轻链可变区的核苷酸序列如SEQ ID NO:10所示，或如SEQ ID NO:10所示的核苷酸序列经缺失、***或替换所获得的具有相同功能的核苷酸序列。A nucleotide sequence encoding the light chain variable region is represented by SEQ ID NO: 10, or a nucleotide having the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown in SEQ ID NO: Glycosidic acid sequence.

另一方面，本发明还提供一种载体，所述载体具有上述核酸分子。In another aspect, the invention also provides a vector having the above nucleic acid molecule.

另一方面，本发明还提供一种基因工程化的宿主细胞，所述宿主细胞包含上述载体；和／或In another aspect, the invention provides a genetically engineered host cell, the host cell comprising the above vector; and/or

所述宿主细胞的基因组中整合有上述核酸分子。The nucleic acid molecule described above is integrated into the genome of the host cell.

另一方面，本发明还提供一种免疫偶联物，所述免疫偶联物含有上述可变区；和／或In another aspect, the present invention provides an immunoconjugate comprising the above variable region; and/or

含有上述抗体；和／或Containing the above antibodies; and/or

含有上述重组蛋白，以及可检测标记物、药物、毒素、细胞因子、放射性核素、酶中的至少一种。The recombinant protein described above, and at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

另一方面，本发明还提供一种药物组合物，所述药物组合物含有上述可变区；和／或In another aspect, the present invention provides a pharmaceutical composition comprising the above variable region; and/or

含有上述抗体；和／或Containing the above antibodies; and/or

含有上述重组蛋白；和／或Containing the above recombinant protein; and/or

含有上述免疫偶联物，以及药学上可接受的载体。Containing the above immunoconjugates, as well as a pharmaceutically acceptable carrier.

另一方面，本发明还提供一种上述可变区、上述抗体、上述重组蛋白、上述免疫偶联物在制备抗DR5蛋白的肿瘤药物，或制备检测DR5蛋白的试剂和／或试剂盒中的应用。In another aspect, the present invention provides a variable region, the above antibody, the above recombinant protein, the above immunoconjugate in the preparation of an anti-DR5 protein tumor drug, or a reagent and/or a kit for preparing a DR5 protein. application.

最后，本发明一种上述抗体或上述重组蛋白的制备方法，所述制备方法包括培养宿主细胞，或采用杂交瘤法利用小鼠腹水生产。Finally, the present invention provides a method for producing the above antibody or the above recombinant protein, which comprises culturing a host cell or using a hybridoma method to produce mouse ascites.

本发明提供的抗体的可变区，含有六个特有的互补决定区CDR（见SEQ ID NO:1－6），可特异性识别抗原DR5，具有很高的亲和力和生物活性和，其可诱导细胞凋亡。The variable region of the antibody provided by the present invention, comprising six unique complementarity determining region CDRs (see SEQ ID NO: 1-6), can specifically recognize the antigen DR5, has high affinity and biological activity, and can induce Apoptosis.

本发明提供的抗体具有上述独特的抗体可变区序列，可与抗原DR5结合，具有很高的亲和力和生物活性，其不需添加交联剂就能诱导细胞凋亡；而且其可以与TRAIL联合协同诱导细胞凋亡。The antibody provided by the invention has the above-mentioned unique antibody variable region sequence, can bind to the antigen DR5, has high affinity and biological activity, can induce apoptosis without adding a cross-linking agent; and can be combined with TRAIL Synergistically induce apoptosis.

本发明提供的抗体或重组蛋白用途广泛，可作为抗肿瘤药物（该肿瘤表达DR5蛋白）单独使用，也可与TRAIL联合使用，还可以与常用的肿瘤放疗药物或化疗药物联合使用，或为基础产生的基因工程抗体作为靶向部分，与放射性核素或化学药物或毒素偶联。另外，该检测DR5蛋白的试剂和／或试剂盒，如ELISA检测、Western blot检测和流式细胞检测等领域的相关试剂盒，其灵敏度显著优于现有技术。The antibody or the recombinant protein provided by the invention can be widely used as an antitumor drug (the tumor expresses DR5 protein), can also be used in combination with TRAIL, and can also be used in combination with commonly used tumor radiotherapy drugs or chemotherapy drugs, or as a basis. The resulting genetically engineered antibody acts as a targeting moiety and is coupled to a radionuclide or chemical drug or toxin. In addition, the reagents and/or kits for detecting DR5 proteins, such as ELISA detection, Western blot detection, and flow cytometry, have significantly higher sensitivity than the prior art.

附图说明DRAWINGS

图1为本发明实施例1中6C93D6抗体的SDS-PAGE电泳图；1 is a SDS-PAGE electrophoresis pattern of the 6C93D6 antibody in Example 1 of the present invention;

图2为本发明实施例1中6C93D6抗体的重链和轻链可变区扩增后电泳图；其中，Y为阴性对照，M为Marker，1为6C93D6抗体的重链，2为6C93D6抗体轻链；2 is an electrophoresis pattern of the heavy chain and light chain variable regions of the 6C93D6 antibody in Example 1 of the present invention; wherein Y is a negative control, M is a Marker, 1 is a heavy chain of 6C93D6 antibody, and 2 is a 6C93D6 antibody light. chain;

图3为本发明实施例2中6C93D6抗体的亚型鉴定图；Figure 3 is a subtype identification diagram of the 6C93D6 antibody in Example 2 of the present invention;

图4为本发明实施例3中6C93D6抗体的亲和力检测图；Figure 4 is a diagram showing the affinity detection of the 6C93D6 antibody in Example 3 of the present invention;

图5为本发明实施例4中无交联剂时6C93D6抗体诱导HepaRG细胞凋亡的结果图；Figure 5 is a graph showing the results of apoptosis of HepaRG cells induced by 6C93D6 antibody in the absence of a cross-linking agent in Example 4 of the present invention;

图6为本发明实施例4中存在交联剂时6C93D6抗体诱导HepaRG细胞凋亡的结果图；Figure 6 is a graph showing the results of apoptosis of HepaRG cells induced by 6C93D6 antibody in the presence of a cross-linking agent in Example 4 of the present invention;

图7为本发明实施例5中6C93D6抗体用于流式检测jurkat细胞的结果图；Figure 7 is a diagram showing the results of flow detection of jurkat cells by the 6C93D6 antibody in Example 5 of the present invention;

图8为本发明实施例6中6C93D6抗体用于ELISA检测的结果图；Figure 8 is a diagram showing the results of the 6C93D6 antibody used in the ELISA assay in Example 6 of the present invention;

图9为本发明实施例7中6C93D6抗体用于Western blot检测的结果图；Figure 9 is a diagram showing the results of Western blot detection of the 6C93D6 antibody in Example 7 of the present invention;

图10为本发明实施例9中6C93D6抗体和3E73F11抗体与抗原DR5的结合表位分析结果图；Figure 10 is a diagram showing the results of binding epitope analysis of the 6C93D6 antibody and the 3E73F11 antibody to the antigen DR5 in Example 9 of the present invention;

图11为本发明实施例10中6C93D6抗体检测人血清中DR5浓度结果图。Figure 11 is a graph showing the results of detecting the concentration of DR5 in human serum by the 6C93D6 antibody in Example 10 of the present invention.

图12为本发明实施例11中6C93D6抗体与人DR4无交叉反应结果图。Figure 12 is a graph showing the results of no cross-reaction of 6C93D6 antibody with human DR4 in Example 11 of the present invention.

具体实施方式Detailed ways

为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clear, the present invention will be further described in detail below with reference to the embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

一方面，本发明实施例提供一种抗体的可变区，包括重链可变区和轻链可变区，所述重链可变区包括以下3个互补决定区CDR：SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2，和SEQ ID NO:3所示的CDR3；In one aspect, the embodiments of the invention provide a variable region of an antibody, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the following three complementarity determining regions CDR: SEQ CDR1 shown by ID NO: 1, CDR2 shown by SEQ ID NO: 2, and CDR3 shown by SEQ ID NO: 3;

所述轻链可变区包括以下3个互补决定区CDR：SEQ ID NO:4所示的CDR1’，SEQ ID NO:5所示的CDR2’，和SEQ ID NO:6所示的CDR3’。The light chain variable region comprises the following three complementarity determining region CDRs: CDR1', SEQ ID NO: 4, SEQ ID NO: CDR2' shown by 5, and CDR3' shown by SEQ ID NO: 6.

即重链可变区的CDR氨基酸序列为：That is, the CDR amino acid sequence of the heavy chain variable region is:

CDR1（SEQ ID NO.1）：GYSFTGHY；CDR1 (SEQ ID NO. 1): GYSFTGHY;

CDR2（SEQ ID NO.2）：VNPNNGGT；CDR2 (SEQ ID NO. 2): VNPNNGGT;

CDR3（SEQ ID NO.3）：ARNGAYYRSDGNYFDY。CDR3 (SEQ ID NO. 3): ARNGAYYRSDGNYFDY.

