CN113444177A - anti-IL-1 beta antibodies, pharmaceutical compositions thereof, and uses thereof - Google Patents
anti-IL-1 beta antibodies, pharmaceutical compositions thereof, and uses thereofDownload PDFInfo
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- CN113444177A CN113444177ACN202110316624.4ACN202110316624ACN113444177ACN 113444177 ACN113444177 ACN 113444177ACN 202110316624 ACN202110316624 ACN 202110316624ACN 113444177 ACN113444177 ACN 113444177A
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Abstract
Translated fromChineseDescription
Translated fromChinese技术领域technical field
本发明属于免疫学领域，涉及一种抗IL-1β的抗体、其药物组合物及其用途。具体地，本发明涉及一种抗人IL-1β的抗体；更具体地，本发明涉及一种抗人IL-1β的单克隆抗体；特别具体地，本发明涉及一种抗人IL-1β的人源化单克隆抗体。The invention belongs to the field of immunology, and relates to an anti-IL-1β antibody, a pharmaceutical composition thereof and the use thereof. Specifically, the present invention relates to an anti-human IL-1β antibody; more specifically, the present invention relates to an anti-human IL-1β monoclonal antibody; particularly, the present invention relates to an anti-human IL-1β antibody Humanized monoclonal antibodies.
背景技术Background technique
IL-1β是IL-1家族成员，其在先天免疫和炎症发生中发挥关键作用。IL-1β家族成员包含7个细胞因子(IL-1α，IL-1β，IL-18，IL-33，IL-36α，IL-36β，IL-36γ，IL-37)和三种特异性受体拮抗物(IL-1Ra，IL-36Ra和IL-38)。IL-1β是由无活性的前体蛋白ProIL-1β酶解生成，这一过程主要由炎性小体NLRP3(NOD-like receptor protein 3)介导(Jesus AAand Goldbach-Mansky R.IL-1 blockade in autoinflammatory syndromes.Annu RevMed 2014)。IL-1β is a member of the IL-1 family, which plays a key role in innate immunity and inflammation. IL-1β family members contain 7 cytokines (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-37) and three specific receptors Antagonists (IL-1Ra, IL-36Ra and IL-38). IL-1β is generated from the inactive precursor protein ProIL-1β, which is mainly mediated by the inflammasome NLRP3 (NOD-like receptor protein 3) (Jesus AA and Goldbach-Mansky R.IL-1 blockade). in autoinflammatory syndromes. Annu RevMed 2014).
IL-1β及IL-1α的受体是IL-1R1和IL-1RAcP异二聚体，其中IL-1R1为IL-1β及IL-1α特异性结合的受体亚基，IL-1RAcP(白介素-1-受体协同蛋白)是IL-1家族其它多个成员如IL-33、IL-36等的受体共有组成部分。IL-1R1为跨膜受体，可与IL-1β结合并与IL-1RAcP结合形成受体复合物，激活下游胞内相关信号通路，介导IL-1β相关生物学效应。IL-1β存在天然的负性调节分子，包括IL-1R2，可溶性IL-1R1和IL-1R2，以及IL-1受体拮抗物(IL-1Ra)。IL-R2可与IL-1β结合，然而由于胞内段较短因此无法激活下游通路，可溶性IL-1R1和IL-1R2结合IL-1阻止其与膜定位受体进一步结合；IL-1Ra可与IL-1R1结合进而阻断IL-1α和IL-1β与相应受体的结合(Boraschi D，ItalianiP，Weil S，Martin MU.The family of theinterleukin-1 receptors.Immunol Rev.2018；281(1)：197-232)。The receptors of IL-1β and IL-1α are IL-1R1 and IL-1RAcP heterodimers, of which IL-1R1 is the receptor subunit that IL-1β and IL-1α specifically bind, and IL-1RAcP (interleukin- 1-receptor cooperatin) is a common component of receptors of other members of the IL-1 family, such as IL-33, IL-36, etc. IL-1R1 is a transmembrane receptor that can bind to IL-1β and IL-1RAcP to form a receptor complex, activate downstream intracellular signaling pathways, and mediate IL-1β-related biological effects. There are naturally occurring negative regulatory molecules for IL-1β, including IL-1R2, soluble IL-1R1 and IL-1R2, and the IL-1 receptor antagonist (IL-1Ra). IL-R2 can bind to IL-1β, but cannot activate downstream pathways due to its short intracellular segment. Soluble IL-1R1 and IL-1R2 bind IL-1 to prevent further binding to membrane-localized receptors; IL-1Ra can bind to IL-1R1 binds to block the binding of IL-1α and IL-1β to the corresponding receptors (Boraschi D, ItalianiP, Weil S, Martin MU. The family of the interleukin-1 receptors. Immunol Rev. 2018; 281(1): 197-232).
IL-1β主要由单核细胞和巨噬细胞产生，IL-1β与其受体结合后激活下游NF-κB和MAPK依赖性的促炎性细胞内信号级联反应。IL-1β是COX-2表达的有效诱导剂，产生大量的前列腺素E2(PGE2)抑制IFN-γ的产生，进而抑制Th1型T细胞主导的针对病毒和胞内寄生菌的Th1型免疫反应(Napolitani G，Acosta-Rodriguez EV，Lanzavecchia A，SallustoF.Prostaglandin E2 enhances Th17 responses via modulation of IL-17 and IFN-gamma production by memory CD4+T cells.European journal of immunology.2009；39(5)：1301-12.)。IL-1β的另一关键效应是促进IL-6的分泌。IL-6是诱导幼稚T细胞分化为Th17型T细胞的关键因素(Bettelli E，Carrier Y，Gao W，Korn T，Strom TB，Oukka M，etal.Reciprocal developmental pathways for the generation of pathogeniceffector TH17 and regulatory T cells.Nature.2006；441(7090)：235-8.)；IL-6还可以抑制调节性T细胞的免疫抑制功能，并阻止Th17型T细胞转化为免疫抑制性的Treg型T细胞(Korn T，Mitsdoerffer M，Croxford AL，Awasthi A，Dardalhon VA，Galileos G，eta1.IL-6 controls Th17 immunity in vivo by inhibiting the conversion ofconventional T cells into Foxp3+regulatory T cells.Proceedings of theNational Academy of Sciences of the United States of America.2008；105(47)：18460-5.Pasare C，Medzhitov R.Toll pathway-dependent blockade of CD4+CD25+Tcell-mediated suppression by dendritic cells.Science.2003；299(5609)：1033-6)。IL-1β介导的Th17型免疫反应抑制了CD8+细胞毒性T细胞对病毒感染细胞的杀伤，并增强了病毒感染细胞的存活率，从而可能降低了机体对病毒的防御(Hou W，Kang HS，Kim BS.Thl7 cells enhance viral persistence and inhibit T cell cytotoxicity in a modelof chronic virus infection.The Journal of experimental medicine.2009；206(2)：313-28.)。此外，IL-1β通过诱导Th17细胞的增殖和活化，增加GM-CSF的分泌(MufazalovIA，Schelmbauer C，Regen T，Kuschmann J，Wanke F，Gabriel LA，Hauptmann J，Müller W，Pinteaux E，Kurschus FC，Waisman A.IL-1 signaling is critical for expansion butnot generation of autoreactive GM-CSF+Th17 cells.EMBO J.20174；36(1)：102-115)；除免疫细胞以外，内皮细胞、成纤维细胞、软骨细胞和平滑肌细胞等在IL-1和TNF-α的诱导下均可产生(GM-CSF S.Matthew Fitzgerald，David S.Chi，H.Kenton Hall，ScottA.Reynolds，Omolola Aramide，Steven A.Lee，and Guha Krishnaswamy.GM-CSFInduction in Human Lung Fibroblasts by IL-1β，TNF-α，and MacrophageContact.Journal of Interferon&Cytokine ResearchVol.23，No.2.A.A.M.A.Baqui，Timothy F.Meiller，Jennifer J.Chon，Been-Foo Turng，and William A.Falkler，Jr.Granulocyte-Macrophage Colony-Stimulating Factor Amplification ofInterleukin-1β and Tumor Necrosis Factor Alpha Production in THP-1 HumanMonocytic Cells Stimulated with Lipopolysaccharide of OralMicroorganisms.Clin Diagn Lab Immunol.1998May；5(3)：341-347)。IL-1β is mainly produced by monocytes and macrophages, and IL-1β binds to its receptors to activate downstream NF-κB and MAPK-dependent pro-inflammatory intracellular signaling cascades. IL-1β is an effective inducer of COX-2 expression, producing a large amount of prostaglandin E2 (PGE2) to inhibit the production of IFN-γ, thereby inhibiting the Th1-type immune response dominated by Th1-type T cells against viruses and intracellular parasites ( Napolitani G, Acosta-Rodriguez EV, Lanzavecchia A, Sallusto F. Prostaglandin E2 enhances Th17 responses via modulation of IL-17 and IFN-gamma production by memory CD4+T cells. European journal of immunology. 2009;39(5):1301- 12.). Another key effect of IL-1β is to promote the secretion of IL-6. IL-6 is a key factor in inducing naive T cells to differentiate into Th17-type T cells (Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.Nature.2006;441(7090):235-8.); IL-6 can also inhibit the immunosuppressive function of regulatory T cells and prevent the transformation of Th17 T cells into immunosuppressive Treg T cells (Korn T, Mitsdoerffer M, Croxford AL, Awasthi A, Dardalhon VA, Galileos G, eta1. IL-6 controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells into Foxp3+regulatory T cells. Proceedings of the National Academy of Sciences of the United States of America. 2008; 105(47): 18460-5. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+Tcell-mediated suppression by dendritic cells. Science. 2003; 299(5609): 1033- 6). IL-1β-mediated Th17-type immune response inhibits the killing of virus-infected cells by CD8+ cytotoxic T cells and enhances the survival rate of virus-infected cells, which may reduce the body's defense against viruses (Hou W, Kang HS, et al. Kim BS. Thl7 cells enhance viral persistence and inhibit T cell cytotoxicity in a model of chronic virus infection. The Journal of experimental medicine. 2009;206(2):313-28.). Furthermore, IL-1β increases GM-CSF secretion by inducing the proliferation and activation of Th17 cells (Mufazalov IA, Schelmbauer C, Regen T, Kuschmann J, Wanke F, Gabriel LA, Hauptmann J, Müller W, Pinteaux E, Kurschus FC, Waisman A. IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+Th17 cells. EMBO J. 20174;36(1):102-115); in addition to immune cells, endothelial cells, fibroblasts, cartilage Cells and smooth muscle cells can be produced under the induction of IL-1 and TNF-α (GM-CSF S.Matthew Fitzgerald, David S.Chi, H.Kenton Hall, ScottA.Reynolds, Omolola Aramide, Steven A.Lee, and Guha Krishnaswamy. GM-CSFInduction in Human Lung Fibroblasts by IL-1β, TNF-α, and MacrophageContact. Journal of Interferon&Cytokine Research Vol.23, No.2.A.A.M.A.Baqui, Timothy F.Meiller, Jennifer J.Chon, Been-Foo Turng, and William A. Falkler, Jr. Granulocyte-Macrophage Colony-Stimulating Factor Amplification of Interleukin-1β and Tumor Necrosis Factor Alpha Production in THP-1 HumanMonocytic Cells Stimulated with Lipopolysaccharide of OralMicroorganisms.Clin Diagn Lab Immunol.1998May;5(3) : 341-347).
