技术领域technical field
本发明属于生物医药领域，具体涉及一种针对PD-1的单克隆抗体及其应用。The invention belongs to the field of biomedicine, and specifically relates to a monoclonal antibody against PD-1 and its application.
背景技术Background technique
跨膜受体PD-1(programmed cell death 1，程序性细胞死亡因子1)是CD28基因家族成员之一，在活化的T细胞、B细胞及骨髓系细胞都有表达。PD-1的配体PD-L1和PD-L2均属于B7超家族，其中PD-L1多种细胞都有表达，包括T细胞、B细胞以及内皮细胞和上皮细胞，PD-L2则仅表达于抗原呈递细胞如树突状细胞和巨噬细胞。The transmembrane receptor PD-1 (programmed cell death 1, programmed cell death factor 1) is a member of the CD28 gene family and is expressed in activated T cells, B cells and myeloid cells. The ligands PD-L1 and PD-L2 of PD-1 belong to the B7 superfamily, in which PD-L1 is expressed in many cells, including T cells, B cells, endothelial cells and epithelial cells, while PD-L2 is only expressed in Antigen presenting cells such as dendritic cells and macrophages.
T细胞对清除病毒感染起着非常重要的作用，但T细胞抗病毒反应通常与免疫病理相关。PD-1在负调节T细胞的活化过程中起着非常重要的作用，PD-1介导的对T细胞负调节作用可减少促感染过程引起的组织损伤，但是阻断或抑制PD-1的负调节作用可导致自身免疫性疾病，例如PD-1基因敲除小鼠更能有效的清楚胰腺病毒感染，但是却导致了更严重的肝脏损伤。另外，高表达PD-1的肿瘤伴随着很难被检测到的癌症。一种实施有效的方法是通过体内注射抗体对PD-1的表达进行调控。T cells play a very important role in clearing viral infections, but T cell antiviral responses are often associated with immunopathology. PD-1 plays a very important role in the process of negatively regulating the activation of T cells. The negative regulation of T cells mediated by PD-1 can reduce the tissue damage caused by the pro-infection process, but blocking or inhibiting the activation of PD-1 Negative regulation can lead to autoimmune diseases, for example, PD-1 knockout mice are more effective in clearing pancreatic virus infection, but lead to more severe liver damage. In addition, tumors with high expression of PD-1 are accompanied by cancers that are difficult to detect. An effective method is to regulate the expression of PD-1 by injecting antibodies in vivo.
由于PD-1抗体的广谱抗肿瘤前景和惊人的药效，业界普遍认为针对PD-1通路的抗体将带来治疗多种肿瘤治疗的突破性的进展；用于治疗非小细胞性肺癌，肾细胞癌，卵巢癌，黑色素瘤，白血病以及贫血病。在2012年和2013年的美国癌症协会(AACR)年会以及美国临床肿瘤协会(ASCO)年会上揭晓的前所未有的临床药效数据后，抗PD-1抗体已成为全球制药行业最炙手可热的在研新药靶点。目前国外已上市的抗PD-1抗体药物是百时美施宝贵的Nivolumab、默沙东的Keytruda。但是由于进口大分子药物对于病人来说需要花费高昂的费用，因此，研发新的抗PD-L1单克隆抗体，减少病人负担，降低治疗费用是一个亟待解决的问题。Due to the broad-spectrum anti-tumor prospects and amazing drug efficacy of PD-1 antibodies, it is generally believed in the industry that antibodies targeting the PD-1 pathway will bring breakthroughs in the treatment of various tumors; for the treatment of non-small cell lung cancer, Renal cell carcinoma, ovarian cancer, melanoma, leukemia and anemia. After the unprecedented clinical efficacy data announced at the American Cancer Society (AACR) annual meeting and the American Society of Clinical Oncology (ASCO) annual meeting in 2012 and 2013, anti-PD-1 antibody has become the hottest topic in the global pharmaceutical industry. Research new drug targets. At present, the anti-PD-1 antibody drugs that have been marketed abroad are Bristol-Myers’ precious Nivolumab and Merck’s Keytruda. However, due to the high cost of imported macromolecular drugs for patients, it is an urgent problem to be solved to develop new anti-PD-L1 monoclonal antibodies to reduce the burden on patients and reduce treatment costs.
发明内容SUMMARY OF THE INVENTION
本发明的目的之一在于提供一种安全的、高效的且特异性的针对PD-1的单克隆抗体，并揭示其示其可变区序列，更具体为其互补决定区(CDR)序列。One of the objectives of the present invention is to provide a safe, efficient and specific monoclonal antibody against PD-1, and reveal its variable region sequence, more specifically its complementarity determining region (CDR) sequence.
本发明的目的之二在于提供上述单克隆抗体的结合物、偶联物、试剂盒或药物组合物。The second object of the present invention is to provide conjugates, conjugates, kits or pharmaceutical compositions of the above-mentioned monoclonal antibodies.
本发明的目的之三在于提供上述单克隆抗体的应用。The third object of the present invention is to provide the application of the above-mentioned monoclonal antibody.
根据本发明的一个方面，本发明提供了一种针对PD-1的单克隆抗体，所述单克隆抗体具有重链可变区和/或轻链可变区：According to one aspect of the present invention, the present invention provides a monoclonal antibody against PD-1, the monoclonal antibody has a heavy chain variable region and/or a light chain variable region:
所述重链可变区包括CDR1、CDR2、CDR3；The heavy chain variable region includes CDR1, CDR2, CDR3;
重链可变区CDR1的氨基酸序列如SEQ ID NO:1所示；或者经取代、缺失或添加一个或多个氨基酸；或与SEQ ID NO:1所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 1; or one or more amino acids are substituted, deleted or added; or at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 1 amino acid sequences with the same or similar functions;
重链可变区CDR2的氨基酸序列如SEQ ID NO:2所示；或者经取代、缺失或添加一个或多个氨基酸；或与SEQ ID NO:2所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 2; or one or more amino acids are substituted, deleted or added; or at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 2 amino acid sequences with the same or similar functions;
重链可变区CDR3的氨基酸序列如SEQ ID NO:3所示；或者经取代、缺失或添加一个或多个氨基酸；或与SEQ ID NO:3所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 3; or one or more amino acids are substituted, deleted or added; or at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 3 amino acid sequences with the same or similar functions;
所述轻链可变区包括CDR1、CDR2、CDR3；The light chain variable region includes CDR1, CDR2, CDR3;
轻链可变区CDR1的氨基酸序列如SEQ ID NO:4所示；或者经取代、缺失或添加一个或多个氨基酸；或与SEQ ID NO:4所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；The amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 4; or one or more amino acids are substituted, deleted or added; or at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 4 amino acid sequences with the same or similar functions;
轻链可变区CDR2的氨基酸序列如SEQ ID NO:5所示；或者经取代、缺失或添加一个或多个氨基酸；或与SEQ ID NO:5所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；The amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 5; or one or more amino acids are substituted, deleted or added; or at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 5 amino acid sequences with the same or similar functions;
轻链可变区CDR3的氨基酸序列如SEQ ID NO:6所示；或者经取代、缺失或添加一个或多个氨基酸；或与SEQ ID NO:6所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列。The amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 6; or one or more amino acids are substituted, deleted or added; or at least 80% homologous to the amino acid sequence shown in SEQ ID NO: 6 amino acid sequences that are identical or similar in function.
在本发明的具体实施方案中，重链可变区CDR1的氨基酸序列如SEQ ID NO:1所示；In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 1;
重链可变区CDR2的氨基酸序列如SEQ ID NO:2所示；The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO:2;
重链可变区CDR3的氨基酸序列如SEQ ID NO:3所示；The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 3;
轻链可变区CDR1的氨基酸序列如SEQ ID NO:4所示；The amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO:4;
轻链可变区CDR2的氨基酸序列如SEQ ID NO:5所示；The amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO:5;
轻链可变区CDR3的氨基酸序列如SEQ ID NO:6所示。The amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO:6.
