CN111643676B - 一种双特异性二聚体、双特异性二聚体-药物偶联物及其应用 - Google Patents
一种双特异性二聚体、双特异性二聚体-药物偶联物及其应用 Download PDFInfo
- Publication number
- CN111643676B CN111643676B CN202010660313.5A CN202010660313A CN111643676B CN 111643676 B CN111643676 B CN 111643676B CN 202010660313 A CN202010660313 A CN 202010660313A CN 111643676 B CN111643676 B CN 111643676B
- Authority
- CN
- China
- Prior art keywords
- bispecific
- medicine
- reaction
- mmol
- added
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明提供了一种能够靶向叶酸受体的双特异性二聚体、双特异性二聚体‑药物缀合物,以及其在制备治疗肿瘤、自身免疫疾病药物中的应用。相较于传统的单独使用抗体介导或受体介导的靶向给药系统,本发明提供的双特异性偶联物能同时靶向肿瘤细胞表面的抗原和受体,达到结合两个抗原或者一个抗原的两个表位的目的,大大增加了与肿瘤细胞的结合能力。不但能将治疗药物最大限度地运送到病变部位,使治疗药物在病灶区浓集,病灶区的药物浓度超出常规制剂的数倍乃至数百倍,疗效显著提高;同时还可以减少药物的用量,从而降低药物的不良反应,而且便于控制给药的速度和方式,达到高效低毒的治疗效果。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种双特异性二聚体、双特异性二聚体-药物偶联物及其应用。
背景技术
恶性肿瘤是一类严重危害人类身心健康、影响社会劳动力的疾病,给众多家庭及社会带来了沉重的经济负担。据统计,2020年全世界癌症发病率将比现在增加50%,全球每年新增癌症患者人数将达到1500万人。我国近20年来癌症发病率上升了69%,死亡率上升了29%,每年癌症新发病例为220万,因癌症死亡人数为160万。每四五个死者中就有一个死于癌症,癌症已成为威胁人类生命健康的主要杀手。
目前,传统治疗恶性肿瘤的方法有手术治疗、放疗和化疗,但是手术治疗只能切除外在的肿瘤,对于通过淋巴途径转移、血液途径转移或浸润的正常组织肿瘤细胞却无能为力;放疗则是只能进行局部照射,而且在杀死肿瘤组织时也会对正常组织有影响;化学治疗是全身治疗,但是对肿瘤细胞不具有肿瘤特异性,在杀死肿瘤细胞时也会杀死正常细胞,而且对于潜伏期的肿瘤细胞无杀伤力。针对这些传统治疗癌症的方法存在的缺陷,一些专家学者提出了靶向给药这一概念。靶向给药系统一般分为主动靶向和被动靶向,主动靶向就是利用靶向分子将药物选择性地定位于肿瘤组织,降低药物对正常组织的毒副作用,包括抗体介导和受体介导的主动靶向给药系统。
近年来,叶酸受体作为抗肿瘤药物的靶点受到了极大的关注,成为新型抗肿瘤药物研究的热点之一。研究发现,叶酸受体在绝大多数恶性肿瘤细胞膜内都过度表达而正常细胞很少表达甚至不表达,而且叶酸与叶酸受体结合力强,能被高效介导进入肿瘤细胞。甲氨蝶呤(Methotrexate),简称MTX,是一种抗叶酸类抗肿瘤药物,目前临床上主要用于急性白血病、乳腺癌、绒毛膜上皮癌、骨肿瘤、类风湿性关节炎、系统性红斑狼疮等的化学治疗。它的结构与叶酸类似,可以与肿瘤细胞上的叶酸受体结合,利用叶酸受体在肿瘤细胞和正常细胞上表达的差异性及叶酸受体与甲氨蝶呤结合的高特异性、高亲和性,通过叶酸受体的介导作用可望实现肿瘤的靶向性。但是,由于肿瘤表面抗原存在异质性,受体配体的结合性、靶向性以及在肿瘤组织中的稳定性都有待提高。
发明内容
为了解决上述问题,本发明提供了一种能够靶向叶酸受体的双特异性二聚体,本发明还进一步提供了一种能够靶向叶酸受体的双特异性二聚体-药物缀合物,以及上述双特异性二聚体、双特异性二聚体-药物缀合物在制备治疗肿瘤、自身免疫疾病药物中的应用。
本发明提供了一种双特异性二聚体,其结构如式I所示:
其中:
A1为任一抗体或其功能性片段,其能够特异性靶向结合免疫检查点分子或肿瘤相关抗原;
A2能够特异性靶向叶酸受体;
L1、L2为任一连接基团,其中L1能与第一靶标单元A1通过氨基或者巯基共价连接;
m选自1、2、3、4、5、6、7、8。