轻链可变区的CDR氨基酸序列为：The CDR amino acid sequence of the light chain variable region is:

CDR1’（SEQ ID NO.4）：ESVDSYGNSF；CDR1' (SEQ ID NO. 4): ESVDSYGNSF;

CDR2’（SEQ ID NO.5）：RAS；CDR2' (SEQ ID NO. 5): RAS;

CDR3’（SEQ ID NO.6）：QQSNEDPYT。CDR3' (SEQ ID NO. 6): QQSNEDPYT.

CDR又称互补决定区（complementarity determining region），在空间结构上可与抗原决定簇形成精密的互补，是抗体识别和结合抗原的区域，直接决定抗体的特异性。本发明提供的抗体的可变区，含有六个特有的互补决定区CDR，可特异性识别抗原DR5，具有很高的亲和力和生物活性和，其可诱导细胞凋亡。The CDR, also known as the complementarity determining region, forms a precise complement to the antigenic determinant in the spatial structure, and is a region in which the antibody recognizes and binds to the antigen, directly determining the specificity of the antibody. The variable region of the antibody provided by the invention contains six unique complementarity determining region CDRs, which can specifically recognize the antigen DR5, has high affinity and biological activity, and can induce apoptosis.

进一步地，在本发明一实施例中，所述重链可变区含有如SEQ ID NO:7所示的氨基酸序列，或如SEQ ID NO:7所示的氨基酸序列经缺失、***或替换所获得的具有相同功能的氨基酸序列；和／或所述轻链可变区含有如SEQ ID NO:8所示的氨基酸序列，或如SEQ ID NO:8所示的氨基酸序列经缺失、***或替换所获得的具有相同功能的氨基酸序列。Further, in an embodiment of the invention, the heavy chain variable region comprises SEQ ID NO: the amino acid sequence shown in 7 or SEQ ID An amino acid sequence having the same function obtained by deleting, inserting or replacing the amino acid sequence shown by NO: 7; and/or the light chain variable region containing SEQ. The amino acid sequence shown by ID NO: 8, or the amino acid sequence having the same function obtained by deletion, insertion or substitution of the amino acid sequence shown in SEQ ID NO: 8.

重链可变区氨基酸序列（SEQ ID NO.7）：Heavy chain variable region amino acid sequence (SEQ ID NO. 7):

EVQLQQSGPDLVKPGASVKISCKASGYSFTGHYMHWVKQSHGQSLEWIGRVNPNNGGTGYNQKFKDKAILTVDKPSSTAYMELRSLTSEDSAVYYCARNGAYYRSDGNYFDYWGQGTTLTVSS。EVQLQQSGPDLVKPGASVKISCKAS GYSFTGHY MHWVKQSHGQSLEWIGR VNPNNGGT GYNQKFKDKAILTVDKPSSTAYMELRSLTSEDSAVYYC ARNGAYYRSDGNYFDY WGQGTTLTVSS.

轻链可变区氨基酸序列（SEQ ID NO.8）：Light chain variable region amino acid sequence (SEQ ID NO. 8):

DIVLTQSTPSLAVSLGQRATISCRASE SVDSYGNSFMHWFQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIK。DIVLTQSTPSLAVSLGQRATISCRAS E SVDSYGNSF MHWFQQKPGQPPKLLIY RAS NLESGIPARFSGSGSRTDFTLTINPVEADDVATYYC QQSNEDPYT FGGGTKLEIK.

具体地，可以是与SEQ ID NO:7或SEQ ID NO:8公开序列至少有80%同源性的氨基酸序列作为替代方案。Specifically, it may be an amino acid sequence having at least 80% homology to the sequence disclosed by SEQ ID NO: 7 or SEQ ID NO: 8 as an alternative.

另一方面，本发明实施例还提供一种抗体，所述抗体包括恒定区以及上述可变区，所述恒定区包括重链恒定区和轻链恒定区。In another aspect, embodiments of the invention also provide an antibody comprising a constant region and a variable region as described above, the constant region comprising a heavy chain constant region and a light chain constant region.

另一方面，本发明实施例还提供一种重组蛋白，所述的重组蛋白含有具有上述可变区的序列；和／或含有上述抗体的序列，以及协助表达和/或纯化的标签序列。In another aspect, the present invention provides a recombinant protein comprising a sequence having the above variable region; and/or a sequence comprising the above antibody, and a tag sequence which facilitates expression and/or purification.

本发明实施例提供的上述抗体或重组蛋白具有上述独特的抗体可变区序列，可与抗原DR5结合，具有很高的亲和力和生物活性，其不需添加交联剂就能诱导细胞凋亡；而且其可以与TRAIL联合协同诱导细胞凋亡。The above antibody or recombinant protein provided by the embodiment of the invention has the above-mentioned unique antibody variable region sequence, can bind to the antigen DR5, has high affinity and biological activity, and can induce apoptosis without adding a crosslinking agent; Moreover, it can synergistically induce apoptosis in combination with TRAIL.

另一方面，本发明实施例提供一种核酸分子，该核酸分子编码本发明实施例的上述可变区；和／或编码上述抗体；和／或编码上述重组蛋白。In another aspect, embodiments of the invention provide a nucleic acid molecule encoding the variable region of the embodiments of the invention; and/or encoding the antibody; and/or encoding the recombinant protein.

进一步地，在一优选实施例中，编码重链可变区氨的核苷酸序列如SEQ ID NO:9所示，或如SEQ ID NO:9所示的核苷酸序列经缺失、***或替换所获得的具有相同功能的核苷酸序列；和/或编码轻链可变区的核苷酸序列如SEQ ID NO:10所示，或如SEQ ID NO:10所示的核苷酸序列经缺失、***或替换所获得的具有相同功能的核苷酸序列。Further, in a preferred embodiment, the nucleotide sequence encoding the heavy chain variable region ammonia is SEQ ID NO: 9 or as SEQ ID a nucleotide sequence having the same function obtained by deletion, insertion or substitution of a nucleotide sequence represented by NO: 9; and/or a nucleotide sequence encoding a variable region of a light chain such as SEQ ID NO: 10, or a nucleotide sequence having the same function obtained by deleting, inserting or replacing the nucleotide sequence shown by SEQ ID NO: 10.

编码重链可变区的核苷酸序列（SEQ ID NO.9）：Nucleotide sequence encoding the heavy chain variable region (SEQ ID NO. 9):

GAGGTCCAGCTGCAGCAGTCTGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCCACTACATGCACTGGGTGAAGCAGAGCCATGGACAGAGCCTTGAGTGGATTGGACGTGTTAATCCTAACAATGGTGGTACTGGCTACAACCAGAAGTTCAAGGACAAGGCCATATTAACTGTAGACAAGCCATCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAACGGAGCCTACTATAGGTCCGATGGGAACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。GAGGTCCAGCTGCAGCAGTCTGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCCACTACATGCACTGGGTGAAGCAGAGCCATGGACAGAGCCTTGAGTGGATTGGACGTGTTAATCCTAACAATGGTGGTACTGGCTACAACCAGAAGTTCAAGGACAAGGCCATATTAACTGTAGACAAGCCATCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAACGGAGCCTACTATAGGTCCGATGGGAACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

编码重链CDR1核苷酸序列为（SEQ ID NO.11）：The nucleotide sequence encoding the heavy chain CDR1 is (SEQ ID NO. 11):

GGTTACTCATTCACTGGCCACTAC；GGTTACTCATTCACTGGCCACTAC;

编码重链CDR2核苷酸序列为（SEQ ID NO.12）：The nucleotide sequence encoding the heavy chain CDR2 is (SEQ ID NO. 12):

GTTAATCCTAACAATGGTGGTACT；GTTAATCCTAACAATGGTGGTACT;

编码重链CDR3核苷酸序列为（SEQ ID NO.13）：The nucleotide sequence encoding the heavy chain CDR3 is (SEQ ID NO. 13):

GCAAGAAACGGAGCCTACTATAGGTCCGATGGGAACTACTTTGACTAC。GCAAGAAACGGAGCCTACTATAGGTCCGATGGGAACTACTTTGACTAC.

编码轻链可变区的核苷酸序列（SEQ ID NO.10）：Nucleotide sequence encoding the light chain variable region (SEQ ID NO. 10):

GACATTGTGCTCACACAATCTACACCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAACAGTTTTATGCACTGGTTCCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。GACATTGTGCTCACACAATCTACACCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAACAGTTTTATGCACTGGTTCCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA.

