CN116640151A - 耐血清性双巯基特异性生物素化试剂 - Google Patents
耐血清性双巯基特异性生物素化试剂 Download PDFInfo
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- CN116640151A CN116640151A CN202310160899.2A CN202310160899A CN116640151A CN 116640151 A CN116640151 A CN 116640151A CN 202310160899 A CN202310160899 A CN 202310160899A CN 116640151 A CN116640151 A CN 116640151A
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- serum
- biotinylation
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Classifications
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
根据式(I)的生物素化试剂其中R1＝烷基、羟基烷基或羧基烷基残基，其包含1至50个碳原子；R2＝烷基、二醇、胺或肽残基，其具有1至50个碳原子；R3＝芳基、杂芳基，其具有3至50个碳原子；R4、R5＝芳基、杂芳基、烷基，其具有3至50个碳原子。该试剂在细胞分离过程中的用途。
Description
背景技术
抗体和抗体片段通常用于体外应用中的全血产品以及用于体内和临床应用，以靶向特定的细胞靶标。
这些生物分子的生物素化通常用于引入标签或柄(handles)，以使它们可以被第二识别系统识别，所述第二识别系统例如基于链霉亲和素、亲和素或抗生物素抗体(仅举几例)。
各种生物素化试剂是已知的，并例如公开在Liberatore等人,BioconjugateChem.1990,1,36-50,doi:10.1021/bc00001a005,EP3620464A1或D.M.Mock&A.Bogusiewicz；第209页；来自:Methods in molecular Biology,第418卷:Avidin-BiotinInteractions,Methods and Applications中。
从不同的技术领域来看，交联抗体的二硫桥是已知的。例如，在1979年的科技出版物Sumita Mitra等人,“Reagents for Cross-Linking of Proteins”,JACS 101211(1979)、Liberatore F等人,Bioconjugate Chem,1,(1990)和Rosario等人,BioconjugateChem,1990,1,51-59中首次描述了识别先前形成了二硫桥的两个相邻的还原硫醇的化学；其中交联剂被称为：平衡转移烷基化交联剂(ETAC)。描述了使完全抗体与交联剂反应，目的是为了用荧光染料标记该抗体。另一科技出版物描述了在Fab上的类似用途(Wilbur等人,Bioconjugate Chem 1994,5,220-235)。
US 2016/0000933 A1公开了在抗体的二硫桥处交联抗体以制备抗体缀合物。
涉及在抗体的二硫桥处交联抗体的其他出版物为例如WO 2016/168769 A1；HANIEH KHALILI等人:“Comparative Binding of DisuIfide-bridged PEG-Fabs”,BIOCONJUGATE CHEMISTRY,第23卷,第11期,2012年9月21日(2012-09-21),第2262-2277页；在“Bioconjugate Techniques”,2013年1月1日(2013-01-01),Academic Press,ISBN:978-0-12-382239-O,第867-920页中的Greg T.Hermanson:“Antibody Modification andConjugation”；WO 2016/154621 A1。