现有证据证实，IL-1β在病毒感染引起的致命性系统炎症反应中发挥关键作用。在严重急性呼吸系统综合症冠状病毒(SARS-CoV)感染中，研究证实SARS-CoV 3a蛋白可直接激活巨噬细胞中的NLRP3炎性小体，介导的IL-1β的分泌，这可能是SARS中观察到的IL-1β，及其下游IL-6升高的机制(Chen IY，Moriyama M，Chang MF，Ichinohe T.Severe AcuteRespiratory Syndrome Coronavirus Viroporin 3a Activates theNLRP3Inflammasome.Front Microbiol.eCollection 2019)。在同属冠状病毒科病毒感染所致的新型冠状病毒肺炎(COVID-19)死亡患者尸检中发现，患者血液中的CD4+和CD8+T细胞数量显著减少，但是Th17细胞比例显著增加(Xu Z，Shi L，Wang YJ，Zhang JY，HuangL，Zhang C et al.Pathological findings of COVID-19 associated with acuterespiratory distress syndrome.The Lancet Respiratory Medicine.2020)。多项研究报道在新型冠状肺炎重症患者中IL-6、GM-CSF在重症及危重症患者中显著升高(Characteristics of lymphocyte subsets and cytokines in peripheral blood of123 hospitalized patients with 2019 novel coronavirus pneumonia(NCP).Wan SX，Yi QJ，Fan SB，Lv JL，Zhang XX，Guo L，Lang CH，Xiao Q，Xiao KH，et a1.medRxiv2020.02.10.20021832；陈蕾，刘辉国，刘威等.2019新型冠状病毒肺炎29例临床特征分析.中华结核和呼吸杂志，2020，43：E005)。以上均提示患者出现过度的、致命性的炎症反应，而IL-1β信号通路抑制则可能用于治疗此类疾病。目前抗IL-6抗体已被批注用于治疗CAT-T疗法发生的细胞因子释放综合征。然而，作为IL-6的上游分子，抑制IL-1β相关信号通路可能对于治疗病毒感染、肿瘤或者细胞免疫疗法引起的过度的、致命性的炎症反应更为有优势。Norelli等人报道，在嵌合抗原受体T细胞(CAR-T)治疗淋巴瘤小鼠模型中，IL-1受体拮抗剂Anakinra，一种重组人IL-1Ra，可有效缓解CAT-T细胞治疗所引起的细胞因子释放综合症临床症状，减轻CAT-T疗法的神经系统毒性，从而大大延长了实验动物的无病生存期；而抗IL-6抗体未能保护小鼠免受以神经系统炎症为特征的延迟致死性神经毒性(Norelli M，Camisa B，Barbiera G，Falcone L，Purevdorj A，Genua M，Sanvito F，Ponzoni M，Doglioni C，Cristofori P，Traversari C，Bordignon C，Ciceri F，Ostuni R，Bonini C，Casucci M，Bondanza A.Monocyte-derived IL-1 and IL-6 are differentiallyrequired for cytokine-release syndrome and neurotoxicity due to CAR Tcells.Nat Med.2018Jun；24(6)：739-748)。Available evidence confirms that IL-1β plays a key role in the lethal systemic inflammatory response to viral infection. In severe acute respiratory syndrome coronavirus (SARS-CoV) infection, studies have confirmed that SARS-CoV 3a protein can directly activate the NLRP3 inflammasome in macrophages to mediate the secretion of IL-1β, which may be a IL-1β observed in SARS, and the mechanism of its downstream IL-6 elevation (Chen IY, Moriyama M, Chang MF, Ichinohe T.Severe AcuteRespiratory Syndrome Coronavirus Viroporin 3a Activates theNLRP3Inflammasome.Front Microbiol.eCollection 2019). In the autopsy of patients with new coronavirus pneumonia (COVID-19) caused by the infection of the same family of coronaviruses, it was found that the number of CD4+ and CD8+ T cells in the blood of the patients was significantly reduced, but the proportion of Th17 cells was significantly increased (Xu Z, Shi L, Wang YJ, Zhang JY, Huang L, Zhang C et al. Pathological findings of COVID-19 associated with acuterespiratory distress syndrome. The Lancet Respiratory Medicine. 2020). Several studies reported that IL-6 and GM-CSF were significantly elevated in severe and critically ill patients with novel coronavirus pneumonia (Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP). Wan SX, Yi QJ, Fan SB, Lv JL, Zhang XX, Guo L, Lang CH, Xiao Q, Xiao KH, et a1.medRxiv2020.02.10.20021832; Chen Lei, Liu Huiguo, Liu Wei, etc. 2019 Novel Coronavirus Analysis of clinical characteristics of 29 cases of pneumonia. Chinese Journal of Tuberculosis and Respiratory Medicine, 2020, 43: E005). All of the above suggest that patients have excessive and fatal inflammatory responses, and IL-1β signaling pathway inhibition may be used to treat such diseases. Anti-IL-6 antibodies have been approved for the treatment of cytokine release syndrome caused by CAT-T therapy. However, as an upstream molecule of IL-6, inhibition of IL-1β-related signaling pathways may be more advantageous for the treatment of excessive and lethal inflammatory responses caused by viral infections, tumors, or cellular immunotherapy. Norelli et al. reported that the IL-1 receptor antagonist Anakinra, a recombinant human IL-1Ra, effectively relieved CAT-T cells in a mouse model of chimeric antigen receptor T cell (CAR-T) therapy of lymphoma. Treatment-induced clinical symptoms of cytokine release syndrome, alleviating the neurotoxicity of CAT-T therapy, thereby greatly prolonging the disease-free survival of experimental animals; while anti-IL-6 antibody failed to protect mice from neurological Delayed lethal neurotoxicity characterized by inflammation (Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M, Sanvito F, Ponzoni M, Doglioni C, Cristofori P, Traversari C, Bordignon C, Ciceri F, Ostuni R, Bonini C, Casucci M, Bondanza A. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR Tcells. Nat Med. 2018 Jun;24(6):739-748).
如前述，病原微生物(病毒、细菌或真菌或其它)感染、烧伤、胰腺炎、输血或异体血细胞移植、外伤、药物(抗体、免疫激动剂、或细胞因子)、嵌合抗原细胞疗法、手术、放疗、化疗等均可导致系统性炎症反应，表现为IL-1β的分泌增加，甚至引起IL-1β分泌增加为特征的细胞因子释放综合征，表现为机体多器官损伤包括急性肾损伤，急性呼吸系统损伤如急性呼吸窘迫综合征(ARDS)，血管内弥散性凝血，高铁血蛋白症，神经系统炎症等等。As mentioned above, pathogenic microorganism (viral, bacterial or fungal or other) infection, burns, pancreatitis, blood transfusion or allogeneic blood cell transplantation, trauma, drugs (antibodies, immune agonists, or cytokines), chimeric antigen cell therapy, surgery, Radiotherapy, chemotherapy, etc. can lead to systemic inflammatory response, manifested as increased secretion of IL-1β, and even cytokine release syndrome characterized by increased secretion of IL-1β, manifested as multi-organ damage, including acute kidney injury, acute respiratory Systemic injury such as acute respiratory distress syndrome (ARDS), disseminated intravascular coagulation, methemoglobinemia, nervous system inflammation, etc.
虽然IL-1受体拮抗剂如Anakinra目前已在临床得到应用，然而由于其体内代谢速率快，半衰期仅为数小时，需要每天注射以维持药效，所以开发具有稳定药代动力学特征的抗IL-1β的抗体药物具有很大临床应用潜力。Although IL-1 receptor antagonists such as Anakinra have been used clinically, due to their fast metabolism in vivo and a half-life of only a few hours, daily injections are required to maintain their efficacy, so anti-ILs with stable pharmacokinetic characteristics are developed. Antibody drugs against -1β have great potential for clinical application.
发明内容SUMMARY OF THE INVENTION
本发明人经过深入的研究和创造性的劳动，利用哺乳动物细胞表达系统表达出重组的IL-1β-His作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过对大量样本进行筛选，得到了如下的杂交瘤细胞株：After in-depth research and creative work, the inventors used a mammalian cell expression system to express recombinant IL-1β-His as an antigen to immunize mice, and obtained hybridoma cells by fusing mouse spleen cells with myeloma cells. The inventors obtained the following hybridoma cell lines by screening a large number of samples:
杂交瘤细胞株LT010，其于2018年6月21日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018133。The hybridoma cell line LT010 was deposited in the China Center for Type Culture Collection (CCTCC) on June 21, 2018, and the deposit number is CCTCC NO: C2018133.
本发明人惊奇地发现：The inventors have surprisingly found that:
杂交瘤细胞株LT010能够分泌产生与人IL-1β特异性结合的特异性单克隆抗体(命名为3H6)，并且该单克隆抗体能够十分有效地阻断IL-1β与IL-1R1的结合；The hybridoma cell line LT010 can secrete and produce a specific monoclonal antibody (named 3H6) that specifically binds to human IL-1β, and the monoclonal antibody can effectively block the combination of IL-1β and IL-1R1;
进一步地，本发明人创造性地制得了抗人IL-1β的人源化抗体(分别命名为3H6H1L1，3H6 H2L2，3H6 H3L3，3H6 H4L1)，能有效地结合人IL-1β，阻断IL-1β与其受体IL-1R1的结合，抑制IL-1β下游信号通路的激活；具有用于制备降低、预防或治疗病原微生物(病毒、细菌或真菌或其它)感染、烧伤、胰腺炎、输血或异体血细胞移植、外伤、药物(抗体、免疫激动剂、或细胞因子)、嵌合抗原细胞疗法、手术、放疗、化疗所引起的系统性炎症反应。Further, the inventors creatively prepared humanized antibodies against human IL-1β (named 3H6H1L1, 3H6 H2L2, 3H6 H3L3, 3H6 H4L1), which can effectively bind to human IL-1β and block IL-1β Binding to its receptor IL-1R1, inhibits the activation of the downstream signaling pathway of IL-1β; has the potential to reduce, prevent or treat pathogenic microbial (virus, bacterial or fungal or other) infections, burns, pancreatitis, blood transfusion or allogeneic blood cells Systemic inflammatory responses caused by transplantation, trauma, drugs (antibodies, immune agonists, or cytokines), chimeric antigen cell therapy, surgery, radiotherapy, and chemotherapy.
由此提供了下述发明：The following invention is thus provided:
本发明的一个方面涉及一种抗IL-1β的抗体或其抗原结合片段，其中，One aspect of the present invention pertains to an anti-IL-1β antibody or antigen-binding fragment thereof, wherein,
所述抗体的重链可变区包含：氨基酸序列分别如SEQ ID NO：17-SEQ ID NO：19所示的HCDR1-HCDR3；和The heavy chain variable region of the antibody comprises: HCDR1-HCDR3 whose amino acid sequences are shown in SEQ ID NO: 17-SEQ ID NO: 19, respectively; and
所述抗体的轻链可变区包含：氨基酸序列分别如SEQ ID NO：20-SEQ ID NO：22所示的LCDR1-LCDR3。The light chain variable region of the antibody comprises: LCDR1-LCDR3 whose amino acid sequences are shown in SEQ ID NO: 20-SEQ ID NO: 22, respectively.
优选地，所述IL-1β为人IL-1β。Preferably, the IL-1β is human IL-1β.
重链可变区和轻链可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Sequences of Proteins of ImmunologicalInterest.Fifth Edition(1991)，第1-3卷，NIH Publication 91-3242，Bethesda Md)。The variable region of the heavy chain and the variable region of the light chain determine the binding of antigen; the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2 , HCDR3, the CDRs of the light chain (L) include LCDR1, LCDR2, LCDR3; named by Kabat et al., see Sequences of Proteins of Immunological Interest. Fifth Edition (1991), Vol. 1-3, NIH Publication 91-3242, Bethesda Md).
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库分析本发明的抗体3H6、3H6H1 L1、3H6H2L2、3H6H3L3和3H6H4L1，可以发现其具有相同的HCDR1-3和LCDR1-3：Through technical means well-known to those skilled in the art, for example, by analyzing the antibodies 3H6, 3H6H1 L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 of the present invention through the VBASE2 database, it can be found that they have the same HCDR1-3 and LCDR1-3:
其重链可变区的3个HCDR区的氨基酸序列如下：The amino acid sequences of the three HCDR regions in the variable region of the heavy chain are as follows:
HCDR1：GFSLSTSGMG(SEQ ID NO：17)，HCDR1: GFSLSTSGMG (SEQ ID NO: 17),
HCDR2：IYWDDDK(SEQ ID NO：18)，HCDR2: IYWDDDK (SEQ ID NO: 18),
HCDR3：ARSAYYSFAY(SEQ ID NO：19)；HCDR3: ARSAYYSFAY (SEQ ID NO: 19);
其轻链可变区的3个CDR区的氨基酸序列如下：The amino acid sequences of the three CDR regions of the light chain variable region are as follows:
LCDR1：QDVDTD(SEQ ID NO：20)，LCDR1: QDVDTD (SEQ ID NO: 20),
LCDR2：WAS(SEQ ID NO：21)，LCDR2: WAS (SEQ ID NO: 21),
LCDR3：QQYSSYPT(SEQ ID NO：22)。LCDR3: QQYSSYPT (SEQ ID NO: 22).
在本发明的一个或多个实施方案中，其中，In one or more embodiments of the present invention, wherein,
所述抗体的重链可变区的氨基酸序列选自SEQ ID NO：2、SEQ ID NO：6、SEQ IDNO：10和SEQ ID NO：14或与SEQ ID NO：2、SEQ ID NO：6、SEQ ID NO：10和SEQ ID NO：14的序列分别具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；和The amino acid sequence of the variable region of the heavy chain of the antibody is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14 or with SEQ ID NO: 2, SEQ ID NO: 6, The sequences of SEQ ID NO: 10 and SEQ ID NO: 14 have sequences of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity, respectively; and
所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO：4和SEQ ID NO：8、SEQ IDNO：12和SEQ ID NO：16或与SEQ ID NO：4和SEQ ID NO：8、SEQ ID NO：12和SEQ ID NO：16的序列分别具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列。The amino acid sequence of the light chain variable region of the antibody is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16 or with SEQ ID NO: 4 and SEQ ID NO: 8, The sequences of SEQ ID NO: 12 and SEQ ID NO: 16 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical, respectively.
在本发明的一些实施方案中，所述抗体选自：In some embodiments of the invention, the antibody is selected from:
(1)如SEQ ID NO：2或与SEQ ID NO：2具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VH和如SEQ ID NO：4或与SEQ ID NO：4具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VL；(1) A VH as represented by SEQ ID NO:2 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO:2 and a VL as set forth in SEQ ID NO:4 or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO:4;
(2)如SEQ ID NO：6或与SEQ ID NO：6具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VH和如SEQ ID NO：8或与SEQ ID NO：8具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VL；(2) A VH as represented by SEQ ID NO:6 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO:6 and a VL as set forth in SEQ ID NO: 8 or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 8;
(3)如SEQ ID NO：10或与SEQ ID NO：10具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VH和如SEQ ID NO：12或与SEQ ID NO：12具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VL；和(3) A VH as set forth in SEQ ID NO: 10 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 10 and a VL as shown in SEQ ID NO: 12 or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 12; and
(4)如SEQ ID NO：14或与SEQ ID NO：14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VH和如SEQ ID NO：16或与SEQ ID NO：16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列所示的VL。(4) VH as shown in SEQ ID NO: 14 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 14 and a VL as set forth in SEQ ID NO: 16 or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 16.
在本发明的一个或多个实施方案中，所述抗体或其抗原结合片段选自Fab、Fab′、F(ab′)2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。In one or more embodiments of the invention, the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragments, single chain antibodies (e.g. , scFv), humanized antibodies, chimeric antibodies or diabodies.