进一步，所述重链可变区的序列如SEQ ID NO:7所示，或者经取代、缺失或添加一个或多个氨基酸；或与SEQ ID NO:7所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；Further, the sequence of the heavy chain variable region is shown in SEQ ID NO: 7, or one or more amino acids are substituted, deleted or added; or at least 80% identical to the amino acid sequence shown in SEQ ID NO: 7 amino acid sequences that are identical or similar in function;
进一步，所述轻链可变区的序列如SEQ ID NO:8所示，或者经取代、缺失或添加一个或多个氨基酸；或与SEQ ID NO:8所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列。Further, the sequence of the light chain variable region is shown in SEQ ID NO: 8, or one or more amino acids are substituted, deleted or added; or at least 80% identical to the amino acid sequence shown in SEQ ID NO: 8 amino acid sequences that are identical or similar in function.
在本发明的具体实施方案中，所述重链可变区的序列如SEQ ID NO:7所示，所述轻链可变区的序列如SEQ ID NO:8所示。In a specific embodiment of the present invention, the sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the sequence of the light chain variable region is shown in SEQ ID NO:8.
优选的，本发明的所述单克隆抗体，其重链类型为IgG1、IgG2、IgG3或Ig4。更优选为IgG1。Preferably, the heavy chain type of the monoclonal antibody of the present invention is IgG1, IgG2, IgG3 or Ig4. More preferably IgG1.
本发明所述的单克隆抗体不仅包括可变区，还包括恒定区。The monoclonal antibodies of the present invention include not only variable regions but also constant regions.
优选的，本发明所述的单克隆抗体的恒定区为人IgG1、IgG2、IgG3或IgG4中的任意一种。Preferably, the constant region of the monoclonal antibody of the present invention is any one of human IgG1, IgG2, IgG3 or IgG4.
根据本发明的另一个方面，本发明提供了一种编码前面所述的单克隆抗体的DNA分子。According to another aspect of the present invention, the present invention provides a DNA molecule encoding the aforementioned monoclonal antibody.
可以利用本领域任一已知的方法得到DNA分子序列，例如，如果已知抗体的核苷酸序列，就可以从化学合成的寡核苷酸装配出编码所述抗体的DNA分子序列。The DNA molecule sequence can be obtained by any method known in the art, for example, if the nucleotide sequence of the antibody is known, the DNA molecule sequence encoding the antibody can be assembled from chemically synthesized oligonucleotides.
或者，可以从来自合适来源的核酸中产生编码抗体的DNA分子。如果无法得到含有编码特定抗体的DNA分子的克隆，但已知所述抗体分子的序列，那么可以通过化学合成得到编码所述免疫球蛋白的DNA分子，或利用与所述序列的3’和5’末端可杂交的合成引物从合适的来源通过PCR扩增得到编码所述免疫球蛋白的DNA分子，或通过利用特异于所述特定基因序列的寡核苷酸探针通过克隆得到编码所述免疫球蛋白的DNA分子，以例如从cDNA文库中鉴定出编码所述抗体的cDNA克隆。然后可以利用本领域任一熟知的方法将PCR所产生的扩增DNA分子克隆到可复制的克隆载体内。Alternatively, antibody-encoding DNA molecules can be produced from nucleic acid from a suitable source. If a clone containing a DNA molecule encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the DNA molecule encoding the immunoglobulin can be obtained by chemical synthesis, or by using the 3' and 5' of the sequence DNA molecules encoding said immunoglobulins are obtained by PCR amplification of synthetic primers whose ends are hybridizable from a suitable source, or by cloning using oligonucleotide probes specific for said particular gene sequence. globulin DNA molecules to identify cDNA clones encoding said antibodies, eg, from cDNA libraries. The amplified DNA molecules generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.
根据本发明的又一个方面，本发明提供了一种表达载体，包括前面所述的DNA分子。According to yet another aspect of the present invention, the present invention provides an expression vector comprising the aforementioned DNA molecule.
根据本发明的又一个方面，本发明提供了一种宿主细胞，所述宿主细胞经由前面所述的表达载体转化或转染而来。According to yet another aspect of the present invention, the present invention provides a host cell, which is transformed or transfected with the aforementioned expression vector.
可用于本发明的宿主细胞包括但不限于微生物，例如经含有抗体编码序列的重组噬菌体DNA、质粒DNA或粘粒DNA表达载体转化的细菌(例如大肠杆菌(E.coli)、枯草芽孢杆菌(B.subtilis))；经含有抗体编码序列的重组酵母表达载体转化的酵母菌例如酵母属(Saccharomyces)、毕赤酵母属(Pichia))；经含有抗体编码序列的重组病毒表达载体(例如杆状病毒)感染的昆虫细胞系统；经重组病毒表达载体(例如花椰菜花叶病毒(CaMV)；烟草花叶病毒(TMV)感染的或经含有抗体编码序列的重组质粒表达载体(例如Ti质粒)转化的植物细胞系统；或携带含有来源于哺乳动物细胞基因组的启动子(例如金属硫蛋白启动子)或来源于哺乳动物病毒的启动子(例如，腺病毒晚期启动子、痘苗病毒7.5K启动子)的重组表达构建体的哺乳动物细胞系统(例如COS、CHO、BHK、293、3T3细胞)。Host cells that can be used in the present invention include but are not limited to microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences (such as Escherichia coli (E. .subtilis)); yeasts transformed with recombinant yeast expression vectors containing antibody coding sequences, such as Saccharomyces, Pichia); recombinant virus expression vectors containing antibody coding sequences (such as baculovirus ) infected insect cell systems; plants infected with recombinant viral expression vectors (e.g. cauliflower mosaic virus (CaMV); tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmids) containing antibody coding sequences Cellular systems; or recombinants containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter) Mammalian cell systems (eg COS, CHO, BHK, 293, 3T3 cells) expressing the construct.
根据本发明的又一个方面，本发明提供了一种结合物，包含与化学标记或生物标记共价连接的前面所述的单克隆抗体。According to yet another aspect of the present invention, the present invention provides a conjugate comprising the aforementioned monoclonal antibody covalently linked to a chemical label or a biological label.
优选的，所述化学标记为同位素标记、免疫毒素标记和/或化学药物标记；所述生物标记为生物素标记、亲和素标记或酶标记。Preferably, the chemical label is isotope label, immunotoxin label and/or chemical drug label; the biomarker is biotin label, avidin label or enzyme label.
根据本发明的又一个方面，本发明提供了一种偶联物，其由前面所述的单克隆抗体和/或前面所述的结合物与固体介质或半固体介质偶联形成。According to yet another aspect of the present invention, the present invention provides a conjugate, which is formed by coupling the above-mentioned monoclonal antibody and/or the above-mentioned conjugate with a solid medium or a semi-solid medium.
固体介质或半固体介质的实例，如，酶标板、磁性或非磁性免疫微球或免疫颗粒，细胞分离介质，所述细胞分离介质的实例，如聚乙烯吡咯烷酮包被的胶质二氧化硅，多聚糖和泛影酸钠或其衍生物。Examples of solid or semi-solid media, such as microtiter plates, magnetic or non-magnetic immunospheres or particles, cell separation media, examples of such cell separation media are polyvinylpyrrolidone-coated colloidal silica , polysaccharides and sodium diatrizoate or its derivatives.