进一步的,所述的A1为单克隆抗体、多克隆抗体、抗体片段、Fab、Fab'、Fab'-SH、F(ab')2、Fv、scFv、线性抗体、嵌合抗体、人源化抗体、人抗体、或包含抗体的抗原结合部分的融合蛋白,所述免疫检查点分子包括但不限于PD-1、PD-L1、CTLA-4、LAG-3、FGL1、TIM-3、Galectin-9、TIGIT、CD155、CD47;所述肿瘤抗原包括但不限于Claudin 18.2、Her-2、Mesothelin、BCMA、SSTR2、GPRC5D、PSMA、FCRH5、CD19、CD33、CD123、CD20、A33、CEA、CD28、DLL3、EGFR、VEGFR、VEGFR2、VEGF-A、Nectin-4、FGFR、c-Met、RANKL、PDGF、PDGFR、PDGFRα、DLL4、Ang-1、Ang-2、DR5。
进一步的,所述的第二靶标单元A2为甲氨蝶呤及其衍生物,优选的所述的衍生物为侧链羧基的衍生物。
优选的,所述的双特异性二聚体选自如下结构:
本发明还提供了一种双特异性二聚体-药物偶联物,其包含权利要求1所述的双特异性二聚体。
进一步的,所述的双特异性二聚体-药物偶联物还包括连接基团L3和活性单元D。
进一步的,所述的双特异性二聚体-药物偶联物结构如式(II)所示:
其中:
A1为任一抗体或其功能性片段,其能够特异性靶向结合免疫检查点分子或肿瘤相关抗原;
A2能够特异性靶向叶酸受体;
L1、L2、L3为任一连接基团,其中L1、L3能与第一靶标单元A1通过氨基或者巯基共价连接;
m选自1、2、3、4、5、6、7、8;
n选自1、2、3、4、5、6、7、8。
更进一步的,所述的连接基团L3选自以下结构中的一种或者它们的任意组合:C1-C9烷基、C2-C9烯基、C2-C9炔基、芳族化合物、杂芳基、C3-C9环烷基、C3-C9杂环基、聚乙二醇、O、S、NR1、C(=O)、C(=O)O、C(=O)NR1、C=NR1、C(=S)O、C(=S)NR1、C(=S)S、NR1(C=O)、NR1(C=S)NR2、O(C=O)NR1、S(=O)2、Val-Cit-PAB、Val-Ala-PAB、Val-Lys(Ac)-PAB、Phe-Lys-PAB、Phe-Lys(Ac)-PAB、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn-PAB、Ala-PAB、PAB,R1和R2独立的选自H、C1-C6烷基、C2-C6烯基、C2-C6炔基。
更进一步的,所述的活性单元D为细胞分化因子、干细胞营养因子、类固醇类药物、治疗自身免疫疾病的药物、抗炎症药物或治疗传染性疾病的药物。更进一步的,所述的活性单元D选自:瓢菌素(amanitins)、蒽环类物(anthracyclines)、澳瑞他汀(auristatins)、浆果赤霉素(baccatins)、加利车霉素(calicheamicins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多卡霉素(duocarmycins)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、多卡霉素(duocarmycins)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、美登木素生物碱(maytansinoids)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、吡咯并[2,1-c][1,4]苯并二氮杂(pyrrolo[2,1-c][1,4]benzodi-azepines;PBDs)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、SN-38、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vincaalkaloids)。
优选的,所述的双特异性二聚体-药物偶联物选自如下结构:
本发明还提供了一种药物组合物,包含有效量的前述任一项所述的双特异性二聚体或其药学上可接受的盐或溶剂合物以及药学上可接受的稀释剂、运载体或赋形剂。
本发明还提供了一种药物组合物,包含有效量的前述任一项所述的双特异性二聚体-药物偶联物或其药学上可接受的盐或溶剂合物以及药学上可接受的稀释剂、运载体或赋形剂。
本发明还提供了前述任一项所述的双特异性二聚体用于制造癌症治疗药物、自身免疫疾病治疗药物的用途。
本发明还提供了前述任一项所述的双特异性二聚体-药物偶联物用于制造癌症治疗药物、自身免疫疾病治疗药物的用途。