在生物素化抗体的血清相关应用(例如体内和全血体外应用)中，生物素标签需要耐受数小时。正常血清(人和小鼠)含有分解生物素酰胺的因子。此类因子的一个实例是天然存在的酶生物素酰胺酶。这种天然酶能够分解生物素酰胺，释放游离的生物素和胺。
因此，本发明的一个目的是提供化学成分明确的耐血清性衔接分子，其在位点特异性位置上具有每个Ab和/或Fab的单变量数量的标签/亲和单元(例如生物素)。
发明内容
在本文中我们描述了合成新的生物素化试剂，所述生物素化试剂耐受人血清和酶促降解((例如尤其是通过生物素酰胺酶的)。不受理论的束缚，这是由于在酰胺键的α中存在羧基基团，其特异性地防止降解。
本发明的试剂特异性地与两个巯基基团反应。这使其对靶向蛋白(例如抗体)中的还原二硫化物而不损失生物分子的结构或稳定性特别有吸引力，因为该试剂能够再桥接巯基基团。因此，此类生物素化抗体或抗体片段适合用于其中生物素缀合物暴露于血液、血液制品、新鲜血清或血浆样品的任何应用，所述应用未以降低或抑制分解生物素酰胺的因子活性的方式治疗，特别是还适合用于其中生物素用作标签的临床应用。
本发明基于使用靶向Ab和/或Fab上的二硫键的定点交联剂。益处依赖于：i)二硫桥的具体位置，已知其在Ab和Fab的序列和结构中的固定位置处；ii)交联剂共价连接到二硫桥的两个还原硫醇上，由此保留了Ab和Fab的天然3D结构和功能，不留下游离硫醇；iii)每个交联剂仅携带一个标签或亲和单元部分(例如生物素或硫胺素)，因此每个二硫桥将被携带一个生物素的连接体修饰，提供已知和可预测的化学计量(即Fab仅在C末端处含有一个二硫桥)；iv)交联反应是定量的，意味着超过95％的Ab和Fab被标记；v)标记程度可以是化学计量控制的(即对于Ab)；vi)交联缀合物对硫醇还原和还原环境不敏感。
因此，本发明的对象是根据式(I)的生物素化试剂
其中R1＝烷基、羟基烷基、羧基烷基残基，其包含1至50个碳原子，优选包含至少一个O或N原子，例如-OH、-CO2H、-CH2CH3、-CH(CH3)2、-OCH3、-CH2OH；
R2＝烷基、二醇、胺或肽残基，其具有1至50个碳原子，优选具有小于150kDa的分子量；
R3＝芳基、杂芳基，其具有3至50个碳原子；
R4、R5＝芳基、杂芳基、烷基，其具有3至50个碳原子。
附图说明
图1显示了设计用于评估所有连接体分子的血清稳定性的工作流程；
图2显示了通过细胞染色进行的原始和未修饰的连接体MS9分子在人血清和鼠血清中的血清稳定性；
图3显示了通过细胞染色进行的所有连接体分子的血清稳定性筛选和修饰的连接体(MS12、MS13、MS14和MS15)的阳性结果；
图4显示了在PBS中温育时对在血清中温育时，具有每种感兴趣的连接体的生物素化FAb产物的LCMS结果，以及取决于血清稳定性性质的靶标m/z信号的存在或丧失。
图5显示了生物素化蛋白产物与生物素酰胺酶(存在于哺乳动物血清中)的温育，证实其在用非耐血清性生物素连接体(例如MS9)标记的生物素化蛋白的降解中的作用。
具体实施方式
生物素化试剂的基团R1-R5可代表以下残基：
R1-CO2H、-CH2CO2H、-CH3、-C2H5、-C3H7、-CH-(CH3)2、-C4H9、-CH2OH、-C2H4OH、-C3H6OH，
R2任选被取代的烷基胺或二胺有机链，如NH-(CH2)4；NH-(CH2)5；NH-(CH2)6；-NH-(CH2)2-NH-CO；-NH-(CH2)3-NH-CO；-NH-(CH2)4-NH-CO；
R3芳族有机化合物和衍生物，优选在对位具有两个羰基残基并任选另外地被取代(优选没有取代)的5元或6元碳环，例如苯，
R4、R5芳族有机化合物和衍生物，通常为任选在邻位、间位和/或对位位置(优选在对位位置)被烷基碳链取代的5元或6元碳环，如苯、甲苯、乙苯、均三甲苯。
更优选地，根据本发明的生物素化试剂具有式(II)、(III)、(IV)或(V)的取代模式：