根据本发明的又一个方面，本发明提供了一种药物组合物，包括前面所述的免疫有效量的单克隆抗体、和/或前面所述的结合物、和/或前面所述的偶联物。According to another aspect of the present invention, the present invention provides a pharmaceutical composition, comprising the above-mentioned immunologically effective amount of the monoclonal antibody, and/or the above-mentioned conjugate, and/or the above-mentioned conjugated things.
本发明的药物组合物可以和其他药物联合应用，其他药物包括抗肿瘤药，所述的抗肿瘤药物包括目前经过FDA批准的所有抗肿瘤药。所述的抗肿瘤药包括经FDA批准的药物或未批准的药物。所述的抗肿瘤药包括已知靶向抗肿瘤药与未知靶向抗肿瘤药。所述的抗肿瘤药还包括任意的化疗药或化合物。The pharmaceutical composition of the present invention can be used in combination with other drugs, and the other drugs include antineoplastic drugs, and the antineoplastic drugs include all antineoplastic drugs currently approved by the FDA. The antineoplastic drugs include FDA-approved drugs or unapproved drugs. The antineoplastic drugs include known targeted antineoplastic drugs and unknown targeted antineoplastic drugs. The antineoplastic drugs also include any chemotherapeutic drugs or compounds.
靶向抗肿瘤药包括(但不限于)17-AAG、2-deoxyglucose、阿比特龙(Abiraterone)、ABT-263、AC-220、AT-406、AZD4547、AZD5363、AZD7762、BI-2536、Birinapant、BMS-754807、硼替佐米(Bortezomib)、BX-795、卡博替尼(Cabozantinib)、CAL-101、卡非佐米(Carfilzomib)、克唑替尼(crizotinib)、danusertib、达沙替尼(Dasatinib)、Dovitinib、Elesclomol、Embelin、恩替诺特(Entinostat)(MS-275)、Enzastaurin、依维莫司(Everolimus)、Foretinib、氟维司群(Fulvestrant)、Ganetespib、GDC0941、GSK429286A、JQ1、KU-55933、KX2-391、Lenvatinib、Linifanib、Linsitinib、LY2157299、Masitinib、MK-1775、MK-2206、MLN-4924、Neratinib、NU7441、Nutlin-3、NVP-BEZ235、NVP-BKM120、Orantinib、PCI-32765、PD-0325901、PD-0332991、PD-173074、PH-797804、PRT062607、R-406、Refametinib、瑞戈非尼(Regorafenib)、SCH900776、sgi-1776、索拉非尼(Sorafenib)、舒尼替尼(Sunitinib)、TAE684、替西莫司(Temsirolimus)、TG-101348、Tideglusib、Tipifarnib、Tivantinib、Toremifene、Tozasertib、曲美替尼(Trametinib)、维甲酸(Tretinoin)、Triptolide、伐地考昔(Valdecoxib)、维莫德吉(Vismodegib)、Volasertib、伏立诺他(Vorinostat)、YM-155、CHIR-99021、NVP-BGJ398、LY2090314。Targeted antineoplastic drugs include (but not limited to) 17-AAG, 2-deoxyglucose, Abiraterone, ABT-263, AC-220, AT-406, AZD4547, AZD5363, AZD7762, BI-2536, Birinapant, BMS-754807, Bortezomib, BX-795, Cabozantinib, CAL-101, Carfilzomib, crizotinib, danusertib, dasatinib ( Dasatinib), Dovitinib, Elesclomol, Embelin, Entinostat (MS-275), Enzastaurin, Everolimus, Foretinib, Fulvestrant, Ganetespib, GDC0941, GSK429286A, JQ1, KU-55933, KX2-391, Lenvatinib, Linifanib, Linsitinib, LY2157299, Masitinib, MK-1775, MK-2206, MLN-4924, Neratinib, NU7441, Nutlin-3, NVP-BEZ235, NVP-BKM120, Oratinib, PCI- 32765, PD-0325901, PD-0332991, PD-173074, PH-797804, PRT062607, R-406, Refametinib, Regorafenib, SCH900776, sgi-1776, Sorafenib, Suni Sunitinib, TAE684, Temsirolimus, TG-101348, Tideglusib, Tipifarnib, Tivantinib, Toremifene, Tozasertib, Trametinib, Tretinoin, Triptolide, Valdecoxib , Vismodegib, Volasertib, Vorinostat, YM-155, CHIR-99021, NVP-BGJ398, LY2090314.
所述的化疗药包括(但不限于)六甲蜜胺、氨鲁米特、阿那罗唑、阿扎胞苷、苯达莫司汀、白消安、卡巴他赛、卡培他滨、卡铂、顺铂、克拉曲滨、氯法拉宾、环磷酰胺、阿糖胞苷、达卡巴嗪、柔红霉素、地西他滨、多西他赛、多柔比星、表柔比星、依托泊甙、伊西美坦、氟尿苷、氟达拉滨、氟尿嘧啶、吉西他滨、羟基脲、依达比星、异环磷酰胺、伊立替康、来那度胺、来曲唑、亚叶酸、洛莫司丁、6-巯基嘌呤、美司钠、甲胺喋呤、米托坦、米托蒽醌、奥沙利铂、紫杉醇、奈拉滨、培美曲塞、喷司他汀、普拉曲沙、甲苄肼、雷替曲噻、链脲霉素、替莫唑胺、替尼泊苷、硫鸟嘌呤、拓扑替康、长春花碱、长春瑞滨、唑来膦酸。The chemotherapeutic drugs include (but not limited to) hexamethylmelamine, aminoglutethimide, anarazole, azacitidine, bendamustine, busulfan, cabazitaxel, capecitabine, capecitabine, Platinum, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, decitabine, docetaxel, doxorubicin, epirubicin , etoposide, exemestane, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, edarubicin, ifosfamide, irinotecan, lenalidomide, letrozole, folinic acid , lomustine, 6-mercaptopurine, mesna, methotrexate, mitotane, mitoxantrone, oxaliplatin, paclitaxel, nelarabine, pemetrexed, pentostatin, Latrexate, procarbazine, raltitrexate, streptozotocin, temozolomide, teniposide, thioguanine, topotecan, vinblastine, vinorelbine, zoledronic acid.
根据本发明的又一个方面，本发明提供了一种试剂盒，包括前面所述的诊断有效量单克隆抗体、和/或前面所述的结合物、和/或前面所述的偶联物。According to yet another aspect of the present invention, the present invention provides a kit, comprising the aforementioned diagnostically effective amount of the monoclonal antibody, and/or the aforementioned conjugate, and/or the aforementioned conjugate.
根据本发明的又一个方面，前面所述的单克隆抗体和/或前面所述的结合物和/或前面所述的偶联物在制备检测PD-1的产品中的应用。According to yet another aspect of the present invention, the application of the above-mentioned monoclonal antibody and/or the above-mentioned conjugate and/or the above-mentioned conjugate in the preparation of a product for detecting PD-1.
优选地，所述检测产品是试剂盒、酶标板、或芯片。Preferably, the detection product is a kit, a microtiter plate, or a chip.
根据本发明的又一个方面，前面所述的单克隆抗体和/或前面所述的结合物和/或前面所述的偶联物在制备治疗免疫障碍的药物中的用途。According to yet another aspect of the present invention, the use of the above-mentioned monoclonal antibody and/or the above-mentioned conjugate and/or the above-mentioned conjugate in the preparation of a drug for treating immune disorders.
根据本发明的又一个方面，所述的单克隆抗体和/或前面所述的结合物和/或前面所述的偶联物在制备治疗免疫障碍的药物中的用途。According to yet another aspect of the present invention, the use of the monoclonal antibody and/or the above-mentioned conjugate and/or the above-mentioned conjugate in the preparation of a drug for treating immune disorders.