在本发明的一个或多个实施方案中，其中，所述的抗体以小于10-5M，例如小于10-6M、小于10-7M、小于10-8M、小于10-9M或小于10-10M或更小的KD结合IL-1β蛋白；优选地，所述KD通过Biacore分子相互作用仪测得；优选地，所述KD通过Fortebio分子相互作用仪测得。In one or more embodiments of the present invention, wherein, the antibody is less than10-5 M, such as less than10-6 M, less than10-7 M, less than10-8 M, less than10-9 M or A KD of less than 10−10 M or less binds to IL-1β protein; preferably, the KD is measured by a Biacore molecular interaction instrument; preferably, the KD is measured by a Fortebio molecular interaction instrument.
在本发明的一些实施方式中，其中，所述的抗体以小于大约100nM，例如小于大约10nM、小于大约1nM、小于大约0.9nM、小于大约0.8nM、小于大约0.7nM、小于大约0.6nM、小于大约0.5nM、小于大约0.4nM、小于大约0.3nM、小于大约0.2nM、小于大约0.1nM或更小的EC50结合IL-1β蛋白。具体地，所述EC50通过间接ELISA方法测得。In some embodiments of the invention, wherein the antibody is present at a concentration of less than about 100 nM, such as less than about 10 nM, less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about EC50 of about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM, less than about 0.1 nM or less binds IL-1 beta protein. Specifically, the EC50 was determined by an indirect ELISA method.
在本发明的一个或多个实施方案中，其中，In one or more embodiments of the present invention, wherein,
所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。The antibody includes non-CDR regions, and the non-CDR regions are from a species other than murine, eg, from a human antibody.
在本发明的一些实施方案中，所述抗体的恒定区是人源化的，例如，重链恒定区均采用Ig gamma-1chain C region，例如ACCESSION：P01857或Ig gamma-4chain C region，例如ACCESSION：P01861.1；轻链恒定区均采用Ig kappa chain C region，例如ACCESSION：P01834。In some embodiments of the invention, the constant regions of the antibodies are humanized, eg, heavy chain constant regions all employ an Ig gamma-1 chain C region, eg, ACCESSION: P01857, or an Ig gamma-4 chain C region, eg, ACCESSION : P01861.1; light chain constant regions all use Ig kappa chain C region, such as ACCESSION: P01834.
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段，其中所述抗体是由杂交瘤细胞株LT010产生的单克隆抗体，所述杂交瘤细胞株LT010保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018133。In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line LT010, which is deposited in China Typical Culture Collection Center (CCTCC), the deposit number is CCTCC NO: C2018133.
在本发明的一个或多个实施方案中，所述抗体为单克隆抗体。In one or more embodiments of the invention, the antibody is a monoclonal antibody.
本发明的另一方面涉及一种抗体药物偶联物(antibody-drug conjugate，ADC)，其包括抗体或其抗原结合片段以及小分子药物，其中，所述抗体或其抗原结合片段为本发明中任一项所述的抗体或其抗原结合片段；优选地，所述小分子药物为小分子细胞毒药物；更优选地，所述小分子药物为化疗药物。Another aspect of the present invention relates to an antibody-drug conjugate (ADC), which includes an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is in the present invention The antibody or antigen-binding fragment thereof of any one; preferably, the small-molecule drug is a small-molecule cytotoxic drug; more preferably, the small-molecule drug is a chemotherapeutic drug.
所述化疗药物可以是常规的肿瘤化疗药物，例如烷化剂、抗代谢药、抗肿瘤抗生素、植物类抗癌药、激素、免疫抑制剂等。The chemotherapeutic drugs can be conventional tumor chemotherapeutic drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immunosuppressants and the like.
在本发明的一个或多个实施方案中，所述抗体或其抗原结合片段通过连接子与小分子药物连接；所述连接子可以是本领域技术人员知悉的连接子，例如，所述连接子为腙键、二硫键或肽键。In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof is linked to the small molecule drug through a linker; the linker may be a linker known to those skilled in the art, for example, the linker For hydrazone bond, disulfide bond or peptide bond.
在本发明的一个或多个实施方案中，所述抗体或其抗原结合片段与小分子药物以一定的摩尔比连接；例如，所述摩尔比为1∶(2-4)。In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof and the small molecule drug are linked in a certain molar ratio; for example, the molar ratio is 1:(2-4).
本发明的再一方面涉及一种双特异性抗体(又称为双功能抗体)，其包括第一蛋白功能区和第二蛋白功能区，其中：Yet another aspect of the present invention relates to a bispecific antibody (also referred to as a diabody) comprising a first protein domain and a second protein domain, wherein:
所述第一蛋白功能区靶向IL-1β，The first protein functional domain targets IL-1β,
所述第二蛋白功能区靶向不同于IL-1β的靶点，例如IL-17A；The second protein functional domain targets a target other than IL-1β, such as IL-17A;
其中，所述第一蛋白功能区为本发明中任一项所述的抗体或其抗原结合片段；Wherein, the first protein functional region is the antibody or antigen-binding fragment thereof of any one of the present invention;
优选地，所述双特异性抗体为IgG-scFv模式；Preferably, the bispecific antibody is in IgG-scFv format;
优选地，Preferably,
(1)所述第一蛋白功能区为本发明中任一项所述的抗体或其抗原结合片段，并且所述第二蛋白功能区为单链抗体；(1) the first protein functional region is the antibody or antigen-binding fragment thereof of any one of the present invention, and the second protein functional region is a single-chain antibody;
或者，or,
(2)所述第一蛋白功能区为单链抗体，其重链可变区包含氨基酸序列如SEQ IDNO：17一SEQ ID NO：19所示的HCDR1-HCDR3，其轻链可变区包含氨基酸序列如SEQ ID NO：20-SEQ ID NO：22所示的LCDR1-LCDR3，并且所述第二蛋白功能区为抗体(例如单克隆抗体)。(2) The first protein functional region is a single-chain antibody, the variable region of its heavy chain comprises HCDR1-HCDR3 whose amino acid sequences are shown in SEQ ID NO: 17-SEQ ID NO: 19, and the variable region of its light chain comprises amino acids The sequences are LCDR1-LCDR3 as shown in SEQ ID NO: 20-SEQ ID NO: 22, and the second protein domain is an antibody (eg, a monoclonal antibody).
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；In some embodiments of the present invention, the bispecific antibody, wherein the first protein functional domain and the second protein functional domain are directly linked or linked by a linker fragment;
优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；Preferably, the connecting fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;
优选地，所述连接片段为SS(GGGGS)n，n为正整数，例如1、2、3、4、5或6。Preferably, the linker segment is SS(GGGGS)n, where n is a positive integer, such as 1, 2, 3, 4, 5 or 6.
在本发明的一些实施方式中，所述的双特异性抗体的第(2)项中，In some embodiments of the present invention, in the item (2) of the bispecific antibody,
所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO：2、SEQ ID NO：6、SEQID NO：10和SEQ ID NO：14或与SEQ ID NO：2、SEQ ID NO：6、SEQ ID NO：10和SEQ ID NO：14的序列分别具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；和The amino acid sequence of the variable region of the heavy chain of the single chain antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14 or the same as SEQ ID NO: 2, SEQ ID NO: 6. A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to the sequences of SEQ ID NO: 10 and SEQ ID NO: 14, respectively; and
所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO：4和SEQ ID NO：8、SEQID NO：12和SEQ ID NO：16或与SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12和SEQ ID NO：16的序列分别具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列。The amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16 or the same as SEQ ID NO: 4, SEQ ID NO: 8. The sequences of SEQ ID NO: 12 and SEQ ID NO: 16 have sequences of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity, respectively.
在本发明的一些实施方式中，所述的双特异性抗体的第(2)项中，In some embodiments of the present invention, in the item (2) of the bispecific antibody,
所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO：2或与SEQ ID NO：2具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO：4或与SEQ ID NO：4具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者The amino acid sequence of the heavy chain variable region of the single chain antibody is SEQ ID NO: 2 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 2 A sequence that is at least 99% identical, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 4 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 4, 96%, 97%, 98% or at least 99% identical sequences; or
所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO：6或与SEQ ID NO：6具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO：8所示或与SEQ ID NO：8具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者The amino acid sequence of the heavy chain variable region of the single chain antibody is SEQ ID NO: 6 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 6 A sequence that is at least 99% identical, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 8 or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 8 %, 96%, 97%, 98% or at least 99% identical to sequences; or
所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO：10或与SEQ ID NO：10具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO：12或与SEQ ID NO：12具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者The amino acid sequence of the heavy chain variable region of the single chain antibody is SEQ ID NO: 10 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or SEQ ID NO: 10 A sequence that is at least 99% identical, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 12 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 12, 96%, 97%, 98% or at least 99% identical sequences; or
所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO：14或与SEQ ID NO：14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO：16或与SEQ ID NO：16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列。The amino acid sequence of the heavy chain variable region of the single chain antibody is SEQ ID NO: 14 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or the same as SEQ ID NO: 14 A sequence that is at least 99% identical, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 16 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 16, Sequences of 96%, 97%, 98% or at least 99% identity.
在本发明的一些实施方式中，所述双特异性抗体的所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。In some embodiments of the present invention, the first protein functional domain and the second protein functional domain of the bispecific antibody are independently 1, 2, or more than 2.
在本发明的一些实施方式中，所述双特异性抗体的(2)项中，所述单克隆抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。In some embodiments of the present invention, in item (2) of the bispecific antibody, the constant region of the monoclonal antibody is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.
在本发明的一些实施方式中，所述双特异性抗体的所述单链抗体连接在抗体或单克隆抗体的重链的C末端。In some embodiments of the invention, the single chain antibody of the bispecific antibody is linked at the C-terminus of the heavy chain of the antibody or monoclonal antibody.
本发明的再一方面涉及一种分离的核酸分子，其包含编码抗体重链可变区的核酸序列和编码抗体轻链可变区的核酸序列，其中，Yet another aspect of the present invention pertains to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody heavy chain variable region and a nucleic acid sequence encoding an antibody light chain variable region, wherein,
所述抗体的重链可变区包含氨基酸序列分别如SEQ ID NO：17-SEQ ID NO：19所示的HCDR1-HCDR3，和所述抗体的轻链可变区包含氨基酸序列分别如SEQ ID NO：20-SEQ IDNO：22所不的LCDR1-LCDR3；The variable region of the heavy chain of the antibody comprises HCDR1-HCDR3 whose amino acid sequences are shown in SEQ ID NO: 17-SEQ ID NO: 19, respectively, and the variable region of the light chain of the antibody comprises the amino acid sequences shown in SEQ ID NO, respectively : LCDR1-LCDR3 shown by 20-SEQ ID NO: 22;
优选地，所述抗体的重链可变区的氨基酸序列选自SEQ ID NO：2、SEQ ID NO：6、SEQ ID NO：10和SEQ ID NO：14或与SEQ ID NO：2、SEQ ID NO：6、SEQ ID NO：10和SEQ IDNO：14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，和所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO：4和SEQ ID NO：8、SEQ ID NO：12和SEQ IDNO：16或与SEQ ID NO：4和SEQ ID NO：8、SEQ JD NO：12和SEQ ID NO：16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；Preferably, the amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 14 or the same as SEQ ID NO: 2, SEQ ID NO: 14 NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical to the antibody's The amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16 or with SEQ ID NO: 4 and SEQ ID NO: 8, SEQ JD NO: 12 and SEQ ID NO: 16 have sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical;
更优选地，所述抗体的重链可变区的氨基酸序列为SEQ ID NO：2或与SEQ ID NO：2具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO：4或与SEQ ID NO：4具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者所述抗体的重链可变区的氨基酸序列为SEQ ID NO：6或与SEQ ID NO：2具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO：8或与SEQID NO：8具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者所述抗体的重链可变区的氨基酸序列为SEQ ID NO：10或与SEQ ID NO：10具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO：12所示或与SEQ ID NO：12具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列；或者所述抗体的重链可变区的氨基酸序列为SEQ ID NO：14或与SEQ ID NO：14具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列，并且所述抗体的轻链可变区的氨基酸序列为SEQ ID NO：16所示或与SEQ ID NO：16具有至少80％，85％，90％，95％，96％，97％，98％或至少99％同一性的序列进一步优选地，所述分离的核酸分子包含：More preferably, the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 2 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% with SEQ ID NO: 2 % or at least 99% identical to the sequence, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 4 or at least 80%, 85%, 90%, 95% with SEQ ID NO: 4, A sequence that is 96%, 97%, 98% or at least 99% identical; or the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 6 or at least 80%, 85% with SEQ ID NO: 2 , 90%, 95%, 96%, 97%, 98% or at least 99% identical to the sequence, and the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 8 or has SEQ ID NO: 8 a sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity; or the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 10 or A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of said antibody is a sequence set forth in SEQ ID NO: 12 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 12; or the antibody The amino acid sequence of the heavy chain variable region is SEQ ID NO: 14 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 14 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 16 or has at least 80%, 85%, 90%, 95%, 96%, 97% with SEQ ID NO: 16 , a sequence of 98% or at least 99% identity further preferably, the isolated nucleic acid molecule comprises:
SEQ ID NO：1和SEQ ID NO：3所示的核酸序列，The nucleic acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3,
SEQ ID NO：5和SEQ ID NO：7所示的核酸序列，The nucleic acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 7,
SEQ ID NO：9和SEQ ID NO：11所示的核酸序列，或者The nucleic acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 11, or
SEQ ID NO：13和SEQ ID NO：15所示的核酸序列。The nucleic acid sequences shown in SEQ ID NO:13 and SEQ ID NO:15.