相较于传统的单独使用抗体介导或受体介导的靶向给药系统,本发明提供的双特异性偶联物能同时靶向肿瘤细胞表面的抗原和受体,达到结合两个抗原或者一个抗原的两个表位的目的,大大增加了与肿瘤细胞的结合能力。不但能将治疗药物最大限度地运送到病变部位,使治疗药物在病灶区浓集,病灶区的药物浓度超出常规制剂的数倍乃至数百倍,疗效显著提高;同时还可以减少药物的用量,从而降低药物的不良反应,而且便于控制给药的速度和方式,达到高效低毒的治疗效果。
附图说明
图1A为Anti-PD-L1抗体和Anti-PD-L1-Mc-Ser-MTX抗体药物偶联物对乳腺癌细胞MDA-MB-231的细胞ELISA实验;图1B为Anti-PD-L1抗体、Anti-PD-L1-NHS-PEG8-MTX对乳腺癌细胞MDA-MB-231的细胞ELISA实验;图1C为Anti-PD-L1抗体、Anti-PD-L1-Mc-EDA-MTX、Anti-PD-L1-Mc-DSDEA-MTX、Anti-PD-L1-Mcc-DSDEA-MTX和Anti-PD-L1-NHS-MTX的对乳腺癌细胞MDA-MB-231的细胞ELISA实验。
图2为Anti-EGFR抗体和Anti-EGFR-MC-Ser-MTX抗体药物偶联物对乳腺癌细胞SK-BR-3的细胞ELISA实验。
图3为Anti-c-Met抗体、Anti-C-MET-MTX、Anti-C-MET-MMAE、Anti-C-MET-MMAE-MTX、Anti-C-MET-MMAD、Anti-C-MET-MMAD-MTX的体外细胞活性评价实验。
具体实施方式
【定义】
与说明书的各方面相关的各种术语在说明书和权利要求书中通篇使用。除非另外指明,否则此类术语被赋予本领域的普通含义。其它具体定义的术语应按照与本文所提供的定义相符的方式理解。
如本文所用,术语“一个”和“一种”和“所述”是按照标准惯例使用的并且意指一个或多个,除非上下文另有指示。因此例如,对“一种抗体药物偶联物”的提及包括两个或更多个抗体药物偶联物的组合等等。
应当理解,无论何处在本文中用语言“包含”描述方面,除此之外还提供了以“由......组成”和/或“基本上由......组成”描述的类似方面。
尽管本发明的广义范围所示的数字范围和参数近似值,但是具体实施例中所示的数值尽可能准确的进行记载。然而,任何数值本来就必然含有一定的误差,其是由它们各自的测量中存在的标准偏差所致。另外,本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围;也就是说,所有以最小值1或更大起始的子范围,例如1至6.1,以及以最大值10或更小终止的子范围,例如5.5至10。另外,任何称为“并入本文”的参考文献应理解为以其整体并入。
【实施例】
下面结合具体实施方式对本发明的技术方案作进一步非限制性的详细说明。需要指出的是,下述实施例仅为说明本发明的技术构思及特点,其目的在于让本领域技术人员能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
实施例1化合物I-1’、I-2’、I-3’、I-4’、I-5’、I-6’、I-7’、I-8’(即L1-L2-A2)的制备
(1)I-1’的制备
N2保护下,称量MTX(227mg,0.5mmol,购自百灵威科技)于50mL单口茄形瓶中,加入TEA(101.2mg,1mmol)和3mL DMF,-5℃搅拌10min。称量TSTU(113.5mg,0.55mmol),溶于1mLDMF,滴加至上述反应液中,搅拌3h。称量化合物1(212mg,0.5mmol),溶于2mL DMF,滴加至上述反应液中,搅拌5h。反应结束后,反应液加入3mL水稀释,制备HPLC分离,制备液经冻干,得288.1mg黄色油状化合物2,收率67%。LC-MS(ESI+)861[M+H]+。
N2保护下,称量化合物2(288.1mg,0.34mmol)于10mL单口瓶中,加入5mL DCM,冰浴搅拌10min,滴加1mL TFA,冰浴搅拌2h。反应液不加热旋蒸浓缩至固体,补加5mL DCM继续旋蒸至固体,重复3次,得256.0mg浅黄色固体化合物3,,收率96%。LC-MS(ESI+)761[M+H]+。
N2保护下,称量化合物4(52.8mg,0.25mmol)、TSTU(90.3mg,0.3mmol)于10mL单口瓶中,加入TEA(75.9mg,0.75mmol)和2mL DMF,冰浴搅拌2.5h。称量I-1-2(199mg,0.25mmol),溶于2mL DMF中,滴加至反应液中,室温搅拌5h。反应结束后,反应液加2mL水稀释,制备HPLC分离,制备液冻干后得185.83mg黄色固体化合物I-1’,收率78%。LC-MS(ESI+)954[M+H]+。
(2)I-2’的制备