本发明的免疫障碍的非限定性实例包括但不限于，类风湿性关节炎、多发性硬化、炎性肠病、克罗恩病、全身性红斑狼疮、I型糖尿病、移植排斥、移植物抗宿主病、过度增生性免疫障碍、肿瘤和感染性疾病。Non-limiting examples of immune disorders of the invention include, but are not limited to, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, systemic lupus erythematosus, type I diabetes, transplant rejection, graft resistance Host diseases, hyperproliferative immune disorders, tumors and infectious diseases.
可以用于治疗的肿瘤种类没有特别限制，任何实体的、非实体的、恶性的或良性的肿瘤均包括在本发明的范围之内。肿瘤的实例包括但不限于：皮肤癌，白血病，肾上腺皮质癌，胆管癌，膀胱癌，骨癌，脑癌，乳腺癌，气管及支气管肿瘤，淋巴瘤，神经系统肿瘤，宫颈癌，肠癌，肛门癌，子宫内膜癌、食管癌，鼻咽癌，卵巢癌，肉瘤，眼癌，恶性纤维组织细胞癌，胆囊癌，胃癌，结直肠癌、胃肠道类癌瘤，胃肠道间质瘤，胚细胞瘤，头颈癌，肝癌，下咽癌，黑色素瘤，胰腺癌，肾癌，喉癌，唇癌，口腔癌，口咽癌，肺癌，间皮瘤，骨髓瘤，甲状旁腺癌，阴茎癌，嗜络细胞瘤，垂体瘤，前列腺癌，视网膜母细胞瘤，涎腺癌，皮肤癌，睾丸癌，喉癌，胸腺瘤，甲状腺癌，尿道癌，阴道癌，外阴癌。The types of tumors that can be used for treatment are not particularly limited, and any solid, non-solid, malignant or benign tumors are included within the scope of the present invention. Examples of tumors include, but are not limited to: skin cancer, leukemia, adrenocortical cancer, cholangiocarcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, trachea and bronchus tumors, lymphoma, nervous system tumors, cervical cancer, bowel cancer, Anal cancer, endometrial cancer, esophageal cancer, nasopharyngeal cancer, ovarian cancer, sarcoma, eye cancer, malignant fibrohistiocellular carcinoma, gallbladder cancer, gastric cancer, colorectal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor Tumor, blastoma, head and neck cancer, liver cancer, hypopharyngeal cancer, melanoma, pancreatic cancer, kidney cancer, laryngeal cancer, lip cancer, oral cavity cancer, oropharyngeal cancer, lung cancer, mesothelioma, myeloma, parathyroid cancer , Penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, retinoblastoma, salivary gland cancer, skin cancer, testicular cancer, laryngeal cancer, thymoma, thyroid cancer, urethral cancer, vaginal cancer, vulvar cancer.
在本发明的其它方面中，还提供了一种抗PD-1的单克隆抗体，其中所述抗体可变区的基因和蛋白质序列的同系物或修饰物；同系物为抗体可变区的蛋白序列，其同系性达到上述序列的60％或以上、70％或以上、80％或以上；修饰物为对所述抗体的氨基酸或其核酸序列通过取代、增加或截短进行改变的产物仍旧保留结合PD-1的能力。In other aspects of the present invention, an anti-PD-1 monoclonal antibody is also provided, wherein the homolog or modification of the gene and protein sequence of the variable region of the antibody; the homolog is the protein of the variable region of the antibody Sequence whose homology reaches 60% or more, 70% or more, 80% or more of the above sequence; the modification is the product of changing the amino acid of the antibody or its nucleic acid sequence through substitution, addition or truncation, and still remains Ability to bind PD-1.
本发明还提供了一种检测PD-1的方法，所述方法包括如下步骤:The present invention also provides a method for detecting PD-1, said method comprising the steps of:
(1)提取含有PD-1的样品；(1) Extract samples containing PD-1;
(2)将步骤(1)的样品与本发明的单克隆抗体、或本发明的结合物、或本发明的偶联物接触；(2) contacting the sample of step (1) with the monoclonal antibody of the present invention, or the conjugate of the present invention, or the conjugate of the present invention;
(3)检测抗原抗体的免疫反应。(3) Detect the immune response of antigen and antibody.
本文所用的术语“单克隆抗体”指从一类基本均一的群体获得的抗体，除少数可能存在的天然发生的突变外，该群体中包含的单个抗体是相同的。修饰语“单克隆”仅表示抗体的特性，是从基本均一的抗体群中获得的，这不能解释成需要用任何特殊方法来生产抗体。As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population comprising individual antibodies that are identical except for a few possible naturally occurring mutations. The modifier "monoclonal" only indicates the characteristics of the antibody, which is obtained from a substantially homogeneous antibody population, which should not be construed as requiring any special method to produce the antibody.
本文所用的术语“单克隆抗体”不仅包括完整的免疫球蛋白，还包括单克隆抗体的免疫学活性部分，包括但不限于Fab、Fab’和F(ab’)2、Fd、单链Fv(scFv)、单链抗体、二硫键连接的Fv(sdFv)和包括VL或VH结构域的单域抗体。包括单链抗体在内的结合抗原的抗体片段可以包括单独的可变区或与以下的全部或一部分组合的可变区：绞链区、CH1、CH2和CH3结构域；术语“单克隆抗体”还包括抗原结合片段，其包括可变区与绞链区、CH1、CH2和CH3结构域的任一组合。The term "monoclonal antibody" as used herein includes not only intact immunoglobulins, but also immunologically active portions of monoclonal antibodies, including but not limited to Fab, Fab' and F(ab')2, Fd, single chain Fv( scFv), single chain antibodies, disulfide-linked Fv (sdFv) and single domain antibodies comprising VL or VH domains. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region alone or in combination with all or a portion of the following: hinge region, CH1, CH2, and CH3 domains; the term "monoclonal antibody" Also included are antigen-binding fragments comprising any combination of variable and hinge regions, CH1 , CH2 and CH3 domains.
本文所述的“样品”涵盖了多种样品类型，包括生物学来源的血液及其它体液样品，实体组织样品如活检组织样品或者组织培养物，或者衍生自其中的细胞或者其后代。该术语还包括在获得后已经通过任何方式处理的样品，例如用试剂处理、溶解、或者富集某些成分如蛋白质或者多核苷酸。该术语涵盖了得自任何物种的各种临床样品，也包括培养的细胞、细胞上清和细胞溶解产物。A "sample" as used herein encompasses a variety of sample types, including blood and other bodily fluid samples of biological origin, solid tissue samples such as biopsy tissue samples or tissue cultures, or cells derived therefrom or progeny thereof. The term also includes samples that have been manipulated in any way after they are obtained, such as treatment with reagents, solubilization, or enrichment for certain components such as proteins or polynucleotides. The term covers a variety of clinical samples from any species and also includes cultured cells, cell supernatants and cell lysates.
附图说明Description of drawings
图1显示pcDNATM5/FRT的物理图谱；Figure 1 shows the physical map of pcDNATM 5/FRT;
图2显示利用琼脂糖电泳检测PCR产物和酶切产物的电泳图，其中：A：Kappa-BGHpoly A片段PCR产物，泳道1代表DL2000，泳道2代表PCR产物，B：Kappa-BGH poly A片段XbaI，BamHI酶切，泳道1代表DL2000，泳道2代表酶切片段，C：PCMV/T7片段PCR产物，泳道1代表DL2000，泳道2代表PCR产物，D：PCMV/T7片段BamHI，NotI酶切，泳道1代表DL2000，泳道2代表酶切片段，E：重链恒定区片段PCR产物，泳道1代表DL2000，泳道2代表PCR产物，F：重链恒定区片段NotI，PmeI酶切，泳道1代表DL2000，泳道2代表酶切产物。Figure 2 shows the electrophoresis of PCR products and restriction products detected by agarose electrophoresis, in which: A: Kappa-BGH poly A fragment PCR product, lane 1 represents DL2000, and lane 2 represents PCR product, B: Kappa-BGH poly A fragment XbaI , BamHI digestion, lane 1 represents DL2000, lane 2 represents the fragment, C: PCMV/T7 fragment PCR product, lane 1 represents DL2000, lane 2 represents the PCR product, D: PCMV/T7 fragment BamHI, NotI digestion, lane 1 represents DL2000, lane 2 represents the enzyme-digested fragment, E: PCR product of heavy chain constant region fragment, lane 1 represents DL2000, lane 2 represents PCR product, F: heavy chain constant region fragment NotI, PmeI digestion, lane 1 represents DL2000, Lane 2 represents the digested product.