根据本发明的生物素化剂尤其可用于具有抗原结合结构域的抗原结合分子的生物素化，所述抗原结合结构域包含通过硫原子-连接体-硫原子桥连接的重链和轻链。
术语“抗原识别结构域”是指针对在感兴趣的生物试样上表达的靶标部分的任何类型的抗体、片段化抗体或片段化抗体衍生物。该术语涉及完全完整的抗体、片段化抗体或片段化抗体衍生物，例如Fab、Fab、F(ab)2、sdAb、scFv、二价scFv(di-scFv)、纳米抗体。此类片段化抗体衍生物可以通过重组程序合成，所述片段化抗体衍生物包括含有这些类型的分子的共价和非共价缀合物。抗原识别部分的其他实例是靶向TCR分子、细胞粘附受体分子、共刺激分子的受体、人工工程化结合分子的肽/MHC复合物，例如靶向例如细胞表面分子的肽或适体。
优选地，术语“抗原识别结构域”是指针对由生物试样(靶细胞)表达的细胞外抗原的部分，所述抗原如IL2、FoxP3、CD154、CD33(Siglec-3)、CD123(IL3RA)、CD135(FLT-3)、CD44(HCAM)、CD44V6、CD47、CD184(CXCR4)、CLEC12A(CLL1)、LeY、FRβ、MICA/B、CD305(LAIR-1)、CD366(TIM-3)、CD96(TACTILE)、CD133、CD56、CD29(ITGB1)、CD44(HCAM)、CD47(IAP)、CD66(CEA)、CD112(结合素2)、CD117(c-Kit)、CD133、CD146(MCAM)、CD155(PVR)、CD171(L1CAM)、CD221(IGF1)、CD227(MUC1)、CD243(MRD1)、CD246(ALK)、CD271(LNGFR)、CD19、CD20、GD2、EGFR和CD33。这些抗原也可以是与TME特异性结合的可溶抗原，例如精氨酸酶、癌胚抗原、CCL11、CCL18、CCL2、CCL5、CD282、循环肿瘤核酸、CXCL10、FAP、GM-CSF、IFN-γ、IL-4、IL-6、IL-7IL-8、IL-10、IL-11、IL-12、Il-13、IL-14、IL-15、IL-17、IL-23、IL-33、IL-1β、IL-1Ra、INF、LAP、M-CSF、MMP12、MMP13、MMP7、NY-ESO-1抗体、前列腺特异性抗原、sCD106、sCD137、sCD152、sCD223、sCD25、sCD27、sCD253、sCD270、sCD273、sCD274、sCD279、sCD28、sCD30、sCD366、sCD40、sCD54、sCD80、sCD86、sGITR、TGFβ-1、TGFβ-2、TGFβ-3、TIMP1或TNF-α、VEGF。sCDx中的“s”通常代表各个CDx分子的可溶形式。
本发明的用途
本发明的其他对象是如上定义的生物素化试剂的用途或使用如上定义的生物素化试剂用于含有至少一个二硫键的蛋白或肽的生物素化的方法。
该蛋白可含有抗原结合部分，产生其中二硫键被源自生物素化剂的3-碳连接桥(3-carbon linker bridge)置换和重构的蛋白或肽。
在另一个变体中，蛋白包含抗体片段(FAb)单元，并且被生物素化剂修饰的二硫键是链间二硫键。优选地，蛋白含有抗原结合部分，产生其中二硫键被源自生物素化剂的3-碳连接桥置换和重构的蛋白或肽。
在另一个变体中，蛋白包含至少两个具有硫原子的氨基酸，并且其中该至少两个氨基酸通过该生物素化剂连接。
生物素化蛋白可以在通过免疫磁性分离的细胞分离工作流程中用作抗原结合分子，或在通过流式分选分离的细胞分离工作流程中用作抗原结合分子。在这种细胞分离工作流程中，可以从全血或全血制品中或者从含有未热灭活血清或血浆或活性生物素酰胺酶的培养基中将靶细胞与非靶细胞分离。
实施例