称取1999.7mg甲氨蝶呤和3004.7mgTSTU,加入100mL反应瓶中,用20mL注射器取20mL N,N-二甲基甲酰胺加入圆底烧瓶,再用10mL注射器取N,N-二异丙基乙胺6.6mL加入反应瓶,放置于磁力搅拌器上,加入搅拌子,室温搅拌2h。称取1866.3mg化合物20加入反应体系,室温搅拌16h。用20μL移液枪取样20μL加入1.5mL离心管,再用1mL移液枪取1mL乙腈加入离心管,再用1mL注射器吸取后用有机相针式滤器过滤后,送样,通过LC-MS检测反应情况。反应完成后,将反应液滴加至500mL二氯甲烷中,边滴加边搅拌,析出固体,用布氏漏斗过滤,用30mL二氯甲烷清洗滤饼,将滤饼取下,用真空油泵抽真空3h,得到黄色固体产品1.8g(即化合物21),收率为69%。LC-MS:(M+H)+:657,LC-MS:(M-H)-:655。
称取106.2mgMc-OH和383.5mgHATU,加入5mL反应瓶中,用5mL注射器取3mLN,N-二甲基甲酰胺加入圆底烧瓶,再用1000μL移液枪取553μLN,N-二异丙基乙胺加入圆底烧瓶,放置于磁力搅拌器上,加入搅拌子,室温搅拌2h。称取655.6mg化合物21,加入反应体系,室温搅拌16h。用20μL移液枪取样20μL加入1.5mL离心管,再用1mL移液枪取1mL乙腈加入离心管,再用1mL注射器吸取后用有机相针式滤器过滤后,送样,通过LC-MS检测反应情况。反应完成后,将反应液滴加至100mL二氯甲烷中,边滴加边搅拌,析出固体,用布氏漏斗过滤,用10mL二氯甲烷清洗滤饼,将滤饼取下,再制备纯化。制备方法为流动相A:H2O,0.1%HCOOH,流动相B:MeCN,0.1%HCOOH,流速15mL/min,梯度:10%B-30%B,30min,在20.1min出峰。将得到的制备液合并到250mL圆底烧瓶中,放-80℃冰箱冷冻3h,放冻干机冷冻干燥后,得到产品(I-2’)106mg。LC-MS:(M+H)+:850,LC-MS:(M-H)-:848。
(3)I-3’的制备
称取500.1mg甲氨蝶呤和902.7mg TSTU,加入50mL反应瓶中,用10mL注射器取6mLN,N-二甲基甲酰胺加入圆底烧瓶,再用3mL注射器取N,N-二异丙基乙胺1.1mL加入反应瓶,放置于磁力搅拌器上,加入搅拌子,室温搅拌16h。反应完成后,将反应液滴加至150mL二氯甲烷中,边滴加边搅拌,析出固体,用布氏漏斗过滤,用10mL二氯甲烷清洗滤饼,将滤饼取下,用真空油泵抽真空3h,得到黄色固体产品540mg(即I-3’),收率为83%。
(4)I-4’的制备
N2保护下,称量化合物4(15.4mg,0.2mmol)和化合物5(104.2mg,0.2mmol)于10mL单口瓶中,加入2mL水溶解,室温下搅拌反应1h。反应结束后,反应液直接冻干,得122mg无色油状化合物6,不经处理直接投下一步反应。LC-MS(ESI+)599[M+H]+。
N2保护下,称量MTX(181.6mg,0.4mmol)和TSTU(120.4mg,0.4mmol),置于25mL单口瓶中,加入TEA(50.5mg,0.5mmol)和3mL DMF,室温搅拌反应3h。将化合物6(122mg)溶于1mLDMF中,滴加到上述反应液中,室温搅拌反应过夜。反应完成后,反应液用2mL水稀释,经制备HPLC分离,制备液冻干,得131.9mg黄色固体化合物7,收率63.7%。LC-MS(ESI+)1036[M+H]+。
N2保护下,称量化合物7(51.8mg,0.05mmol)和TSTU(36.1mg,0.12mmol),置于10mL单口瓶中,加入TEA(15.2mg,0.15mmol)和2mL DMF。反应液室温搅拌反应3h。反应结束后,反应液中加入5mL EA和5mL PE,析出固体。过滤,滤饼用10mL PE:EA=1:1洗涤3次,烘干,得19.1mg深黄色固体化合物I-4’。收率31.1%。LC-MS(ESI+)1230[M+H]+。
(5)I-5’的制备
N2保护下,称量化合物7(51.8mg,0.05mmol)和化合物8(33.6mg,0.12mmol),置于10mL单口瓶中,加入TEA(15.2mg,0.15mmol)和2mL DMF。反应液室温搅拌反应3h。反应结束后,反应液加入0.5mL水,制备HPLC分离,制备液冻干,得17.8mg深黄色固体化合物I-5’。收率26.1%。LC-MS(ESI+)1368[M+H]+。
(6)I-6’的制备