图3显示利用显示利用琼脂糖电泳检测构建载体的酶切产物电泳图，其中泳道1代表DL2000，泳道2代表EcoRI酶切片段，泳道3代表HindIII酶切片段，泳道4代表Bsiwl酶切片段，泳道5代表ClaI酶切片段，泳道6代表NheI酶切片段，泳道7代表XbaI酶切片段，泳道8代表BamHI+NotI酶切片段，泳道9代表NotI+PmeI酶切片段；Figure 3 shows the electrophoresis diagram of the digested product of the constructed vector by agarose electrophoresis, wherein lane 1 represents DL2000, lane 2 represents EcoRI fragments, lane 3 represents HindIII fragments, and lane 4 represents Bsiwl fragments, lane 5 represents the ClaI digested fragment, lane 6 represents the NheI digested fragment, lane 7 represents the XbaI digested fragment, lane 8 represents the BamHI+NotI digested fragment, and lane 9 represents the NotI+PmeI digested fragment;
图4显示PRT/KIgGl载体的结构示意图；Fig. 4 shows the structural representation of PRT/KIgG1 carrier;
图5显示利用双夹心ELISA检测抗体表达量的统计图；Figure 5 shows a statistical graph of detecting antibody expression by double sandwich ELISA;
图6显示利用间接ELISA检测抗体结合能力的曲线图；Figure 6 shows a graph of detecting antibody binding ability by indirect ELISA;
图7显示利用间接ELISA检测抗体结合特异性的统计图；Figure 7 shows the statistical graph of detecting antibody binding specificity by indirect ELISA;
图8显示利用Biacore检测抗体亲和能力的曲线图；Figure 8 shows a graph utilizing Biacore to detect antibody affinity;
图9显示利用ELISA检测抗体竞争结合抗原的结合曲线图；Fig. 9 shows the binding curve of detecting antibody competition binding antigen by ELISA;
图10显示利用ELISA检测抗体对T细胞IFN-γ表达的影响的统计图。Fig. 10 is a statistical graph showing the effect of antibodies on T cell IFN-γ expression detected by ELISA.
具体实施方式Detailed ways
下面通过实施例进一步说明本发明。应该理解的是，本发明的实施例是用于说明本发明而不是对本发明的限制。根据本发明的实质对本发明进行的简单改进都属于本发明要求保护的范围。The present invention is further illustrated below by way of examples. It should be understood that the embodiments of the present invention are used to illustrate the present invention rather than limit the present invention. The simple improvements made to the present invention according to the essence of the present invention all belong to the protection scope of the present invention.
实施例1抗PD-1抗体制备Example 1 Preparation of anti-PD-1 antibody
1、PRT/KIgG1全长抗体表达载体构建1. Construction of PRT/KIgG1 full-length antibody expression vector
构建全长抗体表达载体需要在pcDNATM5/FRT载体骨架(图1)的基础上依次引入轻链可变区克隆位点、CK基因、BGH polyA、PCMV/T7启动子基因序列、抗体重链可变区克隆位点、抗体重链恒定区基因等。To construct a full-length antibody expression vector, it is necessary to sequentially introduce the light chain variable region cloning site, CK gene, BGH polyA, PCMV/T7 promoter gene sequence, and antibody heavy chain on the basis of the pcDNATM 5/FRT vector backbone (Figure 1). Variable region cloning sites, antibody heavy chain constant region genes, etc.
采用overlap PCR、定点克隆等分子生物学手段构建全长抗体表达载体，所用引物如下：The full-length antibody expression vector was constructed by overlapping PCR, site-directed cloning and other molecular biology methods, and the primers used were as follows:
Kappa-BGH poly A基因序列引物Kappa-BGH poly A gene sequence primer
P-Kappa-TY：P-Kappa-TY:
5’-CCTCTAGAGAATTCAAGCTTTAGCGTACGGTGGCTGCACCATC-3’5'-CCTCTAGAGAATTCAAGCTTTAGCGTACGGTGGCTGCACCATC-3'
Kappa-BGH3：Kappa-BGH3:
5’-GTCGAGGCTGATCAGCGGCTAACACTCTCCCCTGTTG-3’5'-GTCGAGGCTGATCAGCGGCTAACACTTCCCCTGTTG-3'
Kappa-BGH5：Kappa-BGH5:
5’-CAACAGGGGAGAGTGTTAGCCGCTGATCAGCCTCGAC-3’5'-CAACAGGGGAGAGTGTTAGCCGCTGATCAGCCTCGAC-3'
Kappa3：Kappa3:
5’-CCGGATCCACATTCCACAGGGGCCATCC-3’5'-CCGGATCCACATTCCACAGGGGCCATCC-3'
PCMV/T7基因序列引物PCMV/T7 gene sequence primer
Pcmv/t7-up:5’-GGGGATCCTAGTTATTAATAGTAATCAA-3’Pcmv/t7-up:5'-GGGGATCCTAGTTATTAATAGTAATCAA-3'
Pcmv/t7-down:5’-GCGGCCGCTTGGGTCTCCCTATAGTGAG-3’Pcmv/t7-down:5'-GCGGCCGCTTGGGTCTCCCTATAGTGAG-3'
Heavy链恒定区基因引物：Heavy chain constant region gene primers:
P-Ig G1H-TY：P-Ig G1H-TY:
5’-ATCGGCGGCCGCGAATTCATCGATTAGGCTAGCACCAAGGGCCCATC-3’5'-ATCGGCGGCCGCGAATTCATCGATTAGGCTAGCACCAAGGGCCCATC-3'
Heavy-3：Heavy-3:
5’-GTTTAAACTCATTTACCCGGAGACAGGGAGAGGCTCTTCTGCGTGTA-3’。5'-GTTTAAACTCATTTACCCGGAGACAGGGAGAGGCTCTTCTGCGTGTA-3'.
借助overlap PCR将轻链可变区克隆位点(酶A、酶B)、Kappa链恒定区、BGH Poly A序列进行拼接，获得大小为650bp目标片段，并构建T载体，酶切测序正确(图2A，B)，通过XbaI，BamHI酶切位点克隆入pcDNATM5/FRT载体。在此基础上，通过BamHI，NotI酶切位点，将测序正确的PCMV/T7启动子基因序列(图2C，D)克隆入载体中。The light chain variable region cloning site (enzyme A, enzyme B), Kappa chain constant region, and BGH Poly A sequence were spliced by overlapping PCR to obtain a 650bp target fragment, and a T vector was constructed, and the enzyme digestion sequence was correct (Fig. 2A, B), cloned into pcDNATM 5/FRT vector through XbaI, BamHI restriction sites. On this basis, the sequenced correct PCMV/T7 promoter gene sequence (FIG. 2C, D) was cloned into the vector through BamHI and NotI restriction sites.