在实验的同一天抽取人和鼠的血液，并收集在血清促凝剂管(serum clothactivator tube)中。在RT(室温)下温育30分钟后，将管在2200×g下离心15分钟。小心地取出含有人血清的顶层，并转移到新鲜的管中。为了从血清中除去将干扰随后的LC-MS分析的抗体，用PBS以1:1稀释血清，并装载到用PBS平衡的蛋白A柱(Thermo Fisher Scientific,Order-Nr:20356)上。将流穿物收集在新鲜的管中。对于H.I.(热灭活)血清对照，将来自不含IgG的血清的一部分转移到Eppendorf管中，并在56℃下温育30分钟。在预温育后，将样品分份，一部分用于染色并经由MACSQuant进行分析，将剩余的样品纯化并经由LC-MS进行分析。
生物素化抗CD33 Fab在PBS中，或在新鲜制备的血清中以0,1μg/μl的浓度在37℃下温育1小时。
为了染色，如上所述，将每孔1×104个表达CD33的OCI-AML-2细胞与10μl预温育的Fab一起在4℃下温育10分钟。进行用PEB缓冲液(1×PBS/5mM EDTA/0,5％血清)的洗涤步骤和后续的第二染色，其中用抗生物素(Miltenyi Biotec:120-000-898)或抗His抗体(Miltenyi Biotec:120-047-112)和7-AAD(Miltenyi Biotec:订单号:130-111-569)对OCI-AML-2细胞进行染色。最后，用PEB缓冲液洗涤细胞，并使用MACSQuant分析仪10进行分析。
图2显示了比较人血清和鼠血清的血清稳定性测试的结果。生物素化的抗CD33FAb在新鲜制备的人血清、鼠血清、热灭活人血清或PBS中温育1小时。通过多色流式细胞仪确定结合抗CD33 Fab的细胞(OCI-AML2)上FAb部分(抗His)和生物素部分(抗生物素-PE,(中图))的存在。显示n＝3个技术重复的MFI(平均荧光强度)。
此外，测试了不同的连接体部分(MS12、MS13、MS14或MS15)，并将预温育期延长至4小时。
因此，将具有不同连接体部分(MS9、MS12、MS13、MS14或MS15)的抗CD33 Fab在PBS中，或在新鲜制备的血清中以0,1μg/μl的浓度在37℃下温育4小时。
为了染色，如上所述，将每孔1×104个表达CD33的OCI-AML-2细胞与10μl预温育的Fab一起在4℃下温育10分钟。进行用PEB缓冲液(1×PBS/5mM EDTA/0,5％血清)的洗涤步骤和后续的第二染色，其中用抗生物素(Miltenyi Biotec:120-000-898)/链霉亲和素-PE(Miltenyi Biotec:120-045-655)或抗His抗体(Miltenyi Biotec:120-047-112)和7-AAD(Miltenyi Biotec:订单号:130-111-569)对OCI-AML-2细胞进行染色。
最后，用PEB缓冲液洗涤细胞，并使用MACSQuant分析仪10进行分析。
为了制备用于经由LC-MS分析的样品，经由IgG-CH1亲和基质(Thermo Fisher:194320005)纯化预温育的衔接子，因此将25μL的CH1树脂转移到1.5mL微量离心管中用于各个样品，所有进一步的步骤根据制造商的方案进行。
图3显示了血清稳定性测试的结果。通过多色流式细胞仪确定结合抗CD33 FAb的细胞(OCI-AML2)上生物素部分(链霉亲和素-PE(图A)和抗生物素-PE(图B))和FAb部分(抗His APC，图C)的存在。显示n＝2个技术重复的MFI(平均荧光强度)。对于未热灭活血清(血清)中温育的MS9修饰的Fab，观察到抗生物素染色强度的显著降低，而对于MS12、MS13、MS14或MS15修饰的FAb则没有。
图4显示了缀合连接体的Fab分子的LC/MS，所述Fab分子在37℃下暴露于PBS(上图)或未热灭活人血清(下图)4小时，其中A)MS9，B)MS12，C)MS13，D)MS14和E)MS15。A)MS9:49924,68(PBS)；49697,89(血清)，B)MS12:49967,33(PBS)；49967,25(血清)，C)……。