N2保护下,称量化合物10(222.2mg,1mmol)和TSTU(903.2mg,3mmol),置于50mL单口瓶中,加入DIPEA(3.87mg,3mmol)和10mL DMF。反应液室温搅拌反应0.5h。随后,称量化合物9(228.1mg,1mmol),加入到反应液中,继续室温搅拌3h。反应结束后,反应液用50mL乙酸乙酯稀释,加入30mL水,萃取,分液,有机层用30mL水洗涤两次,30mL饱和氯化钠洗涤1次,无水硫酸钠干燥。干燥液过滤,减压浓缩至小体积,经正向柱层析,得330mg淡黄色目标化合物11,收率76.3%。LC-MS(ESI+)433.2[M+H]+。
N2保护下,称量MTX(454.4mg,1mmol)和TSTU(301.2mg,1mmol),置于50mL单口瓶中,加入DIPEA(3.87mg,3mmol)和10mLDMF。反应液室温搅拌反应0.5h。随后,称量化合物12(200mg,1mmol),加入到反应液中,继续室温搅拌3h。反应结束后,反应液用5mL水稀释,制备液相分离,制备液经冻干后,得252mg深黄色目标化合物13,收率39.5%。LC-MS(ESI+)640.4[M+H]+。
N2保护下,称量化合物13(252mg,0.4mmol)置于10mL反应瓶中,加入3mL 4M HCl/Dioxane,反应液室温搅拌2h。反应结束后,过滤,粗品用DMSO溶解,经制备液相纯化。制备液经冻干后,得80mg深黄色目标化合物14,收率37.1%。LC-MS(ESI+)540.4[M+H]+。
N2保护下,称量化合物14(80mg,0.15mmol)和11(130mg,0.3mmol),置于50mL单口瓶中,加入5mL DMSO。反应液室温搅拌反应过夜。反应结束后,反应液用5mL水稀释,经制备液相分离。制备液经冻干后,得19mg淡黄色目标化合物I-6’,收率13.3%。LC-MS(ESI+)955.0[M+H]+。
(7)I-7’的制备
N2保护下,称量化合物15(308.3mg,1mmol)和化合物16(270.2mg,1.2mmol),置于50mL单口瓶中,加入DIPEA(465mg,3.6mmol)和5mL DCM。反应液室温搅拌反应3h。反应结束后,反应液减压浓缩得粗品,粗品用15mL乙腈和1mL甲酸溶解,经制备液相纯化。制备液经冻干后,得63.8mg白色化合物17,收率26%。LC-MS(ESI+)346.5[M+H]+。
N2保护下,称量MTX(222.7mg,0.5mmol)和TSTU(150mg,0.5mmol),置于50mL单口瓶中,加入DIPEA(129.2mg,1mmol)和20mL DMSO,室温搅拌0.5h。随后,称量化合物17(63.8mg,0.26mmol),加入到反应体系中,继续室温搅拌6h。反应结束后,反应液用10mL乙腈稀释,经制备液相纯化。制备液经冻干后,得14.2mg深黄色目标化合物I-7’,收率7%。LC-MS(ESI+)782.9[M+H]+。
(8)I-8’的制备
N2保护下,称量化合物18(334.3mg,1mmol)和化合物16(225.2mg,1mmol),置于50mL单口瓶中,加入DIPEA(387mg,3mmol)和5mL DCM。反应液室温搅拌反应3h。反应结束后,反应液减压浓缩得粗品,粗品用15mL乙腈和1mL甲酸溶解,经制备液相纯化。制备液经冻干后,得93.1mg白色化合物19,收率25%。LC-MS(ESI+)372.5[M+H]+。
N2保护下,称量MTX(222.7mg,0.5mmol)和TSTU(150mg,0.5mmol),置于50mL单口瓶中,加入DIPEA(129.2mg,1mmol)和20mL DMSO,室温搅拌0.5h。随后,称量化合物19(93.1mg,0.25mmol),加入到反应体系中,继续室温搅拌6h。反应结束后,反应液用10mL乙腈稀释,经制备液相纯化。制备液经冻干后,得10.3mg深黄色目标化合物I-8’,收率5%。LC-MS(ESI+)808.9[M+H]+。
实施例2双特异性二聚体通用制备方法
将抗体(10mg/mL)、DTPA(10mM)以及2.5eq.的TCEP(5mM)加入PCR管中,室温搅拌反应2h。再于冰浴中加入25%的DMSO以及5eq.的甲氨蝶呤-L1(5mM)(如I-1’、I-2’、I-3’、I-4’、I-5’、I-6’、I-7’、I-8’等化合物),室温搅拌反应10h。反应结束后,用PBS缓冲液离心超滤3次,纯化去除残余未反应药物以及DMSO等游离小分子。
我们采用了上述方法制备了如下双特异性二聚体:
实施例3双特异性二聚体-药物偶联物通用制备方法
将抗体(10mg/mL)、DTPA(10mM)以及2.5eq.的TCEP(5mM)加入PCR管中,室温搅拌反应2h。再于冰浴中加入25%的DMSO以及5eq.的相应的连接子-毒素,反应2h。再加入2.5eq.的TCEP(5mM),室温搅拌反应2h后,加入linker-MTX(5mM),室温搅拌反应10h。反应结束后,用PBS缓冲液离心超滤3次,纯化去除残余未反应药物以及DMSO等游离小分子。