PCR拼接重链可变区克隆位点(酶C，酶D)，IgGl重链恒定区基因序列(CH1，CH2，CH3获得大小约1000bp的目标片段，克隆T载体，测序正确(图2E，F)，并通过NotI，PmeI酶切位点定向克隆入前述载体中。PCR splicing heavy chain variable region cloning sites (enzyme C, enzyme D), IgG1 heavy chain constant region gene sequence (CH1, CH2, CH3 to obtain a target fragment of about 1000bp in size, clone T vector, sequenced correctly (Fig. 2E, F ), and through NotI, PmeI restriction site directional cloning into the aforementioned vector.
根据载体构建过程，以及载体自身的酶切位点特征，选择EcoRI，HinIII，BsiwI，C1aI，NheI，Xbai对目标载体进行酶切鉴定(图3)。理论上，该载体中一共含有3个EcoRI酶切位点，EcoRI单酶切将载体切成三条线性片段，结果与预期相符；HinIII，BsiwI，C1aI，NheI，Xbai均为载体中单一酶切位点，将载体切成单一的线性片段，结果与预期相符。进一步分别用酶BamHI+NotI以及NotI+PmeI进行酶切(图3泳道8,9)，分别获得载体大片段与PCMV/T7(约650bp)以及Heavy表达盒(约1000bp)小片段，与预期一致。According to the vector construction process and the restriction site characteristics of the vector itself, EcoRI, HinIII, BsiwI, C1aI, NheI, and Xbai were selected for enzyme digestion identification of the target vector (Figure 3). Theoretically, the vector contains a total of 3 EcoRI restriction sites. EcoRI single digestion cuts the vector into three linear fragments, and the result is in line with expectations; HinIII, BsiwI, C1aI, NheI, and Xbai are all single restriction restriction sites in the vector point, the vector was cut into single linear fragments, and the results were as expected. Further digestion with enzymes BamHI+NotI and NotI+PmeI (lanes 8 and 9 in Figure 3), the large fragment of the vector and the small fragment of PCMV/T7 (about 650bp) and Heavy expression cassette (about 1000bp) were obtained respectively, which were consistent with expectations .
上述结果表明，目标载体构建成功，命名为PRT/KIgGl，其结构示意图见图4。理论而言，该载体适用于各种全长抗体的表达。The above results show that the target vector was successfully constructed and named as PRT/KIgG1, and its structural schematic diagram is shown in FIG. 4 . In theory, this vector is suitable for the expression of various full-length antibodies.
2、抗PD-1抗体表达量测定2. Determination of the expression level of anti-PD-1 antibody
(1)利用基因合成的方法合成抗体重链可变区序列(序列如SEQ ID NO.7所示)、轻链可变区序列(序列如SEQ ID NO.8所示)，并将其利用分子克隆的方法将片段克隆入PRT/KIgGl中。(1) Synthesize antibody heavy chain variable region sequence (sequence as shown in SEQ ID NO.7) and light chain variable region sequence (sequence as shown in SEQ ID NO.8) by using gene synthesis method, and use them Methods of Molecular Cloning Fragments were cloned into PRT/KIgG1.
(2)瞬时转染(2) Transient transfection
将处于对数生长期的293T细胞以5*105个/孔密度接种于6孔板中，12h后弃换新鲜培养基。4μg抗体表达质粒加入250μL MEM培养基中，充分混匀后往里加8μLLipofectamineTM 2000脂质体，混匀室温放置20min，将混合物加入对应的6孔板中，培养48h后收获上清，同时以PRT/KIgGl-MIL75载体(MIL75的重链可变区序列为：QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS；轻链可变区序列为：EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK)作为对照。293T cells in the logarithmic growth phase were seeded in 6-well plates at a density of 5*105 cells/well, and the fresh medium was discarded after 12 hours. Add 4 μg of antibody expression plasmids to 250 μL of MEM medium, mix well, add 8 μL of LipofectamineTM 2000 liposomes, mix well and place at room temperature for 20 minutes, add the mixture to the corresponding 6-well plate, harvest the supernatant after 48 hours of culture, and at the same time use PRT /KIgGl-MIL75载体(MIL75的重链可变区序列为：QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS；轻链可变区序列为：EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK)作为对照。
(3)双夹心ELISA测定瞬时表达上清中抗体表达量(3) Determination of antibody expression in transient expression supernatant by double sandwich ELISA
用包被液稀释GAH-IgG抗体至2μg/mL，100μL每孔加入酶联板中，置于湿盒中4℃过夜。洗板机清洗酶联板3次，1.5％酪蛋白封闭，每孔200μL，湿盒37℃封闭lh。1xPBS稀释Herceptin至0.5μg/mL，在此基础上进行2倍稀释获得8个浓度梯度，同时用1xPBS稀释瞬时表达上清至625倍、3125倍、15625倍。将Herceptin与瞬时表达上清按照100μL/每孔加入酶联板中37℃反应1h，清洗酶联板3次，加入羊抗人酶标二抗室温反应45min，清洗酶联板5次并加入100μLTMB底物显色，反应3min并用100μL 2N H2SO4终止反应，酶联免疫检测仪450nm读数。以标准品浓度LN值为横坐标，相应的OD值为纵坐标，绘制一条标准曲线并添加线性回归曲线及其相关系数R2(R2>0.95)，并用y＝kx+b直线方程计算样品中抗体浓度。Dilute the GAH-IgG antibody to 2 μg/mL with coating solution, add 100 μL per well to the enzyme-linked plate, and place it in a humid box at 4 °C overnight. Wash the ELISA plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. 1xPBS was used to dilute Herceptin to 0.5 μg/mL, and 2-fold dilution was performed on this basis to obtain 8 concentration gradients. At the same time, 1xPBS was used to dilute the transient expression supernatant to 625 times, 3125 times, and 15625 times. Add 100 μL/well of Herceptin and transient expression supernatant to the enzyme-linked plate for 1 hour at 37°C, wash the enzyme-linked plate 3 times, add goat anti-human enzyme-labeled secondary antibody to react at room temperature for 45 minutes, wash the enzyme-linked plate 5 times and add 100 μL TMB The substrate was colored, reacted for 3 minutes and terminated with 100 μL 2N H2 SO4 , and read at 450 nm with an enzyme-linked immunosorbent detector. Take the standard concentration LN value as the abscissa, and the corresponding OD value as the ordinate, draw a standard curve and add a linear regression curve and its correlation coefficient R2 (R2 >0.95), and use the y=kx+b linear equation to calculate the sample Medium antibody concentration.
(4)结果(4) Results
结果如图5所示，利用上述方法能够表达出抗PD-1抗体。The results are shown in Figure 5, and the anti-PD-1 antibody can be expressed by the above method.
实施例2抗PD-1抗体结合能力测定Example 2 Determination of Anti-PD-1 Antibody Binding Ability
1、抗体大量表达与纯化鉴定1. Massive expression and purification of antibodies
将抗体表达质粒按照以上的转染方法大量转染293T细胞并利用Protein A纯化获得抗体的纯化样品。The antibody expression plasmid was transfected into 293T cells in large quantities according to the transfection method above, and purified by Protein A to obtain purified samples of the antibody.