所述分离的核酸分子，可以是一个核酸分子，也可以是多个核酸分子，例如两个核酸分子。当是一个核酸分子时，抗体的重链可变区和轻链可变区可以通过同一个核酸分子表达，例如通过位于同一核酸分子上的相同或不同的表达框来表达。当是多个核酸分子例如两个核酸分子时，抗体的重链可变区和轻链可变区可以通过不同的核酸分子分别表达。The isolated nucleic acid molecule may be one nucleic acid molecule or multiple nucleic acid molecules, such as two nucleic acid molecules. When one nucleic acid molecule, the heavy and light chain variable regions of the antibody can be expressed by the same nucleic acid molecule, eg, by the same or different expression cassettes on the same nucleic acid molecule. In the case of multiple nucleic acid molecules, eg, two nucleic acid molecules, the heavy and light chain variable regions of the antibody may be expressed separately by different nucleic acid molecules.
本发明的再一方面涉及一种重组载体，其包含本发明的分离的核酸分子。所述的重组载体可以是一个或多个。当所述核酸分子是多个(例如两个)时，多个(例如两个)核酸分子可以通过同一重组载体表达，也可以分别通过不同的重组载体来表达。Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention. The recombinant vector can be one or more. When the nucleic acid molecules are multiple (eg, two), the multiple (eg, two) nucleic acid molecules can be expressed by the same recombinant vector, or they can be expressed by different recombinant vectors.
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者包含本发明的重组载体。Yet another aspect of the present invention relates to a host cell comprising an isolated nucleic acid molecule of the present invention, or a recombinant vector of the present invention.
本发明的再一方面涉及一种制备本发明中任一项所述的抗体或其抗原结合片段的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述抗体或其抗原结合片段的步骤。Yet another aspect of the present invention relates to a method of preparing the antibody or antigen-binding fragment thereof of any one of the present invention, comprising culturing the host cell of the present invention under suitable conditions, and recovering the resulting antibody from the cell culture described antibody or antigen-binding fragment thereof.
本发明的再一方面涉及一种杂交瘤细胞株LT010，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018133。Another aspect of the present invention relates to a hybridoma cell line LT010, which is deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number of CCTCC NO: C2018133.
本发明的再一方面涉及一种药物组合物，其包含有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体；可选地，其还包括药学上可接受的载体和/或赋形剂。Yet another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof of any one of the present invention, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention ; Optionally, it also includes a pharmaceutically acceptable carrier and/or excipient.
本发明的再一方面涉及有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体在制备治疗和/或预防IL-1β异常分泌增加为特征的系统性炎症的药物中的用途；Yet another aspect of the present invention relates to an effective amount of the antibody or antigen-binding fragment thereof of any one of the present invention, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention in the preparation of the treatment and/or prevention of IL - Use in the medicament of systemic inflammation characterized by increased abnormal secretion of 1β;
优选地，所述系统性炎症包括系统性炎症综合征，细胞因子释放综合征、多器官功能障碍综合征(multiple organ dysfunction syndrome，MODS)、急性呼吸窘迫综合征；Preferably, the systemic inflammation includes systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), acute respiratory distress syndrome;
优选地，所述系统性炎症是由病原微生物感染所引起的；Preferably, the systemic inflammation is caused by infection with pathogenic microorganisms;
优选地，所述病原微生物为病毒、细菌、真菌、立克次体、衣原体、支原体、寄生虫、朊病毒；Preferably, the pathogenic microorganisms are viruses, bacteria, fungi, rickettsia, chlamydia, mycoplasma, parasites, prions;
优选地，所述病毒包括RNA病毒及DNA病毒；Preferably, the virus includes RNA virus and DNA virus;
优选地，所述RNA病毒包括冠状病毒科病毒；Preferably, the RNA virus comprises a Coronaviridae virus;
优选地，所述冠状病毒科病毒包括2019新型冠状病毒(2019-nCoV或SARS-CoV-2，引发新型冠状病毒肺炎COVID-19)，HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)。Preferably, the coronaviruses include 2019 novel coronavirus (2019-nCoV or SARS-CoV-2, which causes novel coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (which causes severe acute respiratory syndrome) and MERS-CoV (which causes Middle East respiratory syndrome).
本发明的再一方面本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体在制备如下药物中的用途：Another aspect of the present invention is the use of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention in the preparation of the following medicines:
阻断人IL-1β与人IL-1R1和/或人IL-1R2结合的药物，Drugs that block the binding of human IL-1β to human IL-1R1 and/or human IL-1R2,
下调人IL-1β活性或水平的药物，或者A drug that downregulates the activity or level of human IL-1β, or
抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活的药物。Drugs that inhibit the activation of downstream signaling pathways mediated by the binding of human IL-1β to human IL-1R1 and/or human IL-1R2.
在本发明的一个实施方案中，所述人IL-1R1和/或人IL-1R2是细胞表面的人IL-1R1和/或人IL-1R2。In one embodiment of the invention, the human IL-1R1 and/or human IL-1R2 is cell surface human IL-1R1 and/or human IL-1R2.
在本发明的一个实施方案中，所述用途是非治疗目的的和/或非诊断目的的。In one embodiment of the invention, the use is non-therapeutic and/or non-diagnostic.
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体，其用于制备治疗和/或预防(IL-1β异常分泌增加为特征的)系统性炎症的药物中的用途；In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention is used for the preparation of therapeutic and/or prophylactic (IL- - Use in the medicament of systemic inflammation characterized by increased abnormal secretion of 1β;
优选地，所述系统性炎症包括系统性炎症综合征，细胞因子释放综合征、多器官功能障碍综合征(multiple organ dysfunction syndrome，MODS)、急性呼吸窘迫综合征；Preferably, the systemic inflammation includes systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), acute respiratory distress syndrome;
优选地，所述系统性炎症是由病原微生物感染所引起的；Preferably, the systemic inflammation is caused by infection with pathogenic microorganisms;
优选地，所述病原微生物为病毒、细菌、真菌、立克次体、衣原体、支原体、寄生虫、朊病毒；Preferably, the pathogenic microorganisms are viruses, bacteria, fungi, rickettsia, chlamydia, mycoplasma, parasites, prions;
优选地，所述病毒包括RNA病毒及DNA病毒；Preferably, the virus includes RNA virus and DNA virus;
优选地，所述RNA病毒包括冠状病毒科病毒；Preferably, the RNA virus comprises a Coronaviridae virus;
优选地，所述冠状病毒科病毒包括2019新型冠状病毒(2019-nCoV或SARS-CoV-2，引发新型冠状病毒肺炎COVID-19)，HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)，Preferably, the coronaviruses include 2019 novel coronavirus (2019-nCoV or SARS-CoV-2, which causes novel coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (which causes severe acute respiratory syndrome) and MERS-CoV (which causes Middle East respiratory syndrome),
优选地，所述抗体或其抗原结合片段为单独使用或者与一种或多种抗病毒药物联用；Preferably, the antibody or antigen-binding fragment thereof is used alone or in combination with one or more antiviral drugs;
优选地，所述抗病毒药物为抗逆转录病毒药物或干扰素；Preferably, the antiviral drug is an antiretroviral drug or interferon;
优选地，所述抗逆转录病毒药物其中：抗逆转录病毒药物是RNA依赖的RNA聚合酶(RdRp)抑制剂，或者是核苷类似物，或者是类肽，或者是人免疫缺陷病毒类型1(HIV-1)整合酶的链转移抑制剂(INSTI)，或者是HIV蛋白酶抑制剂。Preferably, the antiretroviral drug wherein: the antiretroviral drug is an RNA-dependent RNA polymerase (RdRp) inhibitor, or a nucleoside analog, or a peptoid, or a human immunodeficiency virus type 1 (HIV-1) integrase strand transfer inhibitor (INSTI), or HIV protease inhibitor.
优选地，所述抗逆转录病毒药物为法匹拉韦(favipiravir)、瑞德西韦(Remdesivir)、Beclabuvir，沙奎那韦(Saquinavir)，比特拉韦(Bictegravir)，洛匹那韦(Lopinavir)，多替拉韦(Dolutegravir)；Preferably, the antiretroviral drug is favipiravir, Remdesivir, Beclabuvir, Saquinavir, Bictegravir, Lopinavir ), Dolutegravir;
更优选地，所述药物为法匹拉韦。More preferably, the drug is favipiravir.
在本发明的一个或多个实施方案中，所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体，其用于：In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention is used for:
阻断人IL-1β与人IL-1R1和/或人IL-1R2结合，Blocks the binding of human IL-1β to human IL-1R1 and/or human IL-1R2,
下调人IL-1β活性或水平，或者down-regulates human IL-1β activity or levels, or
抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活。Inhibits activation of downstream signaling pathways mediated by the binding of human IL-1β to human IL-1R1 and/or human IL-1R2.
在本发明的一个实施方案中，所述人IL-1R1和/或人IL-1R2是细胞表面的人IL-1R1和/或人IL-1R2。In one embodiment of the invention, the human IL-1R1 and/or human IL-1R2 is cell surface human IL-1R1 and/or human IL-1R2.
本发明的再一方面涉及一种在体内或体外方法，包括施加细胞以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体的步骤，所述方法选自如下：Yet another aspect of the present invention relates to an in vivo or in vitro method comprising administering to cells an effective amount of an antibody or antigen-binding fragment thereof of any one of the present invention, an antibody drug conjugate of the present invention, or an The steps of the bispecific antibody, the method is selected from the following:
阻断人IL-1β与人IL-1R1和/或人IL-1R2结合的方法，A method of blocking the binding of human IL-1β to human IL-1R1 and/or human IL-1R2,
下调人IL-1β活性或水平的方法，或者A method of downregulating the activity or level of human IL-1β, or
抑制由人IL-1β与人IL-1R1和/或人IL-1R2结合介导的下游信号转导通路激活的方法。Methods of inhibiting activation of downstream signaling pathways mediated by the binding of human IL-1β to human IL-1R1 and/or human IL-1R2.
在本发明的一个实施方案中，所述人IL-1R1和/或人IL-1R2是细胞表面的人IL-1R1和/或人IL-1R2。In one embodiment of the invention, the human IL-1R1 and/or human IL-1R2 is cell surface human IL-1R1 and/or human IL-1R2.
在本发明的一个实施方案中，所述体外方法是非治疗目的的和/或非诊断目的的。In one embodiment of the invention, the in vitro method is non-therapeutic and/or non-diagnostic.
本发明的再一方面涉及一种治疗和/或预防治疗和/或预防IL-1β异常分泌增加为特征的系统性炎症的方法，包括给予有需求的受试者或患者以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的抗体药物偶联物或者本发明的双特异性抗体的步骤；Yet another aspect of the present invention pertains to a method of treating and/or preventing systemic inflammation characterized by abnormally increased secretion of IL-1β, comprising administering to a subject or patient in need thereof an effective amount of the present invention any one of the steps of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention, or the bispecific antibody of the present invention;
优选地，所述系统性炎症包括系统性炎症综合征，细胞因子释放综合征、多器官功能障碍综合征(multiple organ dysfunction syndrome，MODS)、急性呼吸窘迫综合征；Preferably, the systemic inflammation includes systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), acute respiratory distress syndrome;
优选地，所述系统性炎症是由病原微生物感染所引起的；Preferably, the systemic inflammation is caused by infection with pathogenic microorganisms;
优选地，所述病原微生物为病毒、细菌、真菌、立克次体、衣原体、支原体、寄生虫、朊病毒；Preferably, the pathogenic microorganisms are viruses, bacteria, fungi, rickettsia, chlamydia, mycoplasma, parasites, prions;
优选地，所述病毒包括RNA病毒及DNA病毒；Preferably, the virus includes RNA virus and DNA virus;
优选地，所述RNA病毒包括冠状病毒科病毒；Preferably, the RNA virus comprises a Coronaviridae virus;
优选地，所述冠状病毒科病毒包括2019新型冠状病毒(2019-nCoV或SARS-CoV-2，引发新型冠状病毒肺炎COVID-19)，HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)。Preferably, the coronaviruses include 2019 novel coronavirus (2019-nCoV or SARS-CoV-2, which causes novel coronavirus pneumonia COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 , SARS-CoV (which causes severe acute respiratory syndrome) and MERS-CoV (which causes Middle East respiratory syndrome).
在本发明的任一个实施方案中，所述的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体可以单独使用或与一种或多种抗病毒药物联合使用。In any of the embodiments of the invention, the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody may be used alone or in combination with one or more antiviral drugs use.
在本发明的一个方面，涉及试剂盒，其包含有效量(例如0.001mg-1000mg)的所述的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体，和任选地，还包含有效量的一种或多种抗病毒药物(例如100-2400mg)，其中所述抗病毒药物所上所述。In one aspect of the present invention, it relates to a kit comprising an effective amount (eg, 0.001 mg-1000 mg) of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody, And optionally, an effective amount of one or more antiviral drugs (eg, 100-2400 mg), wherein the antiviral drugs are described above.