我们采用了上述方法制备了如下双特异性二聚体-药物偶联物:
实施例4双特异性偶联物的细胞结合活性检测
采用ELISA法检测双特异性偶联物与乳腺癌细胞MDA-MB-231、乳腺癌细胞SK-BR-3的结合活性。
其通用试验过程为:
(1)包被:用多聚左旋赖氨酸0.01%(W/V)包被96孔细胞培养板,100μl/孔,4℃过夜;
(2)洗板:96孔细胞板用灭菌超纯水洗涤2遍,200μl/孔,37℃、5%CO2培养箱烘干1.5-2.5小时;
(3)铺板:将细胞用2mlPBS洗涤2遍,用2ml胰酶消化,2ml培养基终止消化后,104个/孔铺于干燥的96孔细胞培养板中,放置30分钟,弃上清,37℃、5%CO2培养箱烘干1小时;
(4)固定:干燥的96孔细胞培养板中加入4%多聚甲醛,150μl/孔,4℃固定15分钟;
(5)洗板:温PBS洗涤2遍,200μl/孔,吸干孔内溶液;
(6)封闭:用3%BSA封闭液200μl/孔封闭,4℃过夜;
(7)洗板:PBST洗涤2遍,300μl/孔,吸干孔内溶液;
(8)加样:用稀释液(1%BSA-PBST溶液)将样品稀释,将稀释好的样品以100μl/孔加入封闭后的细胞培养板中,设置三个复孔,样品稀释液作为空白对照,37℃、5%CO2培养箱孵育2小时(稀释方案可根据实际情况进行改变);洗板PBST洗涤2遍,200μl/孔,吸干孔内溶液;加酶联抗体用1%BSA-PBST稀释液以1:5000稀释Goat anti-Human IgG-HRPconjugate,100μl/孔,37℃、5%CO2培养箱孵育1小时;洗板PBST洗涤4遍,200μl/孔,吸干孔内溶液;
(9)显色:加入TMB substrate(100μl/孔),显色2分钟;
(10)终止:加入2M H2SO4(50μl/孔),终止反应;
(11)读数:酶标仪测定450/655nm处光密度吸收值,实验结果用prism分析软件进行分析,以浓度为横坐标,光密度吸收值为纵坐标,由软件自动计算EC50等值。
表1双特异性偶联物的细胞结合活性检测结果
表1第一组A、B、C及图1A、图1B、图1C给出了Anti-PD-L1、Anti-PD-L1-Mc-Ser-MTX、Anti-PD-L1-NHS-PEG8-MTX、Anti-PD-L1-Mc-EDA-MTX、Anti-PD-L1-Mc-DSDEA-MTX、Anti-PD-L1-Mcc-DSDEA-MTX、Anti-PD-L1-NHS-MTX对乳腺癌细胞MDA-MB-231结合活性的ELISA结果。结果显示,和Anti-PD-L1相比,Anti-PD-L1-Mc-Ser-MTX、Anti-PD-L1-NHS-PEG8-MTX、Anti-PD-L1-Mc-EDA-MTX、Anti-PD-L1-Mc-DSDEA-MTX、Anti-PD-L1-Mcc-DSDEA-MTX、Anti-PD-L1-NHS-MTX均表现出良好的结合活性。
表1第二组及图2给出了Anti-EGFR、Anti-EGFR-Mc-Ser-MTX对乳腺癌细胞SK-BR-3结合活性的ELISA结果。结果显示,和Anti-EGFR相比,Anti-EGFR-Mc-Ser-MTX表现出良好的结合活性。
实施例4双特异性偶联物的细胞活性检测
采用CCK8法检测双特异性偶联物对结肠癌HT29的增殖抑制作用。
其通用试验过程为:
(1)铺板,将人肿瘤细胞株SK-BR-3细胞悬液以100μL/孔,5000个/孔的密度加到96孔板中,置于水饱和的37℃CO2培养箱培养过夜。
(2)加药,将ADC浓度进行梯度稀释并加到含有细胞的96孔板中,100μL/孔。初始的偶联药浓度为50000ng/ml,浓度稀释到0.59ng/ml。继续置于37℃培养箱中培养72小时。
(3)检测,细胞活性由细胞活性试剂盒(Cell Counting Kit-8)(DOJINDO,CK04)检测,利用酶标仪检测吸光值。IC50值为某种药物在一定浓度诱导肿瘤细胞凋亡50%,该浓度称为50%抑制浓度,即凋亡细胞与全部细胞数之比等于50%时所对应的浓度,该值由曲线拟合软件Graphpad计算得到。
表2双特异性偶联物的细胞活性检测结果
表2、图3的结果显示,对于anti-c-Met ADC和anti-EGFR ADC,在偶联MTX后几乎不会影响原ADC的细胞活性,这表明偶联MTX可以在不改变原ADC抑制细胞增殖能力的同时,提高ADC与肿瘤细胞的结合能力。因此,偶联MTX使抗体或者ADC变为双特异性的方法是极具优势的。