2、间接ELISA检测抗体抗原结合能力2. Indirect ELISA detection of antibody-antigen binding ability
2.1步骤2.1 steps
包被液稀释PD-1/Fc抗原至1μg/mL，100μL每孔加入酶联板中，置于湿盒中4℃过夜。洗板机清洗酶联板3次，1.5％酪蛋白封闭，每孔200μL，湿盒37℃封闭1h。用1xPBS稀释抗体至3μg/mL，并以100μL每孔加入酶联板中，湿盒中37℃反应1h，清洗酶联板3次，加入羊抗人(Fab')2-HRP二抗室温反应45min，清洗酶联板5次并加入100μL TMB底物显色，反应3min并用100μL 2N H2SO4终止反应，酶联免疫检测仪450nm读数。并以抗体浓度为横坐标，OD值为纵坐标绘制抗体-抗原结合曲线。利Graphpad分析软件拟合四参数方程曲线，方程为y＝(A-D)/(1+(X/C)^B)+D，其中B代表斜率，C代表EC50。Dilute the PD-1/Fc antigen to 1 μg/mL in the coating solution, add 100 μL per well to the enzyme-linked plate, and place it in a humid box at 4 °C overnight. Wash the enzyme-linked plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute the antibody to 3 μg/mL with 1xPBS, add 100 μL per well to the enzyme-linked plate, react in a wet box at 37°C for 1 hour, wash the enzyme-linked plate 3 times, add goat anti-human (Fab') 2-HRP secondary antibody to react at room temperature After 45 minutes, wash the enzyme-linked plate 5 times and add 100 μL TMB substrate for color development, react for 3 minutes and stop the reaction with 100 μL 2N H2 SO4 , and read at 450 nm with an enzyme-linked immunosorbent detector. And the antibody-antigen binding curve was drawn with the antibody concentration as the abscissa and the OD value as the ordinate. Using Graphpad analysis software to fit the four-parameter equation curve, the equation is y=(AD)/(1+(X/C)^B)+D, where B represents the slope and C represents EC50.
2.2结果2.2 Results
结果如图6所示，本发明制备的抗PD-1抗体结合活性与MIL75相近，能够特异性识别固相载体上的PD-1抗原分子，且随着抗体浓度的增加抗原一抗体之间结合具有良好的剂量依赖性。The results are shown in Figure 6, the binding activity of the anti-PD-1 antibody prepared by the present invention is similar to that of MIL75, and it can specifically recognize the PD-1 antigen molecule on the solid phase carrier, and with the increase of antibody concentration, the combination of antigen and antibody Has good dose dependence.
3、抗体结合抗原特异性检测3. Detection of antibody-binding antigen specificity
3.1步骤3.1 steps
包被液稀释PD-1/Fc，CD28/Fc，CTLA4/Fc，BTLA/Fc，ICOS/Fc抗原至1μg/mL，100μL每孔加入酶联板中，以平行孔作为复孔。置于湿盒中4℃过夜。洗板机清洗酶联板3次，1.5％酪蛋白封闭，每孔200μL，湿盒中37℃封闭1h。用1xPBS稀释抗体至1μg/mL，并以100μL每孔加入酶联板中，湿盒中37℃反应1h，清洗酶联板3次，加入羊抗人(Fab')a-HRP二抗室温反应45min，清洗酶联板5次并加入100μL TMB底物显色，反应5min并用100μL 2N H2SO4终止反应，酶联免疫检测仪450nm读数。并以抗体名称为横坐标，OD值为纵坐标绘制柱形图。Dilute PD-1/Fc, CD28/Fc, CTLA4/Fc, BTLA/Fc, ICOS/Fc antigens to 1 μg/mL in the coating solution, add 100 μL to each well of the enzyme-linked plate, and use parallel wells as duplicate wells. Place in a humid box overnight at 4°C. Wash the enzyme-linked plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a wet box. Dilute the antibody to 1 μg/mL with 1xPBS, add 100 μL per well to the enzyme-linked plate, react in a wet box at 37°C for 1 hour, wash the enzyme-linked plate 3 times, add goat anti-human (Fab') a-HRP secondary antibody to react at room temperature After 45 minutes, wash the enzyme-linked plate 5 times and add 100 μL TMB substrate for color development, react for 5 minutes and stop the reaction with 100 μL 2N H2 SO4 , and read at 450 nm with an enzyme-linked immunosorbent detector. And draw a histogram with the antibody name as the abscissa and the OD value as the ordinate.
3.2结果3.2 Results
结果如图7所示，本发明制备的抗PD-1抗体只特异性识别PD-1分子，而与其他分子不存在交叉识别现象。The results are shown in Figure 7, the anti-PD-1 antibody prepared by the present invention only specifically recognizes PD-1 molecules, and there is no cross-recognition phenomenon with other molecules.
4、抗PD-1抗体亲和力测定4. Anti-PD-1 antibody affinity determination
4.1步骤4.1 steps
利用Biacore测定抗体的亲和力。Antibody affinity was determined using Biacore.
将抗人Fab抗体偶联到CM5芯片上，将抗体样品稀释到1-2μg/ml并以30ml/min流速进样60-150秒。将PD-1/Fc融合蛋白进行梯度稀释至浓度为3.125，6.25，12.5，25，50，100nM并以30μL/min流速进样抗原结合120秒，解离1200S。实验结束后用10mM Glycine-HClpH2.1进行再生60秒。利用Biacore T200分析软件分析结果。The anti-human Fab antibody was coupled to the CM5 chip, the antibody sample was diluted to 1-2 μg/ml and injected at a flow rate of 30ml/min for 60-150 seconds. The PD-1/Fc fusion protein was serially diluted to a concentration of 3.125, 6.25, 12.5, 25, 50, 100nM and injected at a flow rate of 30μL/min for 120 seconds for antigen binding and 1200S for dissociation. Regeneration was performed for 60 seconds with 10 mM Glycine-HCl pH 2.1 after the experiment. The results were analyzed using Biacore T200 analysis software.
4.2结果4.2 Results
结果如表1所示，本发明制备的抗PD-1抗体与MIL75的亲和力相当，图8是抗体的亲和力动力学曲线(选择3.125nM、6.25nM、12.5nM、25nM、50nM五个浓度进行拟合)。The results are shown in Table 1. The anti-PD-1 antibody prepared by the present invention has the same affinity as MIL75. combine).
表1 抗体亲和力测定结果Table 1 Results of antibody affinity determination
实施例3抗PD-1抗体阻断PD-1/PD-L1作用Example 3 Anti-PD-1 antibody blocks the effect of PD-1/PD-L1
1、ELISA检测PDL1/Fc-Biotin与PD-1结合活性1. ELISA detection of PDL1/Fc-Biotin and PD-1 binding activity
1.1步骤1.1 steps
包被液稀释PD-1/Fc抗原至1μg/mL，100μL每孔加入酶联板中，置于湿盒中4℃过夜。洗板机清洗酶联板3次，1.5％酪蛋白封闭，每孔200μL，湿盒37℃封闭1h。用1xPBS稀释PD-L1/Fc-Biotin至48μg/mL，进行3倍稀释共获得12个浓度点。以100μL每孔加入酶联板中，湿盒中37℃反应1h，清洗酶联板3次，加入Peroxidase-Labeled Streptavidin(1:4000)室温反应45min，清洗酶联板5次并加入100μL TMB底物显色，反应3min并用100μL 2N H2SO4终止反应，酶联免疫检测仪450nm读数。以PD-L1/Fc-Biotin浓度为横坐标，OD值为纵坐标绘制PD-1/PD-L1结合曲线，选取PD-L1结合PD-1最低饱和浓度进行抗体阻断实验。Dilute the PD-1/Fc antigen to 1 μg/mL in the coating solution, add 100 μL per well to the enzyme-linked plate, and place it in a humid box at 4 °C overnight. Wash the enzyme-linked plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute PD-L1/Fc-Biotin to 48 μg/mL with 1xPBS, and perform 3-fold dilution to obtain 12 concentration points in total. Add 100 μL per well to the enzyme-linked plate, react in a wet box at 37°C for 1 hour, wash the enzyme-linked plate 3 times, add Peroxidase-Labeled Streptavidin (1:4000) for 45 minutes at room temperature, wash the enzyme-linked plate 5 times and add 100 μL TMB bottom The color was developed, reacted for 3 minutes and terminated with 100 μL 2N H2 SO4 , and read at 450 nm with an enzyme-linked immunosorbent detector. Draw the PD-1/PD-L1 binding curve with the PD-L1/Fc-Biotin concentration as the abscissa and the OD value as the ordinate, and select the lowest saturation concentration of PD-L1 binding to PD-1 for antibody blocking experiments.