在本发明的另一个方面，涉及单次药物剂量单元，其包含0.001mg-1000mg的本发明的抗体或其抗原结合片段，优选0.001mg-900mg、0.001mg-800mg、0.001mg-700mg、0.001mg-600mg、0.001mg-500mg、0.001mg-400mg、0.001mg-300mg、0.001mg-200mg、0.001mg-100mg，更优选为100mg、200mg、300mg、400mg、500mg、600mg、700mg、800mg、900mg或1000mg的本发明的抗体或其抗原结合片段。In another aspect of the invention, it relates to a single pharmaceutical dosage unit comprising 0.001 mg-1000 mg of the antibody or antigen-binding fragment thereof of the invention, preferably 0.001 mg-900 mg, 0.001 mg-800 mg, 0.001 mg-700 mg, 0.001 mg - 600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, more preferably 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg The antibody or antigen-binding fragment thereof of the present invention.
在本发明的上述任一实施方案中，本发明的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体的有效量为0.001mg-1000mg，优选0.001mg-900mg、0.001mg-800mg、0.001mg-700mg、0.001mg-600mg、0.001mg-500mg、0.001mg-400mg、0.001mg-300mg、0.001mg-200mg、0.001mg-100mg，更优选为100mg、200mg、300mg、400mg、500mg、600mg、700mg、800mg、900mg或1000mg。或者，在本发明的上述任一实施方案中，基于受试者的体重，本发明的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体的有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。In any of the above embodiments of the present invention, the effective amount of the antibody or antigen-binding fragment thereof, the antibody drug conjugate or the bispecific antibody of the present invention is 0.001mg-1000mg, preferably 0.001mg- 900mg, 0.001mg-800mg, 0.001mg-700mg, 0.001mg-600mg, 0.001mg-500mg, 0.001mg-400mg, 0.001mg-300mg, 0.001mg-200mg, 0.001mg-100mg, more preferably 100mg, 200mg, 300mg , 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg. Alternatively, in any of the above embodiments of the present invention, based on the body weight of the subject, the effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the bispecific antibody of the present invention is 0.1-100mg/kg, preferably 1-90mg/kg, 1-80mg/kg, 1-70mg/kg, 1-60mg/kg, 1-50mg/kg, 1-40mg/kg, 1-30mg/kg, 1 -20mg/kg or 1-10mg/kg.
在本发明的上述任一实施方案，一种或多种抗病毒药物(例如法匹拉韦)的有效量为100-2400mg，优选100mg-2300mg、100mg-2200mg、100mg-2100mg、100mg-2000mg、100mg-1900mg、100mg-1800mg、100mg-1700mg、100mg-1600mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg、100mg-1800mg，更优选为100mg，200mg，300mg，400mg，500mg，600mg，700mg，800mg，900mg，1000mg。或者，在本发明的上述任一实施方案中，基于受试者或患者的体重，所述抗病毒药物的有效量为0.1-100mg/kg，优选1-90mg/kg、1-80mg/kg、1-70mg/kg、1-60mg/kg、1-50mg/kg、1-40mg/kg、1-30mg/kg、1-20mg/kg或1-10mg/kg。In any of the above embodiments of the present invention, the effective amount of one or more antiviral drugs (eg favipiravir) is 100-2400mg, preferably 100mg-2300mg, 100mg-2200mg, 100mg-2100mg, 100mg-2000mg, 100mg-1900mg, 100mg-1800mg, 100mg-1700mg, 100mg-1600mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, 100mg-1800mg, more preferably 100mg, 200mg, 300mg, 400mg, 600mg, 500mg , 700mg, 800mg, 900mg, 1000mg. Or, in any of the above-mentioned embodiments of the present invention, based on the body weight of the subject or patient, the effective amount of the antiviral drug is 0.1-100 mg/kg, preferably 1-90 mg/kg, 1-80 mg/kg, 1-70 mg/kg, 1-60 mg/kg, 1-50 mg/kg, 1-40 mg/kg, 1-30 mg/kg, 1-20 mg/kg or 1-10 mg/kg.
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本发明中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory operation steps of cell culture, molecular genetics, nucleic acid chemistry, and immunology used in the present invention are all routine steps widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.
在本发明中，术语“单次药物剂量单元”表示在给药方案的时刻待给药于受试者或患者的本发明所述的抗体或其抗原结合片段、所述的抗体药物偶联物或者所述的双特异性抗体(或包含其的药物组合物)的单次药物剂型，如以一个安瓿瓶为单位。In the present invention, the term "single drug dosage unit" refers to the antibody or antigen-binding fragment thereof, the antibody-drug conjugate of the present invention to be administered to a subject or patient at the moment of the dosing regimen Or a single-dose pharmaceutical dosage form of the bispecific antibody (or a pharmaceutical composition comprising the same), such as in one ampoule.
如本文中所使用的，当提及IL-1β的氨基酸序列时，其包括IL-1β蛋白的全长(GenBank ID：NP_000567.1)，还包括IL-1β的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)或者多个His进行融合的片段。然而，本领域技术人员理解，在IL-1β的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-1β”应包括所有此类序列，及其天然或人工的变体。并且，当描述IL-1β蛋白的序列片段时，其不仅包括序列片段，还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of IL-1β, it includes the full length of the IL-1β protein (GenBank ID: NP_000567.1), and also includes fusion proteins of IL-1β, eg, with mouse or The Fc protein fragment of human IgG (mFc or hFc) or a fragment of multiple His fusions. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced in the amino acid sequence of IL-1β without affecting its biological function. Therefore, in the present invention, the term "IL-1β" shall include all such sequences, as well as natural or artificial variants thereof. And, when describing a sequence fragment of IL-1β protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variants.
如本文中所使用的，当提及IL-1R1的氨基酸序列时，其包括IL-1R1蛋白的全长(GenBank ID：NP_000868)，还包括IL-1R1的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)或者多个His进行融合的片段。然而，本领域技术人员理解，在IL-1R1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-1R1”应包括所有此类序列以及其天然或人工的变体。并且，当描述IL-1R1蛋白的序列片段时，包括IL-1R1序列片段，还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of IL-1R1, it includes the full length of the IL-1R1 protein (GenBank ID: NP_000868), and also includes fusion proteins of IL-1R1, eg, with mouse or human IgG Fc protein fragments (mFc or hFc) or fragments fused with multiple His. However, those skilled in the art understand that in the amino acid sequence of IL-1R1 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "IL-1R1" shall include all such sequences as well as natural or artificial variants thereof. Also, when describing a sequence fragment of an IL-1R1 protein, it includes the IL-1R1 sequence fragment, and also includes the corresponding sequence fragment in its natural or artificial variants.
如本文中所使用的，当提及IL-1R2的氨基酸序列时，其包括IL-1R2蛋白的全长(GenBank ID：CAA42441.1)，还包括IL-1R2的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)或者多个His进行融合的片段。然而，本领域技术人员理解，在IL-1R2蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-1R2”应包括所有此类序列以及其天然或人工的变体。并且，当描述IL-1R2蛋白的序列片段时，包括IL-1R2序列片段，还包括其天然或人工变体中的相应序列片段。As used herein, when referring to the amino acid sequence of IL-1R2, it includes the full length of the IL-1R2 protein (GenBank ID: CAA42441.1), and also includes fusion proteins of IL-1R2, eg, with mouse or The Fc protein fragment of human IgG (mFc or hFc) or a fragment of multiple His fusions. However, those skilled in the art understand that in the amino acid sequence of IL-1R2 protein, mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "IL-1R2" shall include all such sequences as well as natural or artificial variants thereof. Also, when a sequence fragment of an IL-1R2 protein is described, the IL-1R2 sequence fragment is included, as well as the corresponding sequence fragment in natural or artificial variants thereof.
如本文中所使用的，术语EC50是指半最大效应浓度(concentration for 50％ofmaximal effect)，是指能引起50％最大效应的浓度。As used herein, the term EC50 refers to the concentration for 50% of maximal effect, which refers to the concentration that elicits 50% of the maximal effect.
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区即CDR)，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of ImmunologicalInterest(National Institutes of Health，Bethesda，Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM抗体。As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain . Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability (called complementarity determining regions or CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair, respectively, form the antibody binding site. The assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al (1989) Nature 342:878-883 definition. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology，Ch.7(Paul，W.，ed.，第2版，Raven Press，N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab′、F(ab′)2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to, and/or compete with, the same antigen to which the full-length antibody binds Specific binding to an antigen, which is also referred to as an "antigen binding moiety". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is hereby incorporated by reference in its entirety for all purposes. Recombinant DNA techniques Or generate antigen-binding fragments of antibodies by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining regions (CDRs). Fragments, single chain antibodies (eg, scFvs), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide.
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如，Bird等人，Science 242：423426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85：58795883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90：6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immunol.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述。In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (eg, scFv) in which the VL and VH domains are paired to form a monovalent molecule by linkers that enable it to be produced as a single polypeptide chain (see, eg, Bird et al. , Science 242: 423426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 58795883 (1988)). Such scFv molecules may have the general structure:NH2 -VL-linker-VH-COOH orNH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker with the amino acid sequence (GGGGS) 4 can be used, but also variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.等人，Proc.Natl.Acad.Sci.USA 90：6444-6448(1993)，和Poljak R.J.等人，Structure 2：1121-1123(1994))。In some cases, the antigen-binding fragment of the antibody is a diabody, i.e., a diabody, in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow for both structures on the same chain Pairing between domains forces the domains to pair with the complementary domains of the other chain and create two antigen-binding sites (see, eg, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2: 1121-1123 (1994)).
在另一些情况下，抗体的抗原结合片段是“双功能抗体”。双功能抗体亦称为双特异性抗体(Bispecific Antibody)，是同时靶向两种不同抗原的特异性药物，其可通过免疫分选纯化生产。另外，也可通过基因工程获得，基因工程在结合位点优化，合成形式的考量以及产量等方面都具有相应的灵活性，所以具有一定的优势。目前，其存在形式已被证明有超过45种(Müller D，Kontermann RE.Bispecific antibodies for cancerimmunotherapy：Current perspectives.BioDrugs 2010；24：89-98)。目前已开发的多种双特异性抗体为IgG-ScFv形式即Morrison模式(1997Coloma MJ，Morrison SL.Design andproduction of novel tetravalent bispecific antibodies.Nature Biotechnology，1997；15：159-163)，由于这种类似于天然存在的IgG形式，其在抗体工程、表达和纯化上所具有的优势，已被证明是双功能抗体的其中一种理想存在形式(Miller BR，Demarest SJ，et al.，Stability engineering of scFvs for the development of bispecific andmultivalent antibodies.Protein Eng Des Sel 2010；23：549-57；Fitzgerald J，Lugovskoy A.Rational engineering of antibody therapeutics targeting multipleoncogene pathways.MAbs 2011；3：299-309)。In other instances, the antigen-binding fragment of an antibody is a "diabody." Bifunctional antibodies, also known as bispecific antibodies (Bispecific Antibodies), are specific drugs that simultaneously target two different antigens, which can be produced by immunosorting and purification. In addition, it can also be obtained through genetic engineering. Genetic engineering has corresponding flexibility in terms of binding site optimization, consideration of synthetic forms, and yield, so it has certain advantages. Currently, more than 45 forms of its existence have been demonstrated (Müller D, Kontermann RE. Bispecific antibodies for cancerimmunotherapy: Current perspectives. BioDrugs 2010; 24: 89-98). A variety of bispecific antibodies have been developed so far in the IgG-ScFv format, the Morrison model (1997Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nature Biotechnology, 1997; 15: 159-163), due to this similarity to The naturally occurring IgG form, with its advantages in antibody engineering, expression and purification, has been shown to be one of the ideal forms for diabodies (Miller BR, Demarest SJ, et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010;23:549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multipleoncogene pathways. MAbs 2011;3:299-309).
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体3H6、3H6H1 L1、3H6H2L2、3H6 H3L3或3H6H4L1)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。From a given antibody, such as the monoclonal antibodies 3H6, 3H6H1 L1, 3H6H2L2, 3H6 H3L3 or 3H6H4L1 provided herein, conventional techniques known to those of skill in the art (eg, recombinant DNA techniques or enzymatic or chemical fragmentation methods) can be used. ) to obtain antigen-binding fragments of the antibody (eg, the antibody fragments described above), and screen the antigen-binding fragments of the antibody for specificity in the same manner as for intact antibodies.
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature，256：495，1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules. Monoclonal antibodies are highly specific for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies are typically obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (eg, see U.S.P 4,816,567).
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.，Nature，321：522 525(1986)；Reichmann etal.，Nature，332：323 329(1988)；Presta，Curr.Op.Struct.Biol.，2：593 596(1992)；和Clark，Immunol.Today 21：397402(2000)。As used herein, the term "humanized antibody" refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody) The antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity. In addition, some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, eg, Jones et al., Nature, 321:522 525 (1986); Reichmann et al., Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593 596 (1992); and Clark, Immunol. Today 21: 397402 (2000).
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。As used herein, the term "isolated" or "isolated" refers to artificially obtained from the natural state. If an "isolated" substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of. The terms "isolated" or "isolated" do not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as λ phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain an origin of replication site.
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
如本发明中所使用的，术语“双特异性”、“双重特异性”或“双功能性”抗原结合蛋白或抗体是分别具有两个不同的抗原结合位点的杂交抗原结合蛋白或抗体。双特异性抗体是一种多特异性抗原结合蛋白或多特异性抗体，并且可通过多种方法产生，包括，但不限于杂交瘤的融合或Fab′片段的连接。参见，例如，Songsivilai和Lachmann，1990，Clin.Exp.Immunol.79：315-321；Kostelny等人.1992，J.Immunol.148：1547-1553。双特异性抗原结合蛋白或抗体的两个结合位点将结合两个不同的表位，所述表位存在于相同或不同的蛋白质靶标上。As used in the present invention, the terms "bispecific", "dual specific" or "bifunctional" antigen binding proteins or antibodies are hybrid antigen binding proteins or antibodies that have two distinct antigen binding sites, respectively. A bispecific antibody is a multispecific antigen binding protein or multispecific antibody, and can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linkage of Fab' fragments. See, eg, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al. 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes, which are present on the same or different protein targets.