编码轻链CDR1’核苷酸序列为（SEQ ID NO.14）：The nucleotide sequence encoding the light chain CDR1' is (SEQ ID NO. 14):

AGTGTTGATAGTTATGGCAACAGTTTT；AGTGTTGATAGTTATGGCAACAGTTTT;

编码轻链CDR2’核苷酸序列为（SEQ ID NO.15）：The nucleotide sequence encoding the light chain CDR2' is (SEQ ID NO. 15):

CGTGCATCC；CGTGCATCC;

编码轻链CDR3’核苷酸序列为（SEQ ID NO.16）：The nucleotide sequence encoding the light chain CDR3' is (SEQ ID NO. 16):

CAGCAAAGTAATGAGGATCCGTACACG。CAGCAAAGTAATGAGGATCCGTACACG.

本发明实施例提供的上述核酸分子因核苷酸序列的互补序列或因遗传密码的简并性，可以为与本发明实施例公开的核苷酸序列不同、但编码相同蛋白的序列；也可以是与本发明实施例公开的核苷酸序列至少有80%同源性的序列作为替代方案。The nucleic acid molecule provided by the embodiment of the present invention may be a sequence different from the nucleotide sequence disclosed in the embodiment of the present invention but encoding the same protein due to the complementary sequence of the nucleotide sequence or the degeneracy of the genetic code; Is a sequence which is at least 80% homologous to the nucleotide sequence disclosed in the examples of the present invention as an alternative.

另一方面，本发明实施例提供一种载体，该重组载体具有上述核酸分子。该重组载体可用来表达含有上述重链可变区和轻链可变区的抗体或重组蛋白。In another aspect, embodiments of the invention provide a vector having the nucleic acid molecule described above. The recombinant vector can be used to express an antibody or recombinant protein comprising the above-described heavy chain variable region and light chain variable region.

另一方面，本发明实施例还提供一种免疫偶联物，所述免疫偶联物含有上述可变区；和／或含有上述抗体；和／或含有上述重组蛋白，以及可检测标记物、药物、毒素、细胞因子、放射性核素、酶中的至少一种。In another aspect, embodiments of the present invention also provide an immunoconjugate comprising the above variable region; and/or comprising the above antibody; and/or comprising the above recombinant protein, and a detectable label, At least one of a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

另一方面，本发明实施例还提供一种药物组合物，所述药物组合物含有上述可变区；和／或含有上述抗体；和／或含有上述重组蛋白；和／或含有上述免疫偶联物，以及药学上可接受的载体。In another aspect, the present invention provides a pharmaceutical composition comprising the above variable region; and/or containing the above antibody; and/or containing the above recombinant protein; and/or containing the above immunoconjugate And a pharmaceutically acceptable carrier.

另一方面，本发明实施例还提供一种本发明实施例中上述可变区、上述抗体、上述重组蛋白、上述免疫偶联物在制备抗DR5蛋白的肿瘤药物，或制备检测DR5蛋白的试剂和／或试剂盒中的应用。In another aspect, the embodiment of the present invention further provides a tumor drug for preparing an anti-DR5 protein, or a reagent for detecting DR5 protein, wherein the variable region, the antibody, the recombinant protein, and the immunoconjugate are used in the embodiment of the present invention. And/or the application in the kit.

本发明实施例提供的抗体或重组蛋白用途广泛，可作为抗肿瘤药物（该肿瘤表达DR5蛋白）单独使用，也可与TRAIL联合使用，还可以与常用的肿瘤放疗药物或化疗药物联合使用，或为基础产生的基因工程抗体作为靶向部分，与放射性核素或化学药物或毒素偶联。进一步地，在一优选实施例中，本发明实施例公开了该抗人DR5的单克隆抗体对肝癌细胞系的杀伤效果，可用于其它有DR5表达的多种肿瘤类型的治疗。另外，该检测DR5蛋白的试剂和／或试剂盒，如ELISA检测、Western blot检测和流式细胞检测等领域的相关试剂盒，其灵敏度显著优于现有技术。The antibody or the recombinant protein provided by the embodiment of the invention can be widely used as an antitumor drug (the tumor expresses the DR5 protein), can also be used in combination with TRAIL, and can also be used in combination with a commonly used tumor radiotherapy or chemotherapy drug, or The genetically engineered antibody produced as a basis is coupled to a radionuclide or a chemical drug or toxin. Further, in a preferred embodiment, the present invention discloses the killing effect of the monoclonal antibody against human DR5 on a liver cancer cell line, and can be used for the treatment of other tumor types having DR5 expression. In addition, the reagents and/or kits for detecting DR5 proteins, such as ELISA detection, Western blot detection, and flow cytometry, have significantly higher sensitivity than the prior art.

本发明实施例提供了抗人DR5的单克隆抗体的可变区氨基酸序列和核苷酸序列，在此基础上利用上述重组载体或宿主细胞可以产生基因工程抗体，其所含的重链和轻链可变区序列与本发明实施例公开的可变区序列是一致的。该基因工程抗体包括但不限于：抗体的功能性片段Fab或者是单链抗体，或者是重链可变区和完整轻链融合的抗体功能性片段VH-L，或者是重链和轻链的一个或多个CDR的排列、串联或组合而成的抗体功能性片段，或者是上述抗体和抗体功能片段和其他各种蛋白或多肽进行连接、拼接、融合而得到的类抗体的功能性的融合蛋白。The present invention provides a variable region amino acid sequence and a nucleotide sequence of a monoclonal antibody against human DR5, and on the basis of the above recombinant vector or host cell, a genetically engineered antibody can be produced, which comprises a heavy chain and a light chain. The chain variable region sequence is identical to the variable region sequences disclosed in the examples of the present invention. The genetically engineered antibody includes, but is not limited to, a functional fragment of the antibody, Fab, or a single-chain antibody, or an antibody functional fragment VH-L, which is a heavy chain variable region and a full light chain fusion, or a heavy chain or a light chain. An antibody functional fragment of one or more CDRs arranged, tandem, or combined, or a functional fusion of an antibody-like antibody obtained by ligation, splicing, and fusion of the above-described antibody and antibody functional fragments and various other proteins or polypeptides protein.

具体在一优选实施例中，该制备方法采用小鼠腹水生产单克隆抗体；或采用培养杂交瘤细胞、CHO细胞或293F细胞生产所述单克隆抗体。本发明一实施例中，提供了小鼠抗人DR5单克隆抗体的可变区氨基酸序列和核苷酸序列，通过抗体人源化改造，可以获得抗人DR5的人鼠嵌合抗体和人源化抗体。抗人DR5的人鼠嵌合抗体将鼠源单克隆抗体基因的可变区和人的恒定区连接在一起，然后在哺乳动物细胞中进行表达产生；而抗人DR5的人源化抗体则是除了将抗体的恒定区换成人源的之外，更进一步的将可变区的FR区转换成人源的，从而降低免疫原性。由此产生的抗人DR5的人鼠嵌合抗体和人源化抗体将同样具有结合人DR5分子诱导癌细胞凋亡，从而***的作用。Specifically, in a preferred embodiment, the preparation method employs mouse ascites to produce a monoclonal antibody; or the cultured hybrid C cell, CHO cell or 293F is used to produce the monoclonal antibody. In an embodiment of the present invention, a variable region amino acid sequence and a nucleotide sequence of a mouse anti-human DR5 monoclonal antibody are provided, and a human mouse chimeric antibody and a human source resistant to human DR5 can be obtained by humanized modification of the antibody. Antibody. The anti-human DR5 human-mouse chimeric antibody ligates the variable region of the murine monoclonal antibody gene to the human constant region and then expresses it in mammalian cells; whereas the humanized antibody against human DR5 is In addition to changing the constant region of the antibody to an adult source, the FR region of the variable region is further converted to an adult source, thereby reducing immunogenicity. The human-human chimeric antibody and humanized antibody against human DR5 thus produced will also have the effect of binding to human DR5 molecules to induce apoptosis of cancer cells, thereby treating tumors.

本发明先后进行过多次试验，现举一部分试验结果作为参考对发明进行进一步详细描述，下面结合具体实施例进行详细说明。The present invention has been subjected to a number of tests in succession, and a part of the test results are now described in further detail as a reference, and will be described in detail below in conjunction with specific embodiments.

实施例1Example 1

抗DR5的单克隆抗体的制备，本实施例采用杂交瘤法利用小鼠腹水生产抗DR5的单克隆抗体。Preparation of a monoclonal antibody against DR5, this example uses a hybridoma method to produce a monoclonal antibody against DR5 using mouse ascites.