1.2结果1.2 Results
摸索出PD-L1/Fc-Biotin参与结合固相抗原PD-1分子最低饱和浓度为5μg/mL。The minimum saturation concentration of PD-L1/Fc-Biotin involved in the binding of solid-phase antigen PD-1 molecules was found to be 5 μg/mL.
2、抗体阻断PD-1/PD-L1相互作用2. Antibody blocking PD-1/PD-L1 interaction
2.1步骤2.1 steps
包被液稀释PD-1/Fc抗原至1μg/mL，100μL每孔加入酶联板中，置于湿盒中4℃过夜。洗板机清洗酶联板3次，1.5％酪蛋白封闭，每孔200μL，湿盒37℃封闭lh。用1xPBS稀释PD-L1/Fc-Biotin至5μg/mL，以上述溶液作为稀释液稀释抗体至抗体浓度500μg/mL，并进行5倍稀释共获得12个浓度梯度。100μL每孔加入酶联板中湿盒中37℃反应1h，清洗酶联板3次，加入Peroxidase-LabeledStreptavidin(1:4000)室温反应45min，清洗酶联板5次并加入100μL TMB底物显色，反应3min并用100μL 2N H2SO4终止反应，酶联免疫检测仪450nm读数。以抗体浓度为横坐标，OD值为纵坐标绘制结合曲线。Dilute the PD-1/Fc antigen to 1 μg/mL in the coating solution, add 100 μL per well to the enzyme-linked plate, and place it in a humid box at 4 °C overnight. Wash the ELISA plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute PD-L1/Fc-Biotin to 5 μg/mL with 1xPBS, dilute the antibody to an antibody concentration of 500 μg/mL with the above solution as the diluent, and perform 5-fold dilution to obtain a total of 12 concentration gradients. Add 100 μL to each well of the enzyme-linked plate and react in a wet box at 37°C for 1 hour, wash the enzyme-linked plate 3 times, add Peroxidase-LabeledStreptavidin (1:4000) to react at room temperature for 45 minutes, wash the enzyme-linked plate 5 times, and add 100 μL TMB substrate for color development , reacted for 3 minutes and terminated the reaction with 100 μL 2N H2 SO4 , and read at 450 nm with an enzyme-linked immunosorbent detector. The binding curve was drawn with the antibody concentration as the abscissa and the OD value as the ordinate.
2.2结果2.2 Results
结果如图9所示，结果表明本发明制备的抗体可以有效阻断PD-1/PD-L1相互作用。The results are shown in Figure 9, and the results show that the antibodies prepared in the present invention can effectively block the PD-1/PD-L1 interaction.
实施例4抗PD-1抗体的体外活性检测Example 4 In vitro activity detection of anti-PD-1 antibody
外周血单个核细胞活性实验Peripheral blood mononuclear cell activity test
取1名健康志愿者全血10-15mL，利用人淋巴细胞分离液分离人PBMC细胞，生理盐水洗细胞2遍，10％FBS 1640培养基重悬细胞并计数，以1*105/孔接种于96孔板中，每孔接种50μL。将梯度的CD3抗体以及CD28抗体以25μL/孔加入对应的培养孔中至二者的终浓度均为1μg/mL，0.8μg/mL，0.6μg/mL，0.4μg/mL，0.2μg/mL，0.1μg/mL。同时在培养体系中以25μL/孔加入梯度PD-L1/Fc融合蛋白至终浓度为0μg/mL，5μg/mL，10μg/mL，20μg/mL。置于37℃细胞培养箱培养72h，收获上清并利用人IFN-γELISA检测试剂盒测定上清中IFN-γ的表达情况。Take 10-15mL of whole blood from a healthy volunteer, use human lymphocyte separation medium to separate human PBMC cells, wash the cells twice with normal saline, resuspend the cells in 10% FBS 1640 medium and count them, inoculate at 1*105 /well Inoculate 50 μL per well in a 96-well plate. Add the gradient CD3 antibody and CD28 antibody into the corresponding culture wells at 25 μL/well to a final concentration of 1 μg/mL, 0.8 μg/mL, 0.6 μg/mL, 0.4 μg/mL, 0.2 μg/mL, 0.1 μg/mL. At the same time, gradient PD-L1/Fc fusion protein was added to the culture system at 25 μL/well to a final concentration of 0 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL. Place them in a 37°C cell culture incubator for 72 hours, harvest the supernatant, and measure the expression of IFN-γ in the supernatant using a human IFN-γELISA detection kit.
基于上述方法测定刺激PBMC活化的最适CD3，CD28浓度以及PD-L1抑制浓度。同样的方法分离人PBMC细胞并以同样的细胞密度接种于96孔板中。配置最适抗体刺激浓度以及PD-L1抑制浓度加入相应的孔中。10％FBS 1640培养基稀释抗体至终浓度为0μg/mL、2.5μg/mL、10μg/mL、40μg/mL加入对应的反应体系，37℃细胞培养箱培养72h，收获上清并利用人IFN-γELISA检测试剂盒测定上清中IFN-γ的表达情况。The optimal CD3, CD28 concentration and PD-L1 inhibitory concentration to stimulate PBMC activation were determined based on the above method. Human PBMC cells were isolated by the same method and seeded in 96-well plates with the same cell density. Configure the optimal antibody stimulating concentration and PD-L1 inhibitory concentration and add them to the corresponding wells. Dilute the antibody in 10% FBS 1640 medium to a final concentration of 0 μg/mL, 2.5 μg/mL, 10 μg/mL, and 40 μg/mL, add to the corresponding reaction system, culture in a cell incubator at 37°C for 72 hours, harvest the supernatant and use human IFN- γELISA detection kit was used to measure the expression of IFN-γ in the supernatant.
结果：结果如图10所示，CD3/CD28抗体能够有效激活T细胞，PD-L1的加入则在一定程度抑制T细胞活性，本发明制备的抗体的加入能够有效逆转T细胞被抑制的状态，进而上调IFN-γ的表达，而且抗体的逆转效果具有剂量依赖性。Results: As shown in Figure 10, the CD3/CD28 antibody can effectively activate T cells, and the addition of PD-L1 can inhibit the activity of T cells to a certain extent. The addition of the antibody prepared by the present invention can effectively reverse the state of T cell suppression. Then up-regulate the expression of IFN-γ, and the reversal effect of the antibody is dose-dependent.
虽然以上仅描述了本发明的具体实施方式范例，但是本领域的技术人员应当理解，这些仅是举例说明，本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更或修改均落入本发明的保护范围。Although the specific implementation examples of the present invention are only described above, those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principle and essence of the present invention, but these changes or modifications all fall within the protection scope of the present invention.
序列表 sequence listing
<110> 中国人民解放军军事科学院军事医学研究院<110> Academy of Military Medical Sciences, Chinese People's Liberation Army
<120> 针对PD-1的单克隆抗体及其应用<120> Monoclonal antibody against PD-1 and its application
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| CN117447596A (en)* | 2019-06-30 | 2024-01-26 | 福州创方医药科技有限公司 | PD-1 targeting monoclonal antibody and application thereof |
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| WO2016205551A2 (en)* | 2015-06-16 | 2016-12-22 | The Regents Of The University Of California | Fzd7 specific antibodies and vaccines to treat cancer and control stem cell function |
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