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、小于大约10-7M、小于大约10-8M、小于大约10-9M或小于大约10-10M或更小的亲和力(KD)结合该抗原。As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as between an antibody and the antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) refers to an antibody that is less than about10-5 M, such as less than about10-6 M, less than about10-7 M, less than about10-8 M, less than about10-9 M, or less than about10-10 M or less binds the antigen with an affinity (KD ).
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体(例如，本发明的单克隆抗体3H6、3H6H1L1、3H6H2L2、或3H6H3L3)以小于大约10-5M，例如小于大约10-6M、小于大约10-7M、小于大约10-8M、小于大约10-9M或小于大约10-10M或更小的解离平衡常数(KD)结合抗原(例如，IL-1β蛋白)。可以使用本领域技术人员知悉的方法测定KD，例如使用Biacore分子相互作用仪测定。As used herein, the term "KD " refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, an antibody (eg, a monoclonal antibody 3H6, 3H6H1L1, 3H6H2L2, or 3H6H3L3 of the invention) is produced at a concentration of less than about10-5 M, such as less than about10-6 M, less than about10-7 M, less than about10-8 M , less than about10-9 M or less than about10-10 M or less with a dissociation equilibrium constant (KD ) that binds an antigen (eg, IL-1β protein).KD can be determined using methods known to those skilled in the art, eg, using a Biacore Molecular Interactometer.
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。例如，当提及杂交瘤细胞株LT010时，其还指杂交瘤细胞株LT010的亚克隆和后代细胞。As used herein, the terms "hybridoma" and "hybridoma cell line" are used interchangeably, and when referring to the terms "hybridoma" and "hybridoma cell line", it also includes subclones of hybridomas and progeny cells. For example, when referring to the hybridoma cell line LT010, it also refers to subclones and progeny cells of the hybridoma cell line LT010.
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者或患者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19thed.Pennsylvania：Mack Publishing Company，1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject or patient and the active ingredient agents, which are well known in the art (see, eg, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19thed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, Ionic strength enhancer. For example, pH adjusting agents include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如RA)有效量是指，足以预防、阻止或延迟疾病(例如RA)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactically effective amount refers to an amount sufficient to prevent, arrest or delay the onset of a disease (eg RA); a therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent the disease in a patient already suffering from the disease and the amount of its complications.
发明的有益效果：Beneficial effects of the invention:
本发明的抗IL-1β抗体特别是人源化的抗IL-1β抗体具有如下技术效果中的一项或多项：The anti-IL-1β antibody of the present invention, especially the humanized anti-IL-1β antibody, has one or more of the following technical effects:
(1)有效地结合人IL-1β，阻断IL-1β与其受体IL-1R1的结合；(1) Effectively bind to human IL-1β and block the binding of IL-1β to its receptor IL-1R1;
(2)抑制IL-1β下游信号通路的激活；(2) Inhibit the activation of IL-1β downstream signaling pathway;
(3)能够特异性抑制IL-1β诱导MRC-5细胞分泌IL-6的活性；(3) Can specifically inhibit the activity of IL-1β-induced MRC-5 cells to secrete IL-6;
(4)能够有效地阻断IL-1β对NF-κB的激活；(4) Can effectively block the activation of NF-κB by IL-1β;
(5)具有用于制备抑制IL-1β的药物的潜力；(5) has the potential to be used for the preparation of drugs that inhibit IL-1β;
(6)具有用于制备预防和/或治疗由病原微生物感染所引起的系统性炎症，包括系统性炎症综合征，细胞因子释放综合征、多器官功能障碍综合征(multiple organdysfunction syndrome，MODS)、急性呼吸窘迫综合征的药物的潜力。(6) It has the ability to prevent and/or treat systemic inflammation caused by pathogenic microorganism infection, including systemic inflammatory syndrome, cytokine release syndrome, multiple organ dysfunction syndrome (MODS), Drug potential for acute respiratory distress syndrome.
附图说明Description of drawings
图1：3H6、3H6H1L1、3H6H2L2和3H6H3L3与人IL-1β-His-Bio的结合活性检测结果。Figure 1: Binding activity test results of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 to human IL-1β-His-Bio.
图2：3H6H4L1与人IL-1β-His-Bio的结合活性检测结果。Figure 2: Binding activity test results of 3H6H4L1 to human IL-1β-His-Bio.
图3：3H6、3H6H1L1、3H6H2L2和3H6H3L3与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果。Figure 3: Activity assay results of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 competing with human IL-1R1(1-332)-his for binding to human IL-1β-hFc.
图4：3H6H4L1与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果。Figure 4: Activity assay results of 3H6H4L1 competing with human IL-1R1(1-332)-his for binding to human IL-1β-hFc.
图5：3H6H4L1与人IL-1β亲和力常数检测结果。注：曲线1～5分别表示分析物的浓度分别为25nM、12.5nM、6.25nM、3.13nM、1.56nM。Figure 5: The results of the affinity constant detection between 3H6H4L1 and human IL-1β. Note:
图6：Canakinumab与人IL-1β亲和力常数检测结果。注：曲线1～5分别表示分析物的浓度分别为25nM、12.5nM、6.25nM、3.13nM、1.56nM。Figure 6: Canakinumab and human IL-1β affinity constant test results. Note:
图7：3H6H4L1对IL-1β诱导MRC-5分泌IL-6的抑制作用。Figure 7: Inhibitory effect of 3H6H4L1 on IL-1β-induced IL-6 secretion by MRC-5.
图8：不同剂量3H6H4L1对IL-1β的中和生物学活性。Figure 8: Neutralizing biological activity of 3H6H4L1 at different doses on IL-1β.
图9：3H6H4L1阻断IL-1β的剂量效应曲线。Figure 9: Dose-response curve of 3H6H4L1 blocking IL-1β.
具体实施方式Detailed ways
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. Those who do not indicate specific technology or conditions in the embodiment, according to the technology or conditions described in the literature in this area (for example, with reference to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang et al., 3rd edition, Science Press) or follow the product instructions. If the reagents or instruments used do not indicate the manufacturer, they are conventional products that can be purchased in the market.
在本发明的下述实施例中，使用的BALB/c小鼠购自广东省医学实验动物中心。In the following examples of the present invention, the BALB/c mice used were purchased from the Guangdong Provincial Medical Laboratory Animal Center.
在本发明的下述实施例中，所用的同靶点已上市药物抗体Canakinumab(商品名
制备例1：融合蛋白人IL-1β-His、IL-1R1(1-332)-His、IL-1β-hFc以及人IL-1β-Preparation Example 1: Fusion proteins human IL-1β-His, IL-1R1(1-332)-His, IL-1β-hFc and human IL-1β-His-Bio的制备Preparation of His-Bio
通过NCBI GenBank蛋白数据库查找人IL-1β(Genbank ID：NP_000567.1)及IL-1R1(Genbank ID：NP_000868，1-332位氨基酸)的蛋白序列。将人IL-1β及IL-1R1的氨基酸序列分别与His标签序列(6个His)，人IgG Fc纯化标签序列(Ig gamma-1 chain C region，GenbankID：P01857位置114-330)进行融合设计；上述的融合蛋白分别简写命名为人IL-1β-His、IL-1R1(1-332)-His、IL-1β-hFc。The protein sequences of human IL-1β (Genbank ID: NP_000567.1) and IL-1R1 (Genbank ID: NP_000868, amino acids 1-332) were searched through the NCBI GenBank protein database. The amino acid sequences of human IL-1β and IL-1R1 were respectively designed by fusion with His tag sequence (6 His) and human IgG Fc purification tag sequence (Ig gamma-1 chain C region, GenbankID: P01857 position 114-330); The above fusion proteins are abbreviated as human IL-1β-His, IL-1R1(1-332)-His, and IL-1β-hFc, respectively.
蛋白样品质量通过SDS-PAGE鉴定合格。The quality of protein samples was qualified by SDS-PAGE.
使用
制得的上述融合蛋白用于下面的实施例。The above-described fusion proteins prepared were used in the following examples.
实施例1：抗IL-1β鼠源抗体3H6的制备Example 1: Preparation of anti-IL-1β murine antibody 3H6
1.杂交瘤细胞株LT010的制备1. Preparation of hybridoma cell line LT010
以人IL-1β-his作为抗原免疫BALB/c小鼠(购自广东医学实验动物中心)，取免疫后小鼠的脾细胞与小鼠骨髓瘤细胞融合，制成杂交瘤细胞。以IL-1β-His-Bio作为抗原，对杂交瘤细胞进行ELISA法筛选，获得能够分泌和IL-1β-His-Bio特异性结合的抗体的杂交瘤细胞。对ELISA筛选得到的杂交瘤细胞，通过竞争ELISA筛选出能够分泌与受体IL-1R1(1-332)-His竞争结合IL-1β-hFc的抗体的杂交瘤细胞，并经过有限稀释法得到稳定的杂交瘤细胞株。杂交瘤细胞制备的方法参考目前已确立的方法(例如，Stewart，S.J.，“MonoclonalAntibody Production”，in Basic Methods in antibody Production andCharacterization，Eds.G.C.Howard and D.R.Bethell，Boca Raton：CRC Press，2000)。BALB/c mice (purchased from Guangdong Medical Laboratory Animal Center) were immunized with human IL-1β-his as an antigen, and the spleen cells of the immunized mice were fused with mouse myeloma cells to prepare hybridoma cells. Using IL-1β-His-Bio as the antigen, the hybridoma cells were screened by ELISA method, and the hybridoma cells that could secrete the antibody specifically binding to IL-1β-His-Bio were obtained. For the hybridoma cells screened by ELISA, the hybridoma cells that can secrete antibodies that compete with the receptor IL-1R1(1-332)-His for binding to IL-1β-hFc were screened by competitive ELISA, and were stabilized by limiting dilution method. of hybridoma cell lines. Methods of hybridoma cell preparation refer to currently established methods (eg, Stewart, S.J., "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. G.C. Howard and D.R. Bethell, Boca Raton: CRC Press, 2000).
发明人将以上杂交瘤细胞株命名为杂交瘤细胞株LT010(IL-1β-3H6)，其分泌的单克隆抗体命名为3H6。The inventors named the above hybridoma cell line as hybridoma cell line LT010 (IL-1β-3H6), and the monoclonal antibody secreted by it was named as 3H6.
杂交瘤细胞株LT010(IL-1β-3H6)，其于2018年6月21日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018133，保藏地址为中国.武汉.武汉大学，邮编：430072。Hybridoma cell line LT010 (IL-1β-3H6), which was deposited in the China Center for Type Culture Collection (CCTCC) on June 21, 2018, the deposit number is CCTCC NO: C2018133, and the deposit address is China. Wuhan. Wuhan University , Zip Code: 430072.
2.抗IL-1β的抗体3H6的制备2. Preparation of Anti-IL-1β Antibody 3H6
用含杂交瘤无血清培养基对上面制得的LT010细胞株进行培养(杂交瘤无血清培养基，内含1％青链霉素和4％的Glutamax，于5％CO2，37℃细胞培养箱中进行培养)，7天后收集细胞培养上清，通过高速离心、微孔滤膜抽真空过滤以及HiTrap protein A HP柱进行纯化，制得抗体3H6。纯化后的3H6样品进行SDS-PAGE电泳检测合格。The LT010 cell line prepared above was cultured with a hybridoma serum-free medium (hybridoma serum-free medium, containing 1% penicillin and 4% Glutamax, in 5% CO2 , 37°C for cell culture The cell culture supernatant was collected after 7 days, and purified by high-speed centrifugation, vacuum filtration with a microporous membrane, and HiTrap protein A HP column to obtain antibody 3H6. The purified 3H6 samples were qualified by SDS-PAGE electrophoresis.
实施例2：抗IL-1β的抗体3H6的序列分析Example 2: Sequence analysis of anti-IL-1β antibody 3H6
按照总RNA提取试剂盒(Tiangen，货号DP430)的方法，从实施例1中培养的LT010细胞株中提取mRNA。按照Invitrogen
将TA克隆的产物直接进行测序，测序结果如下：The products of the TA clones were directly sequenced, and the sequencing results were as follows:
编码抗体3H6的重链可变区的核酸序列：(354bp)Nucleic acid sequence encoding the heavy chain variable region of antibody 3H6: (354bp)
抗体3H6的重链可变区的氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)The amino acid sequence of the heavy chain variable region of antibody 3H6 is as follows: (118aa, wherein the underlined amino acid sequence is the CDR region)
编码抗体3H6的轻链可变区的核酸序列：(318bp)Nucleic acid sequence encoding the light chain variable region of antibody 3H6: (318bp)
抗体3H6的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)The amino acid sequence of the light chain variable region of antibody 3H6 is as follows: (106aa, wherein the underlined amino acid sequence is the CDR region)
实施例3：抗IL-1β的人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1的设计和制Example 3: Design and manufacture of anti-IL-1β humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1备prepare
1.抗IL-1β人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1的轻链和重链序列的设计1. Design of light and heavy chain sequences of anti-IL-1β humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1
根据IL-1β蛋白的三维晶体结构(van Oostrum J，Priestle JP，Grütter MG，Schmitz A.The structure of murine interleukin-1beta at 2.8A resolution.JStruct Biol.1991，107(2)：189-95.)以及实施例2获得的序列信息，设计得到人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1重链可变区的序列和轻链可变区的序列(3H6H1L1、3H6H2L2和3H6H3L3抗体恒定区序列，来自NCBI的数据库，重链恒定区为Ig gamma-1chain Cregion，ACCESSION：P01857，轻链恒定区为Ig kappa chain C region，ACCESSION：P01834；3H6H4L1抗体恒定区序列，来自NCBI的数据库，重链恒定区为Ig gamma-4chain C region，ACCESSION：P01861.1；轻链恒定区为Ig kappa chain C region，ACCESSION：P01834)。According to the three-dimensional crystal structure of IL-1β protein (van Oostrum J, Priestle JP, Grütter MG, Schmitz A. The structure of murine interleukin-1beta at 2.8A resolution. JStruct Biol. 1991, 107(2): 189-95.) And the sequence information obtained in Example 2, the sequence of the variable region of the heavy chain and the variable region of the light chain of the humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 (3H6H1L1, 3H6H2L2 and 3H6H3L3 antibody constant region sequences, from NCBI database, the heavy chain constant region is Ig gamma-1chain Cregion, ACCESSION: P01857, the light chain constant region is Ig kappa chain C region, ACCESSION: P01834; 3H6H4L1 antibody constant region sequence, from the NCBI database, the heavy chain constant region is Ig gamma-4chain C region, ACCESSION: P01861.1; light chain constant region is Ig kappa chain C region, ACCESSION: P01834).