用sDR5-Fc蛋白抗原（深圳市中科艾深医药有限公司提供，制备方法参考专利201610067931.2）免疫雌性BALB/c小鼠（6周龄），首次免疫使用弗氏完全佐剂进行乳化抗原，第二次免疫开始，使用弗氏不完全佐剂乳化抗原，皮下5~6点注射，每只小鼠注射的抗原量约为100ug。在第3次免疫后10天，对小鼠剪尾采集少量血液进行血清效价ELISA检测，选择抗体滴度高（>1:100000）的小鼠进行加强第4次免疫，通过腹腔注射抗原蛋白，每只小鼠注射约100ug。第4次免疫后3~5天，处死小鼠取其脾细胞与SP2/0细胞融合，通过HAT培养基培养获得稳定的杂交瘤细胞。通过ELISA法筛选得到能分泌DR5抗体的杂交瘤细胞，通过有限稀释的方法进行亚克隆，ELISA法筛选得到能分泌DR5抗体的单克隆杂交瘤细胞株（本说明书中，该抗DR5的单克隆抗体命名为6C93D6抗体），通过逐级扩大培养，液氮冻存保种。Female BALB/c mice (6 weeks old) were immunized with sDR5-Fc protein antigen (provided by Shenzhen Zhongke Ai Shen Pharmaceutical Co., Ltd., preparation method reference patent 201610067931.2), and the first immunization was performed using Freund's complete adjuvant for emulsification antigen. At the beginning of the second immunization, the antigen was emulsified using Freund's incomplete adjuvant, subcutaneously injected at 5-6 points, and the amount of antigen injected per mouse was about 100 ug. 10 days after the third immunization, a small amount of blood was collected from the tail of the mouse for serum titer ELISA, and mice with high antibody titer (>1:100000) were selected for the fourth immunization, and the antigenic protein was injected intraperitoneally. Each mouse was injected about 100 ug. Three to five days after the fourth immunization, the mice were sacrificed and their spleen cells were fused with SP2/0 cells, and stable hybridoma cells were obtained by culturing with HAT medium. Hybridoma cells capable of secreting DR5 antibody were screened by ELISA, subcloned by limiting dilution, and monoclonal hybridoma cell lines capable of secreting DR5 antibody were screened by ELISA (in this specification, the monoclonal antibody against DR5) Named 6C93D6 antibody), the culture was expanded by stepwise, and the liquid nitrogen was frozen and preserved.

腹水抗体制备：雌性BALB/c小鼠（8周龄）腹腔注射弗氏不完全佐剂，每只小鼠注射0.5ml，3~5天后腹腔注射处于对数生长期的杂交瘤细胞，每只小鼠注射1~5×10⁵个细胞（0.5ml），注射杂交瘤细胞约11天后处死小鼠，获得腹水。3000rpm，4℃离心10min，去除沉淀，用10倍体积1×PB溶液稀释腹水，混匀后过0.45μm滤膜。通过Protein G（Protein G Sepharose 4 Fast Flow,GE Healthcare）亲和纯化腹水，得到纯化的DR5抗体蛋白，用BCA法测抗体浓度。将纯化抗体跑SDS-PAGE（上样量8ug），考马斯亮蓝染色，结果如图1所示：6C93D6抗体含有两条带，一条为重链，一条为轻链。Preparation of ascites antibody: Female BALB/c mice (8 weeks old) were injected intraperitoneally with Freund's incomplete adjuvant, 0.5 ml per mouse, and intraperitoneal injection of hybridoma cells in logarithmic growth phase after 3 to 5 days, each Mice were injected with 1~5×10 ⁵ cells (0.5 ml), and the mice were sacrificed about 11 days after the injection of the hybridoma cells to obtain ascites. Centrifuge at 3000 rpm for 10 min at 4 ° C to remove the precipitate, dilute the ascites with 10 volumes of 1×PB solution, mix and pass through a 0.45 μm filter. The ascites was affinity-purified by Protein G (Protein G Sepharose 4 Fast Flow, GE Healthcare) to obtain a purified DR5 antibody protein, and the antibody concentration was measured by the BCA method. The purified antibody was run on SDS-PAGE (loading amount 8 ug) and stained with Coomassie brilliant blue. The results are shown in Figure 1. The 6C93D6 antibody contains two bands, one for the heavy chain and one for the light chain.

DR5单克隆抗体杂交瘤细胞的抗体基因可变区测序：收获处于对数生长期的单克隆抗体杂交瘤细胞，TRIZOL裂解进行RNA提取，反转录后获得cDNA，扩增并获得重链和轻链可变区的DNA序列（电泳图如图2所示），非功能性VK基因去除，克隆至pMD18-T载体，测序，使用数据库进行测序结果比对；测序结果和分析如下。DR5 monoclonal antibody hybridoma cell antibody gene variable region sequencing: harvesting monoclonal antibody hybridoma cells in logarithmic growth phase, TRIZOL cleavage for RNA extraction, reverse transcription to obtain cDNA, amplification and obtaining heavy chain and light The DNA sequence of the variable region of the chain (electropherogram shown in Figure 2), the non-functional VK gene was removed, cloned into the pMD18-T vector, sequenced, and sequenced using the database; the sequencing results and analysis are as follows.

编码重链可变区的核苷酸序列为SEQ ID NO.9；The nucleotide sequence encoding the heavy chain variable region is SEQ ID NO. 9;

编码轻链可变区的核苷酸序列为SEQ ID NO.10；The nucleotide sequence encoding the light chain variable region is SEQ ID NO. 10;

重链可变区氨基酸序列SEQ ID NO.7：轻链可变区氨基酸序列SEQ ID NO.8。Heavy chain variable region amino acid sequence SEQ ID NO. 7: light chain variable region amino acid sequence SEQ ID NO.

经与IMGT/V-QUEST数据库进行比对，重链的CDR氨基酸序列为：After alignment with the IMGT/V-QUEST database, the CDR amino acid sequence of the heavy chain is:

CDR1-IMGT：SEQ ID NO.1；CDR1-IMGT: SEQ ID NO. 1;

CDR2-IMGT：SEQ ID NO.2；CDR2-IMGT: SEQ ID NO. 2;

CDR3-IMGT：SEQ ID NO.3；CDR3-IMGT: SEQ ID NO. 3;

重链的FR区氨基酸序列为：The amino acid sequence of the FR region of the heavy chain is:

FR1-IMGT：EVQLQQSGPDLVKPGASVKISCKAS；FR1-IMGT: EVQLQQSGPDLVKPGASVKISCKAS;

FR2-IMGT：MHWVKQSHGQSLEWIGR；FR2-IMGT: MHWVKQSHGQSLEWIGR;

FR3-IMGT：GYNQKFKDKAILTVDKPSSTAYMELRSLTSEDSAVYYC；FR3-IMGT: GYNQKFKDKAILTVDKPSSTAYMELRSLTSEDSAVYYC;

FR4-IMGT：WGQGTTLTVSS。FR4-IMGT: WGQGTTLTVSS.

轻链的CDR氨基酸序列为：The CDR amino acid sequence of the light chain is:

CDR1’-IMGT：SEQ ID NO.4；CDR1'-IMGT: SEQ ID NO. 4;

CDR2’-IMGT：SEQ ID NO.5；CDR2'-IMGT: SEQ ID NO. 5;

CDR3’-IMGT：SEQ ID NO.6。CDR3'-IMGT: SEQ ID NO. 6.

轻链的FR区氨基酸序列为：The amino acid sequence of the FR region of the light chain is:

FR1’-IMGT：DIVLTQSTPSLAVSLGQRATISCRAS；FR1’-IMGT: DIVLTQSTPSLAVSLGQRATISCRAS;

FR2’-IMGT：MHWFQQKPGQPPKLLIY；FR2’-IMGT: MHWFQQKPGQPPKLLIY;

FR3’-IMGT：NLESGIPARFSGSGSRTDFTLTINPVEADDVATYYC；FR3’-IMGT: NLESGIPARFSGSGSRTDFTLTINPVEADDVATYYC;

FR4’-IMGT：FGGGTKLEIK。FR4’-IMGT: FGGGTKLEIK.

实施例2Example 2

抗DR5的单克隆抗体的亚型鉴定。Subtype identification of monoclonal antibodies against DR5.

使用商品化小鼠单克隆抗体Isotyping试剂盒（sino biological，SEK003）进行单抗亚型鉴定，结果如图3所示，从图中可知：该6C93D6抗体为IgG2b型抗体。The monoclonal antibody subtype was identified using a commercial mouse monoclonal antibody Isotyping kit (sino 003), and the results are shown in Fig. 3. As is clear from the figure, the 6C93D6 antibody is an IgG2b type antibody.

实施例3Example 3

抗DR5的单克隆抗体的亲和力测定。Affinity determination of monoclonal antibodies against DR5.

使用10ug/ml sDR5-Fc蛋白（深圳市中科艾深医药有限公司提供）与传感器进行固化结合，使用SD buffer（PBS+0.02% Tween 20+0.1% BSA）配制不同浓度的6C93D6抗体（76nM、38nM、19nM、9.5nM、4.75nM）作为流动相，使用分子间相互作用仪（OCTET K2，PALL life science）进行亲和力检测，程序设定为：Baseline 240s，Loading 360s，Baseline2 180s，Association 480s，Dissociation 480s，使用AHC（Anti-hIgG Fc Capture）传感器。结果如图4所示，结果显示：6C93D6抗体与DR5的亲和力为6.77E-09（M），即6C93D6抗体与DR5具有高亲和力。Use 10ug/ml sDR5-Fc protein (provided by Shenzhen Zhongke Ai Shen Pharmaceutical Co., Ltd.) combined with sensor curing, using SD Buffer (PBS+0.02% Tween 20+0.1% BSA) Prepare different concentrations of 6C93D6 antibody (76nM, 38nM, 19nM, 9.5nM, 4.75nM) as mobile phase, using intermolecular interaction instrument (OCTET) K2, PALL life science) for affinity testing, the program is set to: Baseline 240s, Loading 360s,Baseline2 180s,Association 480s,Dissociation For 480 s, use an AHC (Anti-hIgG Fc Capture) sensor. The results are shown in Fig. 4. The results showed that the affinity of the 6C93D6 antibody to DR5 was 6.77E-09 (M), that is, the 6C93D6 antibody had high affinity with DR5.

实施例4Example 4

抗DR5的单克隆抗体的体外细胞功能测定。In vitro cellular function assay of monoclonal antibodies against DR5.

1、取对数生长期的HepaRG细胞，胰酶消化，含10%FBS的培养基终止，吹散计数，调整密度至8×10⁴/ml，按100ul/孔加入96孔细胞培养板，即每孔8000个细胞。共接种6×10=60孔，周围的36孔每孔加入200ul PBS进行液封，防止培养基蒸发。将细胞放置培养箱培养22~24h。1. Take HepaRG cells in logarithmic growth phase, trypsinize, stop the medium containing 10% FBS, blow the count, adjust the density to 8×10 ⁴ /ml, and add 96-well cell culture plate at 100 ul/well, ie 8000 cells per well. A total of 6 × 10 = 60 wells were inoculated, and the surrounding 36 wells were filled with 200 ul of PBS for liquid sealing to prevent evaporation of the medium. The cells were placed in an incubator for 22 to 24 hours.

2、取10ml培养液，加2ul 5mg/ml放线菌D，即放线菌素D浓度为1ug/ml。2. Take 10 ml of culture solution and add 2 ul of 5 mg/ml actinomycete D, ie the actinomycin D concentration is 1 ug/ml.

3、抗体稀释：3. Antibody dilution:

不加交联剂情况：用稀释液将6C93D6抗体稀释至200ug/ml，以此浓度为起点，按2倍比稀释19个浓度梯度，每个梯度1个复孔，每孔100ul加入板中。In the absence of cross-linking agent: 6C93D6 antibody was diluted to 200 ug/ml with a diluent, and 19 concentration gradients were diluted at a concentration of 2 times. One gradient per well, 100 ul per well was added to the plate.

交联剂（anti-mouse lgG Fc antibody）稀释：6ul anti-mouse lgG Fc antibody(1mg/ml)+3ml稀释液，稀释至2ug/ml。Dilution of anti-mouse lgG Fc antibody: 6ul Anti-mouse lgG Fc antibody (1 mg/ml) + 3 ml dilution, diluted to 2 ug/ml.

加交联剂情况：Add cross-linking agent:

取30个EP管，每管分别加入125ul6C93D6抗体梯度稀释溶液（1ug/ml~0.001862pg/ml），每管再加入125ul 2ug/ml交联剂稀释液，混匀。每孔100ul加入板中，设一个复孔。Take 30 EP tubes, add 125ul 6C93D6 antibody gradient dilution solution (1ug/ml~0.001862pg/ml) to each tube, add 125ul 2ug/ml crosslinker dilution to each tube and mix. 100 ul per well was added to the plate and a double hole was provided.

4、放置培养箱培养18~22小时。4. Place the incubator for 18~22 hours.

5、以20:1的比例配制MTS/PMS溶液（Promega，G5430），每孔加20ul，不加盖子放置培养箱3~4h，酶标仪检测A490-A630，最终结果如图5和图6所示。5. Prepare MTS/PMS solution (Promega, G5430) in a ratio of 20:1, add 20ul per well, place the incubator for 3~4h without a lid, and measure the A490-A630 with a microplate reader. The final result is shown in Figure 5 and 6 is shown.

图5显示，在无交联剂情况下，6C93D6抗体能诱导HepaRG细胞凋亡，EC50=1.1μg/ml。图6显示，在1ug/ml交联剂情况下，6C93D6抗体诱导HepaRG细胞凋亡的EC50下降为68.51pg/ml。Figure 5 shows that 6C93D6 antibody induced apoptosis in HepaRG cells with no cross-linking agent, EC50 = 1.1 μg/ml. Figure 6 shows that the EC50 of apoptosis induced by 6C93D6 antibody in HepaRG cells was 68.51 pg/ml in the case of 1 ug/ml crosslinker.

实施例5Example 5

抗DR5的单克隆抗体在流式细胞检测中的应用。Application of anti-DR5 monoclonal antibody in flow cytometry.

1、收集jurkat细胞（急性T细胞白血病细胞系中一种），吹散计数，分装5个离心管，每管3×10⁵个细胞，500g离心5min，去上清，分别加入1ml PBS，500g离心5min，去上清。1. Collect jurkat cells (one of the acute T cell leukemia cell lines), blow up the count, dispense 5 centrifuge tubes, 3×10 ⁵ cells per tube, centrifuge for 5 min at 500 g, remove the supernatant, and add 1 ml PBS, respectively. Centrifuge at 500g for 5min and remove the supernatant.

2、按下表1加入试剂：2. Add reagents as shown in Table 1 below:

阴性对照Negative control	DMEMDMEM	100ul DMEM培养液100ul DMEM medium
	PBSPBS	100ul PBS100ul PBS
	同型对照PE Mouse lgG1,κ isotype control antibody（biolegend,401207）Isotype control PE Mouse lgG1, κ isotype control antibody (biolegend, 401207)	100ul DMEM培养液+2ul lgG2b-PE100ul DMEM medium + 2ul lgG2b-PE
样品sample	6C93D66C93D6	100ul DMEM培养液+ 3.5ul 6C93D6100ul DMEM medium + 3.5ul 6C93D6
阳性对照Positive control	PE anti-human CD262（DR5，TRAIL-R2）Antibody（biolegend，307405）PE anti-human CD262(DR5,TRAIL-R2)Antibody(biolegend,307405)	100ul DMEM培养液+4ul anti-DR5 antibody-PE100ul DMEM medium + 4ul anti-DR5 antibody-PE

冰上孵育30min。Incubate on ice for 30 min.

3、各加入1ml PBS+5%FBS，500g离心5min，去上清，各加入100ul DMEM培养液+1ul PE goat anti-mouse lgG（minimal x-reactivity）antibody（biolegend,405307），冰上孵育20min。3. Add 1ml PBS + 5% FBS, centrifuge at 500g for 5min, remove the supernatant, add 100ul each DMEM medium +1 ul PE goat anti-mouse lgG (minimal x-reactivity) antibody (biolegend, 405307), incubated on ice for 20 min.

4、各加入1ml PBS+5%FBS，500g离心5min，去上清。4. Add 1 ml PBS + 5% FBS, centrifuge at 500 g for 5 min, and remove the supernatant.

5、每管加入300ul PBS+5%FBS，重悬，使用流式细胞仪（Beckman CytoFLEX CM）检测。5. Add 300ul PBS + 5% FBS to each tube, resuspend, using flow cytometry (Beckman CytoFLEX CM) detection.

结果如图7所示，从图可知：该6C93D6抗体能与jurkat细胞表面的DR5蛋白结合，应用于流式细胞检测，且检测灵敏度优于商品化阳性对照。因此，其可以用于制备流式细胞检测试剂盒。The results are shown in Fig. 7. As can be seen from the figure, the 6C93D6 antibody can bind to the DR5 protein on the surface of jurkat cells, and is applied to flow cytometry, and the detection sensitivity is superior to the commercial positive control. Therefore, it can be used to prepare a flow cytometry kit.

实施例6Example 6

抗DR5的单克隆抗体在ELISA检测中的应用。Application of anti-DR5 monoclonal antibody in ELISA assay.

酶标板每孔包被25ng sDR5蛋白，4℃包被过夜，经封闭洗板后，加入2倍比稀释的6C93D6抗体，最高浓度为40ug/ml，最低浓度为19.5ng/ml，每孔100ul，37度孵育1h。PBST洗板5次，加入1:5000稀释的HRP-羊抗小鼠IgG抗体，37℃孵育45min。PBST洗板5次，每孔加入100ul TMB，室温避光反应3min，每孔加入50ul 终止液终止，酶标仪测OD450。The ELISA plate is coated with 25ng per well. The sDR5 protein was coated overnight at 4 °C. After blocking the plate, 2 times diluted 6C93D6 antibody was added. The highest concentration was 40 ug/ml, the lowest concentration was 19.5 ng/ml, 100 ul per well, and incubated at 37 degrees for 1 h. The plate was washed 5 times with PBST, and a 1:5000 dilution of HRP-goat anti-mouse IgG antibody was added and incubated at 37 ° C for 45 min. Wash the plate 5 times with PBST and add 100ul per well TMB, room temperature and light-proof reaction for 3 min, 50 ul of stop solution was added to each well, and OD450 was measured by a microplate reader.

结果如图8所示，该6C93D6抗体可用于ELISA检测，其可用于制备ELISA检测的试剂盒。As a result, as shown in Fig. 8, the 6C93D6 antibody can be used for ELISA assay, which can be used to prepare a kit for ELISA assay.

实施例7Example 7

抗DR5的单克隆抗体在Western blot检测中的应用。Application of anti-DR5 monoclonal antibody in Western blot detection.

sDR5蛋白加5×loading buffer混合后，煮沸变性6min，跑SDS-PAGE，每孔上样20ul，~500ng蛋白/孔，400mA转膜1h，5%脱脂奶粉室温封闭1h，加入6C93D6抗体（3ml 5%脱脂奶粉中加入~10ug抗体）4度孵育过夜，PBST洗3遍后加入1:5000稀释羊抗鼠IgG二抗，室温孵育45min，PBST洗3遍后加入Western HRP底物（密理博，WBLUR0500）显影。sDR5 protein plus 5×loading After buffer mixing, boil and denature for 6 min, run SDS-PAGE, load 20ul per well, ~500ng protein/well, 400mA transfer membrane for 1h, 5% skim milk powder for 1h at room temperature, add 6C93D6 antibody (3ml 5% skim milk powder was added with ~10ug antibody) and incubated overnight at 4 degrees. After washing with PBST for 3 times, add 1:5000 diluted goat anti-mouse IgG secondary antibody, incubate for 45 min at room temperature, wash PBST for 3 times and add Western HRP substrate (Millipore, WBLUR0500) Development.

结果如图9所示，该6C93D6抗体能用于Western blot检测，可用于制备Western blot检测的试剂盒。另外在日常Western blot实验中应用DR5抗体，发现6C93D6抗体的实验效果优于GeneTex公司和Santa cruz公司商品化DR5抗体，货号分别为GTX21675和sc166624。Results as shown in Figure 9, the 6C93D6 antibody can be used in Western The blot assay can be used to prepare a kit for Western blot detection. Also in everyday Western The application of DR5 antibody in the blot assay showed that the 6C93D6 antibody was superior to the commercial DR5 antibody of GeneTex and Santa Cruz, and the numbers were GTX21675 and sc166624, respectively.

实施例8Example 8

抗DR5的单克隆抗体的抗原结合表位与TRAIL结合位点的关系验证。The relationship between the antigen binding epitope of the anti-DR5 monoclonal antibody and the TRAIL binding site was verified.

1、包被人TRAIL（sino biological公司，10409-HANE，250ug/ml）：4ml包被液+80ul人TRAIL得到浓度为5ug/ml的人TRAIL，用其包被ELISA板，每孔100ul，37℃包被2h。1. TRAIL (sino biological, 10409-HANE, 250 ug/ml): 4 ml of coating solution + 80 ul of human TRAIL to obtain human TRAIL at a concentration of 5 ug/ml, and coated with an ELISA plate, 100 ul per well, 37 °C is coated for 2h.

2、取出ELISA板，甩净孔内液体，PBST（磷酸盐吐温缓冲液）振荡洗涤5次，30s/次。2. The ELISA plate was taken out, and the liquid in the well was cleaned, and PBST (phosphate tween buffer) was shaken and washed 5 times for 30 s/time.

3、每孔加入300ul封闭液，37℃孵育2h。3. Add 300 ul of blocking solution to each well and incubate for 2 h at 37 °C.

4、配制6C93D6和sDR5-Fc-biotin混合液。4. Prepare a mixture of 6C93D6 and sDR5-Fc-biotin.

60ul 6C93D6（2ug/ul）+2ml PBS（磷酸盐缓冲液），得到2ml 60ug/ml 6C93D6。60ul 6C93D6 (2ug/ul) + 2ml PBS (phosphate buffer), get 2ml 60ug/ml 6C93D6.

取32支EP管，分为2份，每份16支，配制sDR-Fc-biotin；Take 32 EP tubes, divide into 2 parts, each 16 pieces, prepare sDR-Fc-biotin;

4ul sDR5-Fc-biotin（1.5ug/ul）+200ul PBS，得到 200ul 30ug/ml sDR5-Fc-biotin；100ul sDR5-Fc-biotin（30ug/ml）+100ul PBS，得到200ul 15ug/ml sDR5-Fc-biotin；依次类推，稀释16个梯度。每个梯度加100ul 6C93D6，混匀。37℃孵育1h。4ul sDR5-Fc-biotin (1.5ug/ul) + 200ul PBS, obtained 200ul 30ug/ml sDR5-Fc-biotin; 100ul sDR5-Fc-biotin (30ug/ml) + 100ul PBS, get 200ul 15ug/ml sDR5-Fc-biotin; and so on, diluted 16 gradients. Add 100ul of 6C93D6 to each gradient and mix. Incubate for 1 h at 37 °C.

5、取出ELISA板，甩净孔内液体，PBST振荡洗涤5次，30s/次。5. Remove the ELISA plate, clean the liquid in the well, and wash it 5 times with PBST for 30 s/time.

6、加入孵育好的6C93D6+sDR5-Fc-biotin混合液，每孔100ul，37℃孵育1h。6. Add the incubated 6C93D6+sDR5-Fc-biotin mixture, 100ul per well, and incubate for 1h at 37°C.

7、取出ELISA板，甩净孔内液体，PBST振荡洗涤5次，30s/次。7. Remove the ELISA plate, clean the liquid in the well, and wash it 5 times with PBST for 30 s/time.

8、每孔加入1：5000稀释的Streptavidin-HRP，37℃孵育30min。8. Add 1:5000 dilution of Streptavidin-HRP to each well and incubate for 30 min at 37 °C.

9、取出ELISA板，甩净孔内液体，PBST振荡洗涤5次，30s/次。9. Remove the ELISA plate, clean the liquid in the well, and wash it 5 times with PBST for 30 s/time.

10、每孔加入100ul TMB，室温避光反应10min。10. Add 100 ul of TMB to each well and allow to react at room temperature for 10 min in the dark.

11、每孔加入50ul终止液终止，酶标仪测OD450。11. Stop 50 ul of stop solution in each well and measure OD450 by microplate reader.

该ELISA设计的工作浓度与对应的OD450结果如表2所示。The working concentration of the ELISA design and the corresponding OD450 results are shown in Table 2.

不同试剂的工作浓度Working concentration of different reagents	OD450OD450
5ug/ml trail30ug/ml 6C93D6+15ug/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP5ug/ml Trail30ug/ml 6C93D6+15ug/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP	0.5860.586
5ug/ml trail30ug/ml 6C93D6+7.5ug/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP5ug/ml Trail30ug/ml 6C93D6+7.5ug/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP	0.1050.105
5ug/ml trail30ug/ml 6C93D6+3.75ug/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP5ug/ml Trail30ug/ml 6C93D6+3.75ug/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP	0.0830.083
5ug/ml trail30ug/ml 6C93D6+1.875ug/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP5ug/ml Trail30ug/ml 6C93D6+1.875ug/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP	0.0780.078
5ug/ml trail30ug/ml 6C93D6+937.5ng/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP5ug/ml Trail30ug/ml 6C93D6+937.5ng/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP	0.0710.071
5ug/ml trail30ug/ml 6C93D6+468.8ng/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP5ug/ml Trail30ug/ml 6C93D6+468.8ng/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP	0.0600.060
5ug/ml trail30ug/ml 6C93D6+234.4ng/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP5ug/ml Trail30ug/ml 6C93D6+234.4ng/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP	0.0640.064
5ug/ml trail30ug/ml 6C93D6+117.2ng/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP5ug/ml Trail30ug/ml 6C93D6+117.2ng/ml sDR5-Fc-biotin1:5000 Streptavidin-HRP	0.0590.059

从以上表3的数据结果可知：当过量的6C93D6抗体（30ug/ml）与少量抗原sDR5（7.5ug/ml）结合后，形成的抗原抗体复合物只有很少量能与包被在酶标板上的配体TRAIL结合，最终产生很小OD值0.105。这说明6C93D6抗体和DR5结合的位点与TRAIL和DR5结合的位点有重叠，也就是说6C93D6抗体和DR5的结合影响TRAIL配体与DR5的结合。From the data in Table 3 above, it can be seen that when an excess of 6C93D6 antibody (30 ug/ml) is combined with a small amount of antigen sDR5 (7.5 ug/ml), the antigen-antibody complex formed has only a small amount of energy and can be coated on the ELISA plate. The ligand TRAIL on the binding eventually produced a very small OD value of 0.105. This indicates that the site of binding of 6C93D6 antibody and DR5 overlaps with the site of TRAIL and DR5 binding, that is, the binding of 6C93D6 antibody and DR5 affects the binding of TRAIL ligand to DR5.

实施例9Example 9

利用实施例1的制备方法筛选出另一种抗DR5的单克隆抗体（命名为3E73F11），对两种抗DR5的单克隆抗体（6C93D6与3E73F11）与抗原DR5的结合表位进行分析。Another anti-DR5 monoclonal antibody (designated 3E73F11) was screened by the preparation method of Example 1, and the binding epitopes of the two anti-DR5 monoclonal antibodies (6C93D6 and 3E73F11) to the antigen DR5 were analyzed.

1、包被6C93D6：用包被液分别配制1ug/ml和0.25ug/ml 6C9-3D6抗体，每孔100ul，4℃包被过夜。1. Coated 6C93D6: 1ug/ml and 0.25ug/ml 6C9-3D6 antibody were prepared with the coating solution, 100ul per well, and coated at 4°C overnight.

2、PBST洗板5次，30s/次。2, PBST wash plate 5 times, 30s / time.

3、每孔加入250ul封闭液，37℃孵育1.5h。3. Add 250 ul of blocking solution to each well and incubate at 37 ° C for 1.5 h.

4、PBST洗板5次，30s/次。5、稀释人DR5（sino biological，10465-H08H）：用抗体稀释液配制得到2ug/ml DR5；以2倍比稀释2ug/ml human DR5，稀释16个梯度。稀释3E7-3F11：用抗体稀释液配制得到20ug/ml 3E73F11。将20ug/ml 3E73F11以1:1比例分别与16个梯度的人DR5混合，放置37℃，孵育1.5h。4, PBST wash plate 5 times, 30s / time. 5, diluted human DR5 (sino Biological,10465-H08H): Prepare 2ug/ml DR5 with antibody dilution; 2ug/ml diluted 2 times Human DR5, diluted 16 gradients. Dilute 3E7-3F11: Prepare 20ug/ml with antibody dilution 3E73F11. 20 ug/ml 3E73F11 was mixed with 16 gradients of human DR5 in a 1:1 ratio, placed at 37 ° C, and incubated for 1.5 h.

6、PBST洗板5次，30s/次。6, PBST wash plate 5 times, 30s / time.

7、将孵育好的DR5+3E73F11，加入ELISA板，每孔100ul，37℃孵育1h。7. The incubated DR5+3E73F11 was added to an ELISA plate and incubated at 100 ul per well for 1 h at 37 °C.

8、取出ELISA板，甩净孔内液体，PBST振荡洗涤5次，30s/次。8. Remove the ELISA plate, clean the liquid in the well, and wash it 5 times with PBST for 30 s/time.

9、每孔加入1ug/ml DR5兔多抗（sino biological,10465-RP02,2.54ug/ul），37℃孵育1h。9. Add 1 ug/ml DR5 rabbit polyclonal antibody (sino biological, 10465-RP02, 2.54 ug/ul) to each well and incubate for 1 h at 37 °C.

10、PBST洗板5次，30s/次。10, PBST wash plate 5 times, 30s / time.

11、每孔加入1:40000稀释的驴抗兔IgG-HRP（abcam,ab6802），37℃孵育45min。11. Add 1:40000 diluted donkey anti-rabbit IgG-HRP (abcam, ab6802) to each well and incubate for 45 min at 37 °C.

12、PBST洗板5次，30s/次。12, PBST wash plate 5 times, 30s / time.

13、每孔加入100ulTMB（北京四正柏生物科技有限公司，4ATMB200），室温避光反应10min。13. Add 100ul of TMB (Beijing Sizhengbai Biotechnology Co., Ltd., 4ATMB200) to each well and let it react at room temperature for 10 minutes.

14、每孔加入50ul 终止液终止，酶标仪测OD450。14. Add 50 ul of stop solution to each well and measure the OD450 with a microplate reader.

该ELISA设计的工作浓度与对应的OD450结果如表3所示。The working concentration of the ELISA design and the corresponding OD450 results are shown in Table 3.

最终的数据图如图10所示，从图中曲线可知：当过量的3E73F11抗体（10ug/ml）与少量抗原sDR5（31.25ng/ml）结合，形成的抗原抗体复合物仍然能与包被在酶标板上的6C93D6抗体（1ug/ml）结合，最终产生明显阳性OD值（1.130）。这说明6C93D6抗体和3E73F11抗体与抗原DR5的结合表位不同，即两种单克隆抗体都具有各自的特异性。The final data plot is shown in Figure 10. From the curve in the figure, when the excess 3E73F11 antibody (10 ug/ml) is combined with a small amount of antigen sDR5 (31.25 ng/ml), the resulting antigen-antibody complex can still be coated with The 6C93D6 antibody (1 ug/ml) on the plate was bound to produce a significantly positive OD value (1.130). This indicates that the 6C93D6 antibody and the 3E73F11 antibody differ from the antigen DR5 binding epitope, that is, both monoclonal antibodies have respective specificities.

实施例10Example 10

使用6C93D6抗体检测人血清中DR5浓度。The concentration of DR5 in human serum was measured using the 6C93D6 antibody.

1、用包被液稀释6C93D6抗体至1ug/ml，包被96孔板（NUNC，468667），每孔100ul，4℃包被过夜。1. Dilute 6C93D6 antibody to 1 ug/ml with coating solution, coat 96-well plates (NUNC, 468667), 100 ul per well, and coat overnight at 4 °C.

2、PBST洗板5次，30s/次。2, PBST wash plate 5 times, 30s / time.

4、PBST洗板5次，30s/次。4, PBST wash plate 5 times, 30s / time.

5、用抗体稀释液稀释人DR5（0.5mg/ml，Sino Biological Inc，10465-H08H），以1000ng/ml为起始浓度，稀释16个梯度，100ul/孔，37℃孵育1h。5. Dilute human DR5 (0.5 mg/ml, Sino Biological Inc, 10465-H08H) with antibody dilution at a concentration of 1000 ng/ml, dilute 16 gradients, 100 ul/well, and incubate for 1 h at 37 °C.

6、PBST洗板5次，30s/次。6, PBST wash plate 5 times, 30s / time.

7、用抗体稀释液稀释DR5兔多抗（1mg/ml，Sino Biological Inc，10465-RP02）至1ug/ml，100ul/孔，37℃，孵育，1h。7. Dilute DR5 rabbit polyclonal antibody (1 mg/ml, Sino Biological Inc, 10465-RP02) to 1 ug/ml with antibody dilution, 100 ul/well, 37 ° C, incubate for 1 h.

8、PBST洗板5次，30s/次。8, PBST wash plate 5 times, 30s / time.

9、用抗体稀释液1:10000稀释Donkey anti-rabbit IgG H&L（HRP）preadsorbed（Abcam，ab7803），每个梯度分别加入100ul，37℃孵育45min。9. Dilute Donkey anti-rabbit IgG H&L (HRP) prereadsorbed (Abcam, ab7803) with antibody dilution 1: 100 ml per gradient and incubate for 45 min at 37 °C.

10、PBST洗板5次，30s/次。10, PBST wash plate 5 times, 30s / time.

11、每孔加入100ul TMB，室温避光反应4min。11. Add 100 ul of TMB to each well and allow to react at room temperature for 4 min.

12、每孔加入50ul终止液终止，酶标仪（Thermo，Multiskan GO）测OD450。12. Stop 50 ul of stop solution per well and measure OD450 with a microplate reader (Thermo, Multiskan GO).

该ELISA设计的工作浓度与对应的OD450结果如表4所示。The working concentration of the ELISA design and the corresponding OD450 results are shown in Table 4.

DR5浓度（ng/ml）DR5 concentration (ng/ml)	OD₄₅₀ OD ₄₅₀	DR5浓度（ng/ml）DR5 concentration (ng/ml)	OD₄₅₀ OD ₄₅₀	DR5浓度（ng/ml）DR5 concentration (ng/ml)	OD₄₅₀ OD ₄₅₀	DR5浓度（ng/ml）DR5 concentration (ng/ml)	OD₄₅₀ OD ₄₅₀
10001000	1.4041.404	3.93.9	1.0221.022	10001000	1.3891.389	3.93.9	1.0551.055
500500	1.4311.431	1.951.95	0.8140.814	500500	1.3531.353	1.951.95	0.7620.762
250250	1.3601.360	0.980.98	0.6710.671	250250	1.2201.220	0.980.98	0.5660.566
125125	1.3411.341	0.4880.488	0.4750.475	125125	1.2051.205	0.4880.488	0.4700.470
62.562.5	1.4191.419	0.2440.244	0.3730.373	62.562.5	1.2341.234	0.2440.244	0.3380.338
31.2531.25	1.3081.308	0.1220.122	0.3160.316	31.2531.25	1.2981.298	0.1220.122	0.3080.308
15.615.6	1.3071.307	0.0610.061	0.2710.271	15.615.6	1.1061.106	0.0610.061	0.2710.271
7.87.8	1.1681.168	0.030.03	0.2140.214	7.87.8	1.1641.164	0.030.03	0.2380.238

最终的数据图如图11所示，从图可知：6C93D6抗体组成的ELISA检测试剂盒检测人血清中sDR5浓度检测下限可以达到30.5pg/ml，线性区间在30.5pg/ml~7.8125ng/ml。The final data plot is shown in Figure 11. It can be seen from the figure that the detection limit of sDR5 in human serum by ELISA kit consisting of 6C93D6 antibody can reach 30.5pg/ml, and the linear range is 30.5pg/ml~7.8125ng/ml.

实施例11Example 11

检测6C93D6抗体是否与人DR4抗原有交叉反应。It was tested whether the 6C93D6 antibody cross-reacted with the human DR4 antigen.

1、用包被液分别稀释DR4和DR5至2.5ug/ml、1.25ug/ml、625ng/ml、312.5ng/ml、156.25ng/ml、78ng/ml、39ng/ml 7个浓度，包被96孔板，每孔100ul，4℃包被过夜。1. Dilute DR4 and DR5 to 2.5 ug/ml, 1.25 ug/ml, 625 ng/ml, 312.5 ng/ml, 156.25 ng/ml, 78 ng/ml, and 39 ng/ml with coating solution, respectively. Orifice plates, 100 ul per well, coated overnight at 4 °C.

2、PBST振荡洗涤3次，30s/次。2. Wash the PBST shake 3 times for 30 s/time.

3、每孔加入250ul 封闭液，37℃孵育1.5h。3. Add 250 ul of blocking solution to each well and incubate at 37 ° C for 1.5 h.

4、PBST振荡洗涤5次，30s/次。4. Wash PBST for 5 times, 30s/time.

5、用PBS稀释6C93D6至2ug/ml，100ul/孔，37℃孵育1h。5. Dilute 6C93D6 to 2 ug/ml with PBS, 100 ul/well, and incubate for 1 h at 37 °C.

6、PBST振荡洗涤5次，30s/次。6. Wash PBST for 5 times, 30s/time.

7、用PBS以1:5000稀释羊抗鼠lgG-HRP，100ul/孔，37℃孵育45min。7. Sheep anti-mouse lgG-HRP was diluted 1:5000 with PBS, 100 ul/well, and incubated at 37 ° C for 45 min.

8、取出板，甩净孔内液体，PBST振荡洗涤5次，30s/次。8. Remove the plate and clean the liquid in the well. Wash it with PBST for 5 times, 30s/time.

9、每孔加入100ul TMB，室温避光反应4min。9. Add 100 ul of TMB to each well and allow to react at room temperature for 4 min in the dark.

10、每孔加入50ul 终止液终止，测OD450。10. Add 50 ul of stop solution to each well and measure OD450.

结果如图12所示：该6C93D6抗体能特异性识别DR5抗原，和DR4抗原没有交叉反应。The results are shown in Figure 12: The 6C93D6 antibody specifically recognizes the DR5 antigen and does not cross-react with the DR4 antigen.

以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

Claims

一种抗体的可变区，包括重链可变区和轻链可变区，其特征在于，所述重链可变区包括以下3个互补决定区CDR：
SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2，和SEQ ID NO:3所示的CDR3；
所述轻链可变区包括以下3个互补决定区CDR：
SEQ ID NO:4所示的CDR1’，SEQ ID NO:5所示的CDR2’，和SEQ ID NO:6所示的CDR3’。A variable region of an antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following three complementarity determining region CDRs:
CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3;
The light chain variable region comprises the following three complementarity determining region CDRs:
CDR1' shown in SEQ ID NO: 4, CDR2' shown in SEQ ID NO: 5, and CDR3' shown in SEQ ID NO: 6.
如权利要求1所述的可变区，其特征在于，所述重链可变区含有如SEQ ID NO:7所示的氨基酸序列，或如SEQ ID NO:7所示的氨基酸序列经缺失、***或替换所获得的具有相同功能的氨基酸序列；和／或
所述轻链可变区含有如SEQ ID NO:8所示的氨基酸序列，或如SEQ ID NO:8所示的氨基酸序列经缺失、***或替换所获得的具有相同功能的氨基酸序列。The variable region according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, or the amino acid sequence set forth in SEQ ID NO: 7 is deleted, Inserting or replacing the obtained amino acid sequence having the same function; and/or the light chain variable region contains the amino acid sequence shown as SEQ ID NO: 8, or the amino acid sequence shown as SEQ ID NO: 8 is deleted The obtained amino acid sequence having the same function is inserted or replaced.
一种抗体，其特征在于，所述抗体包括恒定区以及权利要求1或2所述的可变区，所述恒定区包括重链恒定区和轻链恒定区。 An antibody comprising a constant region and the variable region of claim 1 or 2, the constant region comprising a heavy chain constant region and a light chain constant region.
一种重组蛋白，其特征在于，所述的重组蛋白含有具有权利要求1或2所述的可变区的序列；和／或
含有权利要求3所述的抗体的序列，以及协助表达和/或纯化的标签序列。A recombinant protein comprising a sequence having the variable region of claim 1 or 2; and/or a sequence comprising the antibody of claim 3, and for facilitating expression and/or Purified tag sequence.
一种核酸分子，其特征在于，所述核酸分子编码如权利要求1或2所述的可变区；和／或
编码权利要求3所述的抗体；和／或
编码权利要求4所述的重组蛋白。A nucleic acid molecule, which encodes the variable region of claim 1 or 2; and/or encodes the antibody of claim 3; and/or encodes the recombination of claim 4. protein.
如权利要求5所述的核酸分子，其特征在于，编码所述重链可变区的核苷酸序列如SEQ ID NO:9所示，或如SEQ ID NO:9所示的核苷酸序列经缺失、***或替换所获得的具有相同功能的核苷酸序列；和/或
编码所述轻链可变区的核苷酸序列如SEQ ID NO:10所示，或如SEQ ID NO:10所示的核苷酸序列经缺失、***或替换所获得的具有相同功能的核苷酸序列。The nucleic acid molecule according to claim 5, wherein the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 9, or the nucleotide sequence shown in SEQ ID NO: a nucleotide sequence having the same function obtained by deletion, insertion or substitution; and/or a nucleotide sequence encoding the light chain variable region is set forth in SEQ ID NO: 10, or as SEQ ID NO: 10 A nucleotide sequence having the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown.
一种载体，其特征在于，所述载体具有如权利要求5或6所述的核酸分子。 A vector, characterized in that the vector has the nucleic acid molecule according to claim 5 or 6.
一种基因工程化的宿主细胞，其特征在于，所述宿主细胞包含如权利要求7所述的载体；和／或
所述宿主细胞的基因组中整合有权利要求5或6所述的核酸分子。A genetically engineered host cell, characterized in that the host cell comprises the vector of claim 7; and/or the nucleic acid molecule of claim 5 or 6 is integrated into the genome of the host cell.
一种免疫偶联物，其特征在于，所述免疫偶联物含有权利要求1或2所述的可变区；和／或
含有权利要求3所述的抗体；和／或
含有权利要求4所述的重组蛋白，以及可检测标记物、药物、毒素、细胞因子、放射性核素、酶中的至少一种。An immunoconjugate comprising the variable region of claim 1 or 2; and/or the antibody of claim 3; and/or comprising the method of claim 4. The recombinant protein, and at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.
一种药物组合物，其特征在于，所述药物组合物含有权利要求1或2所述的可变区；和／或
含有权利要求3所述的抗体；和／或
含有权利要求4所述的重组蛋白；和／或
含有权利要求9所述的免疫偶联物，以及药学上可接受的载体。A pharmaceutical composition comprising the variable region of claim 1 or 2; and/or the antibody of claim 3; and/or the method of claim 4. a recombinant protein; and/or comprising the immunoconjugate of claim 9, and a pharmaceutically acceptable carrier.
如权利要求1或2所述的可变区、如权利要求3所述的抗体、如权利要求4所述的重组蛋白、如权利要求9所述的免疫偶联物在制备抗DR5蛋白的肿瘤药物，或制备检测DR5蛋白的试剂和／或试剂盒中的应用。The variable region according to claim 1 or 2, the antibody according to claim 3, the recombinant protein according to claim 4, and the immunoconjugate according to claim 9 in the preparation of an anti-DR5 protein tumor The drug, or the application in the preparation of reagents and/or kits for detecting DR5 protein.
一种如权利要求3所述的抗体或权利要求4所述的重组蛋白的制备方法，其特征在于，所述制备方法包括培养宿主细胞，或采用杂交瘤法利用小鼠腹水生产。 An antibody according to claim 3 or a method for producing a recombinant protein according to claim 4, wherein the preparation method comprises culturing a host cell or using a hybridoma method to produce mouse ascites.