为了验证生物素酰胺酶(一种在人体中，尤其是在人血清中发现的酶)引起生物素酰胺键的断裂，用生物素酰胺酶抑制剂((+)生物素4酰胺基苯甲酸((+)Biotin 4Amidobenzoic Acid),Cayman,CAS.102418-74-6)进行实验。因此将(+)生物素4酰胺基苯甲酸以1mM的浓度加入到含有人血清的预温育混合物中。不加入抑制剂作为对照。将具有MS9的抗CD33 Fab在37℃下预温育4小时。随后，在温育后，如前所述经由IgG-CHI亲和基质纯化样品。
图5显示了作为未热灭活血清中连接体不稳定的成因剂的生物素酰胺酶。在存在(左图)或不存在(右图)1mM生物素4-酰胺基苯甲酸的情况下，将生物素化的(MS9)抗CD33FAb在新鲜制备的人血清中在37℃下温育4小时。生物素酰胺酶抑制剂保护生物素酰胺键不被降解(含有抑制剂的分子量：49922.91Da，不含有抑制剂的分子量：49696.58Da)。
合成
合成双砜NHS-酯，2
Liberatore等人报道了(4-[2,2-双([对甲苯磺酰基)甲基]乙酰基]苯甲酸)1的合成(Bioconjugate Chem.1990,1,36-50,doi:10.1021/bc00001a005)。在氮气覆盖下将双砜1(1当量)溶解在干燥的四氢呋喃(THF，100mg/mL)中。加入N-羟基琥珀酰亚胺(NHS，1.5当量)，并且将混合物在冰浴中冷却。在0℃下逐滴加入二异丙基碳二亚胺(DIC，1.5当量)。大约10分钟后，让混合物达到室温。产物沉淀为白色固体，形成悬浮液。大约30分钟后反应完成，随后进行薄层色谱。
将产物溶解在二氯甲烷中并用水萃取。有机相在无水硫酸钠上干燥，随后过滤并真空浓缩，得到作为白色粉末的2。
合成Nε-(生物素酰胺基)-L-赖氨酸，4
在氮气气氛中将Nε-叔丁氧羰基-L-赖氨酸(1.0当量)溶解在干燥的二甲基甲酰胺(DMF，20mg/mL)中。加入1.5当量的三乙胺(TEA)。将生物素-NHS(1.5当量)溶解在干燥的DMF(20mg/mL)中，并在氮气覆盖下在搅拌下逐滴加入赖氨酸溶液中。将混合物在室温下搅拌16小时，得到化合物3。
3通过添加三氟乙酸(TFA，60当量)脱保护，并在室温下搅拌3小时，随后真空浓缩。生物素化的赖氨酸4通过沉淀其铵盐来分离。因此，在氢氧化铵存在的情况下用乙醇稀释浓缩的粗反应混合物。通过过滤收集白色沉淀。
合成Nε-(生物素酰胺基)-Nα-(4-[2,2-双[(对甲苯磺酰基)甲基]乙酰基]苯甲酰胺
基)-L-赖氨酸，5(MS 12)
在氮气气氛中将生物素化的赖氨酸4(1.0当量)溶解在干燥的DMF(20mg/mL)中。将双砜NHS酯2(1.10当量)溶解在干燥的DMF(50mg/mL)中，并在氮气覆盖下在搅拌下逐滴加入生物素溶液中。将混合物在室温下搅拌16小时，随后真空浓缩并通过制备型反相(C18)液相色谱，使用100％水+0.1％TFA→100％乙腈+0.1％TFA的梯度纯化。将目标级分冷冻干燥以获得作为白色粉末的5。
Claims (6)
1.根据式(I)的生物素化试剂
其中
R1＝烷基、羟基烷基或羧基烷基残基，其包含1至50个碳原子；
R2＝烷基、二醇、胺或肽残基，其具有1至50个碳原子；
R3＝芳基、杂芳基，其具有3至50个碳原子；
R4、R5＝芳基、杂芳基、烷基，其具有3至50个碳原子。
2.根据权利要求1所述的生物素化试剂，其特征在于所述生物素化试剂具有式(II)、(III)、(IV)或(V)的取代模式：
3.根据权利要求1和2所述的生物素化剂用于含有至少一个二硫键的蛋白或肽的生物素化的用途。
4.根据权利要求3所述的生物素化剂的用途，其中所述蛋白含有抗原结合部分，产生其中二硫键被源自所述生物素化剂的3-碳连接桥(3-carbon linker bridge)置换和重构的蛋白或肽。
5.根据权利要求3所述的生物素化剂的用途，其中所述蛋白包含抗体片段(Fab)单元，并且被所述生物素化剂修饰的所述二硫键是链间二硫键。
6.根据权利要求3所述的生物素化剂的用途，其中所述蛋白包含至少两个具有硫原子的氨基酸，并且其中所述至少两个氨基酸通过所述生物素化剂连接。
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