人源化抗体3H6H1L1、3H6H2L2、3H6H3L3和3H6H4L1的重链可变区序列和轻链可变区序列如下：The heavy chain variable region sequences and light chain variable region sequences of the humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 are as follows:
(1)人源化单克隆抗体3H6H1L1(1) Humanized monoclonal antibody 3H6H1L1
编码抗体3H6H1L1的重链可变区的核酸序列：(354bp)Nucleic acid sequence encoding the heavy chain variable region of antibody 3H6H1L1: (354bp)
抗体3H6H1L1的重链可变区氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)The heavy chain variable region amino acid sequence of antibody 3H6H1L1 is as follows: (118aa, wherein the underlined amino acid sequence is the CDR region)
编码抗体3H6H1L1的轻链可变区的核酸序列：(318bp)Nucleic acid sequence encoding the light chain variable region of antibody 3H6H1L1: (318bp)
抗体3H6H1L1的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)The amino acid sequence of the light chain variable region of antibody 3H6H1L1 is as follows: (106aa, wherein the underlined amino acid sequence is the CDR region)
(2)人源化单克隆抗体3H6H2L2(2) Humanized monoclonal antibody 3H6H2L2
编码抗体3H6H2L2的重链可变区的核酸序列：(354bp)Nucleic acid sequence encoding the heavy chain variable region of antibody 3H6H2L2: (354bp)
抗体3H6H2L2的重链可变区的氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)The amino acid sequence of the heavy chain variable region of antibody 3H6H2L2 is as follows: (118aa, wherein the underlined amino acid sequence is the CDR region)
编码抗体3H6H2L2的轻链可变区的核酸序列：(318bp)Nucleic acid sequence encoding the light chain variable region of antibody 3H6H2L2: (318bp)
抗体3H6H2L2的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)The amino acid sequence of the light chain variable region of antibody 3H6H2L2 is as follows: (106aa, wherein the underlined amino acid sequence is the CDR region)
(3)人源化单克隆抗体3H6H3L3(3) Humanized monoclonal antibody 3H6H3L3
编码抗体3H6H3L3的重链可变区的核酸序列：(354bp)Nucleic acid sequence encoding the heavy chain variable region of antibody 3H6H3L3: (354bp)
抗体3H6H3L3的重链可变区的氨基酸序列如下：(118aa，其中下划线标记的氨基酸序列为CDR区域)The amino acid sequence of the heavy chain variable region of antibody 3H6H3L3 is as follows: (118aa, wherein the underlined amino acid sequence is the CDR region)
编码抗体3H6H3L3的轻链可变区的核酸序列：(318bp)Nucleic acid sequence encoding the light chain variable region of antibody 3H6H3L3: (318bp)
抗体3H6H3L3的轻链可变区的氨基酸序列如下：(106aa，其中划线标记的氨基酸序列为CDR区域)The amino acid sequence of the light chain variable region of antibody 3H6H3L3 is as follows: (106aa, wherein the underlined amino acid sequence is the CDR region)
(4)人源化单克隆抗体3H6H4L1(4) Humanized monoclonal antibody 3H6H4L1
编码抗体3H6H4L1的重链可变区的核酸序列如SEQ ID NO：5所示。The nucleic acid sequence encoding the heavy chain variable region of antibody 3H6H4L1 is shown in SEQ ID NO:5.
抗体3H6H4L1的重链可变区的氨基酸序列如SEQ ID NO：6所示。The amino acid sequence of the heavy chain variable region of antibody 3H6H4L1 is shown in SEQ ID NO:6.
编码抗体3H6H4L1的轻链可变区的核酸序列如SEQ ID NO：7所示。The nucleic acid sequence encoding the light chain variable region of antibody 3H6H4L1 is shown in SEQ ID NO:7.
抗体3H6H4L1的轻链可变区的氨基酸序列如SEQ ID NO：8所示。The amino acid sequence of the light chain variable region of antibody 3H6H4L1 is shown in SEQ ID NO:8.
2.人源化抗体3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1的制备2. Preparation of humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1
3H6H1L1、3H6H2L2和3H6H3L3的重链恒定区均采用Ig gamma-1chain C region，ACCESSION：P01857；轻链恒定区均采用Ig kappa chain C region，ACCESSION：P01834；The heavy chain constant regions of 3H6H1L1, 3H6H2L2 and 3H6H3L3 all use the Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant regions all use the Ig kappa chain C region, ACCESSION: P01834;
3H6H4L1的重链恒定区为Ig gamma-4chain C region，ACCESSION：P01861.1；轻链恒定区为Ig kappa chain C region，ACCESSION：P01834。The heavy chain constant region of 3H6H4L1 is Ig gamma-4chain C region, ACCESSION: P01861.1; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834.
将3H6H1L1重链cDNA和轻链的cDNA、3H6H2L2的重链cDNA和轻链的cDNA、3H6 H3L3重链cDNA和轻链的cDNA，以及3H6H4L1重链cDNA和轻链的cDNA分别克隆到pUC57simple载体(金斯瑞公司提供)中，分别获得8个重组质粒即pUC57simple-3H6H1和pUC57simple-3H6L1；pUC57simple3H6H2和pUC57simple-3H6L2；pUC57simple-3H6H3和pUC57simple-3H6L3；pUC57simple-3H6H4和pUC57simple-3H6L1。并分别亚克隆到pcDNA3.1载体中。将包含重链的质粒和包含轻链的重组质粒共转染293F细胞后收集培养液进行纯化获得人源化抗体3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1；并经SDS-PAGE检测，结果正确。3H6H1L1 heavy chain cDNA and light chain cDNA, 3H6H2L2 heavy chain cDNA and light chain cDNA, 3H6 H3L3 heavy chain cDNA and light chain cDNA, and 3H6H4L1 heavy chain cDNA and light chain cDNA were cloned into pUC57simple vector (gold 8 recombinant plasmids were obtained, namely pUC57simple-3H6H1 and pUC57simple-3H6L1; pUC57simple3H6H2 and pUC57simple-3H6L2; pUC57simple-3H6H3 and pUC57simple-3H6L3; pUC57simple-3H6H4 and pUC57simple-3H6L1. And subcloned into pcDNA3.1 vector respectively. The plasmid containing the heavy chain and the recombinant plasmid containing the light chain were co-transfected into 293F cells, and the culture medium was collected and purified to obtain humanized antibodies 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1; and the results were correct by SDS-PAGE.
实施例4：抗体3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1与人IL-1β-his-bio结合Example 4: Binding of antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 to human IL-1β-his-bio的活性研究(ELISA法)Activity study (ELISA method)
以2μg/mL的SA(链霉亲和素)，每孔50μL包被酶标板，4℃孵育过夜。洗板一次并除去残留液体，每孔用300μL 1％BSA溶液(用PBS溶解)封闭，37℃孵育2小时。洗板三次并除去残留液体。用PBST将人IL-1β-His-Bio稀释成0.2μg/mL，50μL/孔，37℃孵育30分钟，洗板三次并除去残留液体。分别将抗体稀释至如表1的1μg/mL或如表2的0.333μg/mL作为起始浓度，进行1∶3的梯度稀释共7个浓度，另设空白对照，均做2个复孔，每孔体积100μL，37℃孵育30分钟。洗板三次后通过拍打将包被好的酶标板除去残余液体，每孔加入50μL辣根过氧化物酶标记的羊抗人IgG(H+L)二抗(货号：109-035-088，厂家：Jackson)工作液或辣根过氧化物酶标记的羊抗鼠IgG(H+L)二抗(货号：115-035-062，厂家：Jackson)工作液，37℃孵育30分钟。其中，50μL辣根过氧化物酶标记的羊抗人IgG(H+L)二抗工作液(对应3H6H1L1、3H6H2L2、3H6H3L3、3H6H4L1、Canakinumab所在的孔)和50μL辣根过氧化物酶标记的羊抗鼠IgG(H+L)二抗工作液(对应3H6所在的孔)。洗板四次并除去残留液体，每孔加入50μL TMB显色液，室温避光显色5分钟后，每孔加入50μL终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。The ELISA plate was coated with 2 μg/mL SA (streptavidin), 50 μL per well, and incubated at 4°C overnight. The plate was washed once and the residual liquid was removed, each well was blocked with 300 μL of 1% BSA solution (dissolved in PBS), and incubated at 37° C. for 2 hours. Wash the plate three times and remove residual liquid. Human IL-1β-His-Bio was diluted with PBST to 0.2 μg/mL, 50 μL/well, incubated at 37°C for 30 minutes, washed three times and removed residual liquid. The antibody was diluted to 1 μg/mL as shown in Table 1 or 0.333 μg/mL as shown in Table 2 as the initial concentration, and a 1:3 gradient dilution was carried out for a total of 7 concentrations. The volume of each well was 100 μL, and the cells were incubated at 37°C for 30 minutes. After washing the plate three times, the coated ELISA plate was tapped to remove the residual liquid, and 50 μL of horseradish peroxidase-labeled goat anti-human IgG (H+L) secondary antibody (Cat. No.: 109-035-088) was added to each well. Manufacturer: Jackson) working solution or horseradish peroxidase-labeled goat anti-mouse IgG (H+L) secondary antibody (Cat. No.: 115-035-062, Manufacturer: Jackson) working solution, incubate at 37°C for 30 minutes. Among them, 50 μL horseradish peroxidase-labeled goat anti-human IgG (H+L) secondary antibody working solution (corresponding to the well of 3H6H1L1, 3H6H2L2, 3H6H3L3, 3H6H4L1, and Canakinumab) and 50 μL horseradish peroxidase-labeled sheep Anti-mouse IgG (H+L) secondary antibody working solution (corresponding to the well of 3H6). The plate was washed four times and the residual liquid was removed, 50 μL of TMB color development solution was added to each well, and after 5 minutes of color development at room temperature in the dark, 50 μL of stop solution was added to each well to stop the color development reaction. Immediately put the ELISA plate into the microplate reader, select 450nm light wavelength to read the OD value of each well of the ELISA plate.
用SoftMax Pro 6.2.1软件对数据进行分析处理。以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图，结果分别如图1及图2所示。3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1与人IL-1β-His-Bio的结合活性检测结果分别见表1和表2。The data were analyzed with SoftMax Pro 6.2.1 software. Taking the antibody concentration as the abscissa and the absorbance value as the ordinate, a 4-parameter fitting curve was drawn. The results are shown in Figure 1 and Figure 2, respectively. The detection results of the binding activities of 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 to human IL-1β-His-Bio are shown in Table 1 and Table 2, respectively.
表1：3H6、3H6H1L1、3H6H2L2以及3H6H3L3与人IL-1β-His-Bio的结合活性检测结果Table 1: Binding activity test results of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 to human IL-1β-His-Bio
表2：3H6H4L1与人IL-1β-His-Bio的结合活性检测结果Table 2: Binding activity test results of 3H6H4L1 and human IL-1β-His-Bio
结果显示：The results show:
3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1均能够有效地结合人IL-1β-His-Bio，并且结合效率呈现剂量依赖关系；3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 could effectively bind human IL-1β-His-Bio, and the binding efficiency showed a dose-dependent relationship;
在相同检测条件下，3H6 H1L1、3H6 H2L2及3H6H4L1与抗原人IL-1β-His-Bio的结合效率呈现剂量依赖关系，并且结合活性优于同靶点已上市药物Canakinumab；3H6H3L3结合活性与Canakinumab相当。Under the same detection conditions, the binding efficiency of 3H6 H1L1, 3H6 H2L2 and 3H6H4L1 to the antigen human IL-1β-His-Bio showed a dose-dependent relationship, and the binding activity was better than the listed drug Canakinumab with the same target; 3H6H3L3 binding activity was comparable to Canakinumab .
实施例5：抗体3H6、3H6H1L1、3H6H2L2、3H6H3L3及3H6H4L1与人IL-1R1(1-332)-hisExample 5: Antibodies 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 and human IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性研究(ELISA法)Competitive binding activity of human IL-1β-hFc (ELISA method)
以4μg/mL的人IL-1β-hFc，每孔50μL包被酶标板，4℃孵育过夜。洗板一次并除去残留液体，每孔加入300μL 1％BSA溶液(用PBS溶解)封闭，37℃孵育2小时，洗板三次并除去残留液体。抗体稀释至2μg/mL(终浓度1μg/mL)作为起始浓度，进行1：3的梯度稀释共7个浓度，另设空白对照，均做2个复孔，每孔体积50μL，室温孵育10分钟。将0.08μg/mL(终浓度0.04μg/ml)或0.1μg/ml(终浓度0.05μg/ml)的人IL-1R1(1-332)-his加入到酶标板中，每孔体积50μL与抗体体积1：1轻柔混匀，每孔终体积100μL，37℃孵育30分钟。洗板三次并除去残留液体，每孔加入50μL抗His标签鼠单克隆抗体(HRP标记)(货号：CW0285M，厂家：康为世纪)工作液，37℃孵育30分钟。洗板四次并除去残留液体，每孔加入50μl的TMB显色液，室温避光显色10分钟或5分钟后，每孔加入50μL终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。The ELISA plate was coated with 4 μg/mL human IL-1β-hFc, 50 μL per well, and incubated at 4°C overnight. Wash the plate once and remove the residual liquid, add 300 μL of 1% BSA solution (dissolved in PBS) to each well to block, incubate at 37° C. for 2 hours, wash the plate three times and remove the residual liquid. The antibody was diluted to 2 μg/mL (
用SoftMax Pro 6.2.1软件对数据进行分析处理，以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图，结果如图3及图4所示。3H6、3H6H1L1、3H6H2L2及3H6H3L3和3H6H4L1与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果见表3及表4。Use SoftMax Pro 6.2.1 software to analyze and process the data, and draw the 4-parameter fitting curve with the antibody concentration as the abscissa and the absorbance value as the ordinate. The results are shown in Figures 3 and 4. The activity detection results of 3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 competing with human IL-1R1(1-332)-his for binding to human IL-1β-hFc are shown in Table 3 and Table 4.
表3：3H6、3H6H1L1、3H6H2L2以及3H6H3L3与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果Table 3: Activity test results of 3H6, 3H6H1L1, 3H6H2L2 and 3H6H3L3 competing with human IL-1R1(1-332)-his for binding to human IL-1β-hFc
表4：3H6H4L1与人IL-1R1(1-332)-his竞争结合人IL-1β-hFc的活性检测结果Table 4: Activity test results of 3H6H4L1 competing with human IL-1R1(1-332)-his for binding to human IL-1β-hFc
结果显示：The results show:
3H6、3H6H1L1、3H6H2L2及3H6H3L3和3H6H4L1均能够有效地阻断抗原人IL-1β-hFc与其受体人IL-1R1(1-332)-his的结合，且阻断效率呈现剂量依赖关系，竞争结合活性优于同靶点已上市药物Canakinumab。3H6, 3H6H1L1, 3H6H2L2, 3H6H3L3 and 3H6H4L1 can effectively block the binding of the antigen human IL-1β-hFc to its receptor human IL-1R1(1-332)-his, and the blocking efficiency is dose-dependent, competing for binding The activity is better than the marketed drug Canakinumab with the same target.
实施例6：抗体3H6H4L1与人IL-1β的亲和力常数测定Example 6: Affinity constant determination of antibody 3H6H4L1 and human IL-1β
采用Biacore分子相互作用仪检测抗体与人IL-1β-his的亲和力常数。以PBST作为缓冲液，采用氨基偶联方式将抗体固定于CM5芯片表面，固定的信号值约为1000RU。抗体与人IL-1β结合，IL-1β浓度为1.56-25nM(两倍梯度稀释)，流速为30μl/min，结合的时间为120s，解离时间为600s。芯片使用3M MgCl2再生，流速为30μl/min，时间为30s。使用BiacoreControl 2.0软件进行数据采集，Biacore T200 Evaluation 2.0软件进行数据分析。结果如表5、图5和图6所示。The affinity constant of antibody and human IL-1β-his was detected by Biacore molecular interaction analyzer. Using PBST as a buffer, the antibody was immobilized on the surface of the CM5 chip by amino coupling, and the immobilized signal value was about 1000RU. The antibody binds to human IL-1β, the IL-1β concentration is 1.56-25nM (two-fold gradient dilution), the flow rate is 30μl/min, the binding time is 120s, and the dissociation time is 600s. Chips were regenerated using 3MMgCl2 at a flow rate of 30 μl/min for 30 s. BiacoreControl 2.0 software was used for data acquisition, and Biacore T200 Evaluation 2.0 software was used for data analysis. The results are shown in Table 5, Figure 5 and Figure 6.
表5：3H6H4L1与人IL-1β亲和力常数检测结果Table 5: Test results of affinity constant between 3H6H4L1 and human IL-1β
结果显示：3H6H4L1与人IL-1β的亲和力常数为8.79E-11M，Canakinumab与人IL-1β的亲和力常数为9.79E-11M，表明3H6H4L1与人IL-1β有较强的结合能力。The results showed that the affinity constant between 3H6H4L1 and human IL-1β was 8.79E-11M, and the affinity constant between Canakinumab and human IL-1β was 9.79E-11M, indicating that 3H6H4L1 had strong binding ability to human IL-1β.
实施例7：抗体3H6 H4L1的细胞生物学活性研究Example 7: Cell biological activity study of antibody 3H6 H4L1
1.3H6 H4L1阻断IL-1β诱导MRC-5细胞分泌IL-6的细胞学活性检测1.3H6 H4L1 blocking IL-1β-induced MRC-5 cells to secrete IL-6 cytological activity detection
人MRC-5细胞(购自中国科学院细胞中心)常规消化、计数，7，500个/孔接种于平底96孔板中，置于细胞培养箱培养；24h后(细胞生长达到80％汇合度时)，进行给药处理：抗体设4个浓度(0.003nM、0.03nM、0.3nM、3nM)，IL-1β(购自Sino Biological，货号：10139-HNAE)设三个浓度(5pM、50pM、500pM)，抗体组IL-1β用50pM的浓度(抗体和IL-1β先在37℃孵育30min)，另外同时设置空白对照组及同型对照组；加药处理后置于细胞培养箱中培养24h；收集细胞上清，用IL-6ELISA Kit(购自达科为生物技术有限公司，货号：1110602)进行检测。检测结果如图7和表6所示。Human MRC-5 cells (purchased from the Cell Center of the Chinese Academy of Sciences) were routinely digested and counted, and 7,500 cells/well were inoculated in a flat-bottom 96-well plate and cultured in a cell incubator; 24 hours later (when the cell growth reached 80% confluence ), for administration treatment: the antibody was set to four concentrations (0.003nM, 0.03nM, 0.3nM, 3nM), and the IL-1β (purchased from Sino Biological, product number: 10139-HNAE) was set to three concentrations (5pM, 50pM, 500pM). ), the concentration of IL-1β in the antibody group was 50 pM (the antibody and IL-1β were first incubated at 37°C for 30 min), and the blank control group and the isotype control group were set at the same time; after adding the drug, they were placed in a cell incubator for 24 hours; Cell supernatant was detected with IL-6 ELISA Kit (purchased from Daktronics Biotechnology Co., Ltd., product number: 1110602). The test results are shown in Figure 7 and Table 6.
表6：3H6 H4L1对IL-1β诱导MRC-5细胞分泌释放IL-6的抑制作用Table 6: Inhibitory effect of 3H6 H4L1 on IL-1β-induced secretion and release of IL-6 from MRC-5 cells
结果表明IL-1β可梯度诱导MRC-5细胞分泌IL-6，3H6 H4L1可显著抑制IL-1β对MRC-5分泌释放IL-6的诱导作用，且具有显著剂量依赖关系。综合表明，3H6 H4L1可通过特异性结合IL-1β，阻断IL-1β介导的免疫因子IL-6的分泌，阻断效能与同靶点对照抗体Canakinumab相当。The results showed that IL-1β could gradiently induce MRC-5 cells to secrete IL-6, and 3H6 H4L1 could significantly inhibit the induction effect of IL-1β on MRC-5 secretion and IL-6 in a dose-dependent manner. Overall, 3H6 H4L1 can specifically bind to IL-1β and block the secretion of IL-1β-mediated immune factor IL-6, and the blocking efficacy is comparable to that of the same target control antibody Canakinumab.
2.3H6 H4L1阻断IL-1β激活NF-κB信号通路2.3H6 H4L1 blocks IL-1β activation of NF-κB signaling pathway
本实验通过荧光报告基因方法检测3H6 H4L1阻断IL-1β激活NF-κB信号通路的中和生物活性。In this experiment, the neutralizing biological activity of 3H6 H4L1 blocking IL-1β-activated NF-κB signaling pathway was detected by fluorescent reporter gene method.
(1)293T-NF-κB-LUC细胞构建(1) Construction of 293T-NF-κB-LUC cells
胰酶消化293T细胞，传代培养；转染前2h，更换opti-DMEM培养基；在无菌EP管中，加入500μL opti-DMEM培养基，再加入3μg的质粒pNF-kB-Luc2P-hygro(Akeso)；随后用转染试剂PEI(货号：23966，Polyscience)和表达载体感染构建293T-NFκB-Luc细胞；转染8h后，更换新鲜培养基；转染24h后，加入Hygromycin B(Invitrogen，货号：10687010)，终浓度100μg/mL筛选，设对照孔293T未转染质粒。7-10天后，对照孔细胞完全死亡为准，收获筛选完的细胞扩增培养。继续加药100μg/mL维持。得到293T-NF-κB-LUC稳转细胞株。293T cells were trypsinized and subcultured; 2 h before transfection, opti-DMEM medium was replaced; 500 μL opti-DMEM medium was added to a sterile EP tube, followed by 3 μg of plasmid pNF-kB-Luc2P-hygro (Akeso ); then infected with the transfection reagent PEI (Cat. No. 23966, Polyscience) and the expression vector to construct 293T-NFκB-Luc cells; 8h after transfection, replace the fresh medium; 24h after transfection, add Hygromycin B (Invitrogen, Item No.: 10687010), the final concentration was 100 μg/mL for screening, and the control well 293T was not transfected with plasmid. After 7-10 days, the cells in the control wells were completely dead, and the selected cells were harvested for expansion and culture. Continue dosing at 100 μg/mL for maintenance. The 293T-NF-κB-LUC stably transfected cell line was obtained.
(2)3H6 H4L1阻断IL-1β激活NF-κB信号通路的中和生物活性检测常规消化293T-NF-κB-Luc细胞，按每孔20,000个细胞接种于96孔板中。待细胞贴壁后，添加IL-1β，使其终浓度1.65ng/mL，设置空白对照。同时添加抗体，Canakinumab，3H6 H4L1，每个抗体8个梯度，终浓度分别为：1600ng/mL，800ng/mL，200ng/mL，50ng/mL，12.5ng/mL，3.125ng/mL，0.3125ng/mL，0.03125ng/mL。共培养4小时后，去上清，加入50μL PBS，再加入50μL Bright-GloTM底物(Promega，货号：E2620)，反应5min，上机检测。(2) Detection of neutralizing biological activity of 3H6 H4L1 blocking IL-1β-activated NF-κB signaling pathway. 293T-NF-κB-Luc cells were routinely digested, and 20,000 cells per well were seeded in 96-well plates. After the cells adhered, IL-1β was added to make the final concentration 1.65ng/mL, and a blank control was set. Antibodies, Canakinumab, 3H6 H4L1, were added at the same time, 8 gradients for each antibody, and the final concentrations were: 1600ng/mL, 800ng/mL, 200ng/mL, 50ng/mL, 12.5ng/mL, 3.125ng/mL, 0.3125ng/mL mL, 0.03125ng/mL. After 4 hours of co-cultivation, the supernatant was removed, 50 μL of PBS was added, and 50 μL of Bright-GloTM substrate (Promega, product number: E2620) was added, and the reaction was performed for 5 minutes, and then detected on the machine.
结果如图8、图9所示。The results are shown in Figures 8 and 9 .
结果显示：The results show:
IL-1β依赖的以293T-NFκB-Luc作为工作细胞的报告基因检测体系，IL-1β可呈剂量依赖性激活293T-NFκB-Luc细胞的NF-κB信号通路，进而启动报告基因的表达。该体系可用于评价3H6H4L1对IL-1β的中和生物活性。本研究结果表明，3H6H4L1特异性与IL-1β结合后可高效抑制IL-1β介导的信号通路的活化，表现3H6 H4L1对IL-1β的中和生物学活性，中和活性EC50为0.0966nM，而同靶点上市药Canakinumab中和活性ECS0为0.0801nM，二者活性相当。IL-1β-dependent reporter gene detection system using 293T-NFκB-Luc as working cells, IL-1β can activate the NF-κB signaling pathway of 293T-NFκB-Luc cells in a dose-dependent manner, thereby initiating the expression of reporter genes. This system can be used to evaluate the neutralizing biological activity of 3H6H4L1 on IL-1β. The results of this study showed that the specific binding of 3H6H4L1 to IL-1β can effectively inhibit the activation of IL-1β-mediated signaling pathways, showing the neutralization biological activity of 3H6H4L1 on IL-1β. The neutralizing activity EC50 is 0.0966nM, The neutralizing activity ECS0 of Canakinumab, a marketed drug with the same target, was 0.0801 nM, and the activities of the two were comparable.
以上对本发明的较佳实施方式进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明精神的前提下还可做出种种的等同变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments, and those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention , these equivalent modifications or substitutions are all included within the scope defined by the claims of the present application.
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