本发明已通过各个具体实施例作了举例说明。但是,本领域普通技术人员能够理解,本发明并不限于各个具体实施方式,普通技术人员在本发明的范文内可以作出各种改动或变型,并且在本说明书中各处提及的各个技术特征可以相互组合,而仍不背离本发明的精神和范围。这样的改动和变型均在本发明的范围之内。
Claims (4)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010660313.5A CN111643676B (zh) | 2020-07-10 | 2020-07-10 | 一种双特异性二聚体、双特异性二聚体-药物偶联物及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010660313.5A CN111643676B (zh) | 2020-07-10 | 2020-07-10 | 一种双特异性二聚体、双特异性二聚体-药物偶联物及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111643676A CN111643676A (zh) | 2020-09-11 |
CN111643676B true CN111643676B (zh) | 2023-06-30 |
Family
ID=72343265
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010660313.5A Active CN111643676B (zh) | 2020-07-10 | 2020-07-10 | 一种双特异性二聚体、双特异性二聚体-药物偶联物及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111643676B (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4175672A1 (en) * | 2020-07-06 | 2023-05-10 | Byondis B.V. | Antifolate linker-drugs and antibody-drug conjugates |
WO2023126297A1 (en) * | 2021-12-30 | 2023-07-06 | Byondis B.V. | Antifolate linker-drugs and antibody-drug conjugates |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4699784A (en) * | 1986-02-25 | 1987-10-13 | Center For Molecular Medicine & Immunology | Tumoricidal methotrexate-antibody conjugate |
US5547668A (en) * | 1995-05-05 | 1996-08-20 | The Board Of Trustees Of The University Of Illinois | Conjugates of folate anti-effector cell antibodies |
CN102827272A (zh) * | 2010-11-08 | 2012-12-19 | 武汉华耀生物医药有限公司 | 一种叶酸偶联抗体药物及其制备方法与应用 |
CN108853514A (zh) * | 2017-08-18 | 2018-11-23 | 四川百利药业有限责任公司 | 具有两种不同药物的抗体药物偶联物 |
-
2020
- 2020-07-10 CN CN202010660313.5A patent/CN111643676B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4699784A (en) * | 1986-02-25 | 1987-10-13 | Center For Molecular Medicine & Immunology | Tumoricidal methotrexate-antibody conjugate |
US5547668A (en) * | 1995-05-05 | 1996-08-20 | The Board Of Trustees Of The University Of Illinois | Conjugates of folate anti-effector cell antibodies |
CN102827272A (zh) * | 2010-11-08 | 2012-12-19 | 武汉华耀生物医药有限公司 | 一种叶酸偶联抗体药物及其制备方法与应用 |
CN108853514A (zh) * | 2017-08-18 | 2018-11-23 | 四川百利药业有限责任公司 | 具有两种不同药物的抗体药物偶联物 |
Non-Patent Citations (2)
Title |
---|
Naoji UMEMOTOY et al..PREPARATION AND IN VITRO CYTOTOXICITY OF A METHOTREXATEANTI-MM46 MONOCLONAL ANTIBODY CONJUGATE VIA AN OLIGOPEPTIDE SPACER.《International Journal of Cancer》.1989,第43卷 * |
PREPARATION AND IN VITRO CYTOTOXICITY OF A METHOTREXATEANTI-MM46 MONOCLONAL ANTIBODY CONJUGATE VIA AN OLIGOPEPTIDE SPACER;Naoji UMEMOTOY et al.;《International Journal of Cancer》;19890415;第43卷;摘要,Scheme 1,第679页左栏第5-6段 * |
Also Published As
Publication number | Publication date |
---|---|
CN111643676A (zh) | 2020-09-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108853514B (zh) | 具有两种不同药物的抗体药物偶联物 | |
CN115925796B (zh) | 一种抗肿瘤化合物及其制备方法和应用 | |
TWI715611B (zh) | 抗體藥物偶聯物的共價鍵連接子及其製備方法與應用 | |
CN114456186B (zh) | 一种喜树碱类衍生物及其配体-药物偶联物 | |
CN111643676B (zh) | 一种双特异性二聚体、双特异性二聚体-药物偶联物及其应用 | |
CN106459055A (zh) | 双功能细胞毒类药剂 | |
JP2008508858A5 (zh) | ||
CN109200291A (zh) | 一种靶向于egfr的抗体偶联药物及其制备方法和其用途 | |
CN116920115A (zh) | 一种用于抗体药物偶联物的连接子及其应用 | |
CN109890843B (zh) | 一种abcg2单克隆抗体及其用途 | |
CN109678923B (zh) | 雷公藤红素(异)阿魏酸酯类衍生物及其制备方法与用途 | |
CN111574498A (zh) | 基于来那度胺靶向降解egfr蛋白小分子化合物及其制备与应用 | |
CN108976172A (zh) | 一类4-嘧啶二胺类小分子有机化合物及其衍生物及其应用 | |
CN115605510A (zh) | B7h3抗体-依喜替康类似物偶联物及其医药用途 | |
CN110194762B (zh) | 酞嗪酮类衍生物、其制备方法和用途 | |
CN106866822A (zh) | 半胱氨酸改造的抗体‑毒素偶联物 | |
CN109180681A (zh) | 一种dna毒性二聚体化合物 | |
CN114053426B (zh) | 一种双药链接组装单元及双药靶向接头-药物偶联物 | |
CN114569739A (zh) | 抗体药物偶联物 | |
CN112876463B (zh) | 一种制备pd-l1拮抗剂的中间体及其制备方法 | |
Li et al. | Discovery of novel antibody-drug conjugates bearing tissue protease specific linker with both anti-angiogenic and strong cytotoxic effects | |
CN108250187B (zh) | 吲哚-1-碳酸酯类化合物、其制备方法和应用 | |
CN114144418B (zh) | 喹唑啉类化合物的晶型、盐型及其制备方法 | |
CN116018343A (zh) | 一种吡啶并嘧啶化合物的晶型 | |
CN106518879B (zh) | 一类2,3-内酰胺环稠合喹唑啉-4(3h)-酮衍生物及其制备方法和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |