CN110536696B - 基于新的肽的pcsk9疫苗 - Google Patents
基于新的肽的pcsk9疫苗 Download PDFInfo
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- CN110536696B CN110536696B CN201880023715.5A CN201880023715A CN110536696B CN 110536696 B CN110536696 B CN 110536696B CN 201880023715 A CN201880023715 A CN 201880023715A CN 110536696 B CN110536696 B CN 110536696B
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- pcsk9
- peptide
- amino acid
- methyl
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Abstract
本发明涉及新的式(I)的短链肽,当其与合适的免疫原性载体和合适的佐剂缀合时可用作疫苗。这些可用于治疗PCSK9介导的疾病。A‑Z1‑Z2‑Z3‑Z4‑Z5‑Z6‑Z7‑Z8‑Z9‑Z10‑Z11‑Z12‑B式(I)。
Description
技术领域
本发明涉及新的式(I)的短链肽、其对映异构体、其非对映异构体、其立体异构体、其可药用盐或前药。在一个实施方案中,式(I)的肽与合适的免疫原性载体缀合,并且可用于治疗或预防通过PCSK9介导的疾病,并且能够在体内诱导针对PCSK9的抗体的形成。
A-Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10-Z11-Z12-B
式(I)
本发明还涉及这些药物、免疫原性组合物及其药物组合物的生产方法及其在医学中的用途。
背景技术
本发明涉及新的短链肽。此外,这些肽与合适的免疫原性载体缀合,并且能够抑制前蛋白转化酶枯草杆菌蛋白酶/Kexin 9型(Proprotein Convertase Subtilisin/KexinType 9,PCSK9)介导的低密度脂蛋白(Low Density Lipoprotein,LDL)受体(Low DensityLipoprotein receptor,LDLR)的降解。将与免疫原性载体偶联的这些新肽配制成疫苗,用于预防和/或治疗PCSK9相关的健康病症,例如高脂血症、高胆固醇血症或动脉粥样硬化。这些并发症导致心血管疾病(cardiovascular disease,CVD),从而引起发病率和死亡率。
早前,诸如他汀类的药剂用作用于降低血浆低密度脂蛋白胆固醇(Low DensityLipoprotein cholesterol,LDLc)的治疗剂。PCSK9及其在调节血浆LDLc(通过增强LDLR的降解)中的作用的发现导致认识到:PCSK9可能是控制健康病症(例如高脂血症、高胆固醇血症或动脉粥样硬化)的可行靶标。
在2003年,发现PCSK9为与常染色体显性高胆固醇血症(Autosomal DominantHypercholesterolemia,ADH)相关的第三个基因座位。PCSK9也称为神经细胞凋亡调节转化酶1(neural apoptosis-regulated convertase 1,NARC-1),是被确定为哺乳动物PCSK家族的第9个成员的蛋白酶k样枯草杆菌蛋白酶。其主要在肝、肠和肾中表达。其被合成为约72kDa的蛋白质,经历自催化切割之后作为约65kDa的成熟蛋白质分泌。
循环的PCSK9与LDLR的EGF-A结构域结合并促进LDLR的降解。肝LDLR是从血浆中消除LDLc的主要途径。循环的LDLc与LDLR结合并形成复合物,其通过受体介导的内吞作用被内化。随后,LDLc先降解,然后LDLR循环回到细胞表面。PCSK9与LDLR相互作用,并且该PCSK9-LDLR相互作用导致LDLR的降解,降低了LDLR用于从血浆清除LDLc的可用性。这清楚地说明了PCSK9作为LDLR的调节剂和在LDLc代谢中的重要性。
尝试了数种方法(例如,单克隆抗体(mAb)、使用小干扰RNA(siRNA)的PCSK9抑制、反义寡核苷酸、PCSK9结合的adnectin、小分子抑制剂和自催化抑制剂)以降低循环PCSK9的量或抑制其与LDLR的相互作用。但是,PCSK9细胞内和细胞外地作用于LDLR,靶向循环PCSK9是降低LDLc水平的有价值的方法。来自关于PCSK9特异性mAb的人临床试验的令人鼓舞的结果表明,靶向PCSK9允许有效且安全地降低LDLc。此外,最近成功完成了评估靶向PCSK9的安全性和效力的数个临床试验。总之,除了他汀类之外,鉴定作为控制LDLc水平的替代靶标的PCSK9也具有重要意义。
除了通过多种组织以抑制通过循环PCSK9介导的LDLR降解的数种方法之外,最近,Pfizer(WO2011027257A2)和Affiris(WO2014033158A2)通过其多项专利公开了抗原肽,其能够诱导与人PCSK9特异性结合并且抑制PCSK9介导的LDLR降解的抗体。
由于发现了PCSK9基因及其在降低胆固醇中的作用,测试了数种方法以抑制PCSK9介导的LDLR降解。目前,评估了靶向PCSK9的单克隆抗体(mAb)用于人的用途。最近,Regeneron/Sanofi已开发了Alirocumab(mAb)并公布了多项专利申请,例如WO/2015/140079、(EP2015/055369)、WO/2015/142668(US2015/020564)&WO/2015/123423(US2015/015633)WO/2014/194111、(US2014/040050)、WO/2014/194168(US2014/040163)、WO/2013/039969(US2012/054756),其包含批准用于人用途的抗PCSK9抗体(例如mAb316P)。除此之外,Sanofi WO/2015/073494(US2014/065149)、WO/2015/054619、(US2014/060109)、&Regeneron Pharmaceuticals WO/2014/197752(US2014/041204)也公开了PCSK9抑制剂,其是抗PCSK9抗体,或抗原结合蛋白。另外的Sanofi WO/2012/101251(EP2012/051318)、WO/2012/101253(EP2012/051321)、WO/2012/101252(EP2012/051320)已开发了PCSK9特异性抗体或抗原结合片段,并优选通过另外施用HMG-CoA还原酶抑制剂。
由Amgen开发的依洛尤单抗(Evolocumab)(AMG-145)是批准用于人用途的能够抑制PCSK9与LDLR结合的完全人mAb或抗原结合蛋白[WO/2014/209384(US2013/048714)、WO/2014/150983(US2014/024702)、WO/2014/144080(US2014/028339)、WO/2013/166448(US2013/039561)、WO/2012/154999(US2012/037394)]。而由Pfizer,[WO/2013/008185(IB2012/053534)]开发的人源化mAb Bococizumab正在研究中。Eli Lilly[LY3015014]正在开发另一PCSK9 mAb,其目前正在进行II期试验[WO/2013/039958(US2012/054737)]。
Eleven Biotherapeutics WO/2015/200438(US2015/037345)、WO/2014/107739(US2014/010536)、Genentech、WO/2013/188855(US2013/046032)、WO/2012/088313(US2011/066593);Alderbio Holdings WO/2013/169886(US2013/040112);Adaerata WO/2013/091103(CA2012/050923);Novartis WO/2012/168491(EP2012/061045)、WO/2012/170607(US2012/041214);IRM LLC WO/2012/109530(US2012/024633)、WO/2011/072263(US2010/059959);和Merck Sharp& Dohme Corp WO/2012/054438(US2011/056649)、WO/2011/053759(US2010/054640)、WO/2011/053783、(US2010/054714)、WO/2011/037791(US2010/048849)已经开发了数种其他的PCSK9拮抗剂抗体,其许多正在研究中。
在另一方法中,使用了使用RNA干扰(RNAi)的PCSK9表达的抑制。数家公司,包括Roche WO/2014/207232(EP2014/063757);Pfizer WO/2014/170786(IB2014/060407);Alnylam Pharmaceuticals WO/2014/089313(US2013/073349)、WO/2012/058693(US2011/058682)WO/2011/038031(US2010/049868)、WO/2011/028938(US2010/047726);KowaCompany LTD WO/2014/017569(JP2013/070128);Osaka University WO/2014/007305(JP2013/068299)、WO/2012/029870(JP2011/069818);New York University WO/2013/154766(US2013/032271)和Santaris Pharma WO/2011/009697(EP2010/059257)正在开发使用RNAi的PCSK9表达抑制剂,这些中的许多处于临床开发的不同阶段。
抗PCSK9主动疫苗接种策略是抑制PCSK9的替代策略。Pfizer的专利申请WO/2012/131504(IB2012/050924),The Secretary,Dept.of Health and Human services USA,WO/2015/123291(US2015/015408),和Istitutodi Ricerche di Biologia Molecolare WO/2011/117401(EP2011/054646)公开了多种疫苗接种策略以降低LDLc水平。
另一有前景的方法是基于肽的PCSK9疫苗用于PCSK9抑制。将源自PCSK9基因的短链肽与能够产生针对PCSK9的抗体的合适的免疫原性载体缀合。Pfizer的专利申请(WO/2011/027257(IB2010/053784)、US2011/0052621;Affiris US2016/0106822、WO/2015/128287(EP2015/053725)、EP2703483、WO/2014/033158(EP2013/067797)、WO/2013/037889(EP2012/067950)公开了控制LDLc水平中的此类免疫原性肽。
然而,到目前为止,尚未报道包含非天然/经修饰的氨基酸的PCSK9肽疫苗,并且未进行如下的重大尝试来改善这些PCSK9肽的代谢稳定性:使用合适的化学修饰以改善这些PCSK9肽的功效和/或效力以及作用时间。本发明是首次尝试之一,以研究并入来自PCSK9基因然后与合适的免疫原性载体缀合的PCSK9肽中的经修饰的氨基酸的作用。令人惊讶地,已经发现许多这些经修饰的肽能够诱导针对PCSK9的抗体的形成。因此,这些疫苗可用于治疗或预防通过PCSK9介导的疾病。
本发明的目的是提供某些新的短链肽式(I),其可与合适的免疫原性载体缀合并且能够产生特异性结合人PCSK9并抑制PCSK9介导的LDLR降解的抗体。该目的是通过以下来实现的:开发某些通过将天然氨基酸替换为非天然氨基酸而经修饰的肽,所述经修饰的肽任选地与合适的免疫原性载体缀合,然后评估特异性针对PCSK9基因从而降低LDLc水平的抗体的产生,其可用于治疗心血管并发症(例如高脂血症、高胆固醇血症或动脉粥样硬化)。
因此,本发明涉及新的式(I)的短链肽,其任选地与合适的免疫原性载体缀合。这样的组合物可用作疫苗或疫苗组合物。在一个优选的实施方案中,所述肽由式(I)的12个氨基酸残基构成,
A-Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10-Z11-Z12-B
式(I)
其中,
‘A’表示基团-NH-R1、R2-CO-NH-,其中‘R1’表示氢或任选地经取代的直链或支链(C1-18)烷基链;‘R2’选自任选地经取代的直链或支链(C1-18)烷基链、(C1-6)烷氧基、(C3-C6)环烷基、芳基、杂芳基或芳烷基基团;在一个优选的实施方案中,芳基选自苯基、萘基、茚满基、芴基或联苯基基团;杂芳基选自吡啶基、噻吩基、呋喃基、咪唑基、苯并呋喃基基团;‘B’表示R3、-COOR3、-CONHR3或CH2OR3,其中R3表示H或合适的氨基酸,例如丝氨酸、半胱氨酸、缬氨酸、α-甲基-缬氨酸、Lys(生物素)、Lys(烷基)、Lys(乙酰基)等;
Z1是选自不带电荷的氨基酸残基的氨基酸残基,优选选自丝氨酸、苏氨酸、缬氨酸和丙氨酸及其衍生物,例如高丝氨酸、O-甲基-苏氨酸、O-甲基-丝氨酸、O-甲基-高丝氨酸等
Z2是选自不带电荷的氨基酸残基的氨基酸残基,优选选自异亮氨酸、亮氨酸、正亮氨酸、甘氨酸、丙氨酸、Aib及其衍生物,例如N-甲基-异亮氨酸、N-甲基-亮氨酸等
Z3是选自以下的氨基酸残基:脯氨酸、1-氨基碳环酸(1-amino carbocyclicacid)及其衍生物,例如羟脯氨酸、硫代脯氨酸、氨基脯氨酸、α-甲基-脯氨酸、3-氟脯氨酸、4-氟脯氨酸、1-氨基-环丙烷羧酸、1-氨基-环戊烷羧酸、1-氨基-环己烷羧酸等
Z4是选自色氨酸、苯丙氨酸、酪氨酸及其衍生物的氨基酸残基;
Z5是选自谷氨酰胺、组氨酸、优选天冬酰胺及其衍生物的氨基酸残基;
Z6是选自不带电荷的氨基酸残基的氨基酸残基,优选选自异亮氨酸、亮氨酸、丙氨酸、苏氨酸、天冬氨酸、Aib及其衍生物,例如正异亮氨酸、N-甲基-亮氨酸、高亮氨酸、α-甲基-天冬氨酸等
Z7是选自亲水性的、带负电荷的氨基酸残基的氨基酸残基,优选选自以下的氨基酸残基:谷氨酸、天冬氨酸及其衍生物,例如α-甲基-天冬氨酸、α-甲基-谷氨酸、高谷氨酸等
Z8是选自以下的氨基酸残基的氨基酸残基:优选选自精氨酸、高精氨酸、瓜氨酸、谷氨酰胺、天冬酰胺、赖氨酸及其衍生物;
Z9是选自不带电荷的氨基酸残基的氨基酸残基,优选选自异亮氨酸、亮氨酸、正亮氨酸、甘氨酸、丙氨酸或Aib及其衍生物,例如N-甲基异亮氨酸等
Z10是选自极性且不带电荷的氨基酸残基的氨基酸残基,优选选自丝氨酸、苏氨酸、缬氨酸、丙氨酸及其衍生物;
Z11是任何氨基酸残基,优选选自以下的氨基酸残基的氨基酸残基:优选选自脯氨酸及其衍生物,例如羟脯氨酸、硫代脯氨酸、α-甲基-脯氨酸、3-氟脯氨酸、4-氟脯氨酸、1-氨基-环丙烷羧酸、1-氨基-环戊烷羧酸、1-氨基-环己烷羧酸等
Z12是任何氨基酸残基,优选极性和不带电荷的氨基酸残基,所述氨基酸残基选自半胱氨酸、高半胱氨酸及其衍生物;前提是Z1至Z12的至少一个总是表示非天然氨基酸。
用于本发明的氨基酸的单字母缩写可在Zubay,G.,Biochemistry 2nd ed.,1988,MacMillan Publishing,New York,p.33中找到。
本文中报道了通式为A-Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10-Z11-Z12-B(I)的PCSK9肽的系列,其中Z1至Z12的每一个(当存在时)表示天然存在的氨基酸或非天然/经修饰的氨基酸序列,前提是Z1至Z12的至少一个将总是表示非天然/经修饰的氨基酸。
发明概述
本发明提供了新的短链PCSK9肽序列,其任选地与合适的免疫原性载体缀合并且能够抑制PCSK9介导的LDLR降解。这些肽用非天然氨基酸适当地修饰,并与合适的免疫原性载体缀合,以改善其免疫原性并由此提高抗体的产生。在此及之前被称为PCSK9肽衍生物/类似物的这些短链肽主要来源于人PCSK9基因的有活性的肽序列。基于这些新的免疫原性短链PCSK9肽类似物的疫苗可用于治疗或预防与血浆LDL-c水平调节相关的病理状况。这些疫苗产生的抗体可防止PCSK9介导的LDLR降解,从而控制健康病症,例如高脂血症、高胆固醇血症或动脉粥样硬化。
优选的实施方案
本发明的一个实施方案是提供新的通式(I)的短链肽、其合成中涉及的新的中间体、其可药用盐和适合于用作对抗PCSK9基因的疫苗的包含其或其混合物的药物组合物。
在另一个实施方案中,式(I)的短链肽与合适的免疫原性载体缀合以充当疫苗,其能够诱导在活体系统中特异性结合PCSK9的抗体的形成。抗体与PCSK9的相互作用抑制LDLR降解并导致血浆LDLc水平的降低。这些疫苗可适合于治疗健康病症,例如高脂血症、高胆固醇血症或动脉粥样硬化。
在另一个优选的实施方案中,提供了药物组合物,所述药物组合物包含与可药用佐剂、免疫原性载体、溶剂、稀释剂、赋形剂和通常在其制备中使用的其他介质组合的通式(I)的短链肽、其可药用盐、溶剂合物及其混合物。
在另一个优选的实施方案中,提供了药物组合物,所述药物组合物包含与合适的溶剂、稀释剂、通常在其制备中使用的其他赋形剂和其他介质组合的与合适的免疫原性载体缀合的通式(I)的短链肽。
在另一个优选的实施方案中,提供了新的式(I)的短链肽单独或当与合适的免疫原性载体缀合时用于治疗健康病症(例如高脂血症、高胆固醇血症或动脉粥样硬化和其他心血管疾病)的用途。
根据一个特别优选的实施方案,本发明的疫苗中的新的短链肽在其N和/或C端包含直接或通过间隔区序列结合的至少一个半胱氨酸残基。该半胱氨酸残基充当反应基团,以使肽与另一分子或载体蛋白结合。
在本发明的另一个优选实施方案中,免疫原性载体选自白喉毒素(diphtheriatoxin,DT)、匙孔血蓝蛋白(keyhole limpet haemocyanin,KLH)、CRM(优选CRM197)、破伤风类毒素(tetanus toxoid,TT)、蛋白D或包含辅助性T细胞表位的任何其他蛋白质或肽。
在另一个优选的实施方案中,佐剂选自明矾、与以下组合的明矾:MF-59、TLR3激动剂例如聚(I:C)、TLR 4激动剂例如单磷酰脂A或GLA等、TLR5激动剂例如鞭毛蛋白、TLR7激动剂例如嘎德莫特(Gardiquimod)和咪喹莫特(Imiquimod)、TLR7/8激动剂例如R848、NOD2激动剂例如N-乙醇酰-MDP、包含CpG的核酸(其中胞嘧啶是未甲基化的)、QS21(皂苷佐剂)、白介素、β-谷固醇等。
在另一个优选的实施方案中,佐剂选自明矾、与其他佐剂(例如MF-59、GLA、单磷酰脂A、包含CpG的核酸(其中胞嘧啶是未甲基化的)、QS21(皂苷佐剂)、白介素、β-谷固醇)组合的明矾。
使用的缩写
在实施例和本文中其他地方使用以下缩写:
Ac=乙酰基
Aib=α-氨基-异丁酸,
Abu(CN)=2-氨基-4-氰基丁酸,
A-Me-APPA=α-甲基-2-氨基苯基戊酸,
α-Me-Asp=α-甲基-天冬氨酸
α-Me-D=α-甲基-天冬氨酸,
α-Me-E或αMe-Glu=α-甲基-谷氨酸,
α-Me-L=α-甲基-亮氨酸,
α-Me-Phe=α-甲基-苯丙氨酸,
α-Me-2F-Phe=α-甲基-2-氟苯丙氨酸,
α-Me-2,6-diF-Phe=α-甲基-2,6-二氟苯丙氨酸,
α-Me-Pro=α-甲基-脯氨酸,
AC3C=1-氨基-环丙烷羧酸,
AC5C=1-氨基-环戊烷羧酸,
AC6C=1-氨基-环己烷羧酸,
ACN=乙腈,
APPA=2-氨基苯基戊酸,
Amp=4-氨基脯氨酸,
3-Amp=3-氨基脯氨酸,
B-Ala=β丙氨酸,
Boc=叔丁氧羰基,
But=O-叔丁基,
Cit=瓜氨酸
DCM=二氯甲烷,
DMF=N,N-二甲基甲酰胺,
DIPCDI=二-异丙基碳二亚胺,
DIPEA=二异丙基乙胺,
Et=乙基,
Et2O=乙醚,
2F-Phe=2-氟苯丙氨酸,
3F-Pro=3-氟脯氨酸,
4F-Pro=4-氟脯氨酸,
Fmoc=芴基甲氧基羰基,
g=克(s),
h=小时(s),
Har=高精氨酸
HoGlu或Homo-Glu=高谷氨酸,
HoLeu=高亮氨酸,
HoSer=高丝氨酸,
Hyp=4-羟脯氨酸,
3-Hyp=3-羟脯氨酸,
HOBt=1-羟基苯并三唑,
HOAt=7-氮杂-羟基苯并三唑,
HBTU=2-(1H-苯并三唑-1-基)-1,1,3,3-四甲基六氟磷酸铵,
HPLC=高效液相色谱法,
K(生物素)=赖氨酸(生物素),
L=升,
LC/MS=液相色谱/质谱法,
Me=甲基,
Min=分钟(s),
mL=毫升,
μl=微升,
mg=毫克(s),
mmol=毫摩尔(s),
MS=质谱,
Nle=正亮氨酸,
NMe-Ile=N-甲基-异亮氨酸
NMe-Leu=N-甲基-亮氨酸
NMe-Nle=N-甲基-正亮氨酸
OMe-Thr或Thr(OMe)=O-甲基-苏氨酸,
OMe-Ser或Ser(OMe)=O-甲基-丝氨酸,
OMe-HoSer或oSer(OMe)=O-甲基-高丝氨酸,
PyBOP=苯并三唑-1-基-氧基-三-吡咯烷基-六氟磷酸
2-Pal=2-吡啶基丙氨酸,
Pal=3-吡啶基丙氨酸,
4-Pal=4-吡啶基丙氨酸,
Sar=肌氨酸,
SPPS=固相肽合成,
2-Thi=(2-噻吩基)-丙氨酸,
Tha=(4-噻唑基)-丙氨酸,
Thz=4-硫代脯氨酸,
2-Thz=2-硫代脯氨酸,
TMS=三甲基甲硅烷基,
TIPS=三异丙基硅烷,
TFA=三氟乙酸,
TBTU=2-(1H-苯并三唑-1-基)-1,1,3,3-四甲基四氟硼酸铵,
Trt=三苯甲基。
发明详述
根据本发明,合成了具有结构式(I)的多种新的短链肽,然后将其与合适的免疫原性载体缀合。这些疫苗可用于治疗健康病症,例如高脂血症、高胆固醇血症或动脉粥样硬化。
因此,在一个方面,本发明公开了具有以下式(I)结构的PCSK9肽衍生物
A-Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10-Z11-Z12-B
式(I)
其中,
‘A’表示基团-NH-R1、R2-CO-NH-,其中‘R1’表示氢或任选地经取代的直链或支链(C1-18)烷基链;‘R2’选自任选地经取代的直链或支链(C1-18)烷基链、(C1-6)烷氧基、(C3-C6)环烷基、芳基、杂芳基或芳烷基基团;其中芳基选自苯基、萘基、茚满基、芴基或联苯基基团;杂芳基选自吡啶基、噻吩基、呋喃基、咪唑基、苯并呋喃基基团;
‘B’表示R3、-COOR3、-CONHR3或CH2OR3,其中R3表示H或合适的氨基酸,例如丝氨酸、半胱氨酸、缬氨酸、α-甲基-缬氨酸、Lys(生物素)、Lys(烷基)、Lys(乙酰基)等;
Z1是选自不带电荷的氨基酸残基的氨基酸残基,优选选自丝氨酸、苏氨酸、缬氨酸和丙氨酸及其衍生物,例如高丝氨酸、O-甲基-苏氨酸、O-甲基-丝氨酸、O-甲基-高丝氨酸等
Z2是选自不带电荷的氨基酸残基的氨基酸残基,优选选自异亮氨酸、亮氨酸、正亮氨酸、甘氨酸、丙氨酸、Aib及其衍生物,例如N-甲基-异亮氨酸和N-甲基-亮氨酸等
Z3是选自以下的氨基酸残基:脯氨酸、1-氨基碳环酸及其衍生物,例如羟脯氨酸、硫代脯氨酸、氨基脯氨酸、α-甲基-脯氨酸、3-氟脯氨酸、4-氟脯氨酸、1-氨基-环丙烷羧酸、1-氨基-环戊烷羧酸、1-氨基-环己烷羧酸等
Z4是选自优选色氨酸、苯丙氨酸、酪氨酸及其衍生物的氨基酸残基;
Z5是选自谷氨酰胺、组氨酸、优选天冬酰胺及其衍生物的氨基酸残基;
Z6是选自不带电荷的氨基酸残基的氨基酸残基,优选选自异亮氨酸、亮氨酸、丙氨酸、苏氨酸、天冬氨酸、Aib及其衍生物,例如正异亮氨酸、N-甲基-亮氨酸、高亮氨酸、α-甲基-天冬氨酸等
Z7是选自亲水性的、带负电荷的氨基酸残基的氨基酸残基,优选选自以下的氨基酸残基:谷氨酸、天冬氨酸及其衍生物,例如α-甲基-天冬氨酸、α-甲基-谷氨酸、高谷氨酸等
Z8是选自以下的氨基酸残基的氨基酸残基:优选选自精氨酸、高精氨酸、瓜氨酸、谷氨酰胺、天冬酰胺、赖氨酸及其衍生物;
Z9是选自不带电荷的氨基酸残基的氨基酸残基,优选选自异亮氨酸、亮氨酸、正亮氨酸、甘氨酸、丙氨酸、Aib及其衍生物,例如N-甲基异亮氨酸等
Z10是选自极性且不带电荷的氨基酸残基的氨基酸残基,优选选自丝氨酸、苏氨酸、缬氨酸、丙氨酸及其衍生物;
Z11是任何氨基酸残基,优选选自以下的氨基酸残基的氨基酸残基:优选选自脯氨酸及其衍生物,例如羟脯氨酸、硫代脯氨酸或α-甲基-脯氨酸、3-氟脯氨酸、4-氟脯氨酸、1-氨基-环丙烷羧酸、1-氨基-环戊烷羧酸、1-氨基-环己烷羧酸等
Z12是任何氨基酸残基,优选极性和不带电荷的氨基酸残基,所述氨基酸残基选自半胱氨酸、高半胱氨酸及其衍生物;前提是Z1至Z12的至少一个总是表示非天然氨基酸。
在任一个优选的实施方案中,‘A’表示基团-NH-R1、R2-CO-NH-,其中‘R1’是氢或经取代的直链或支链(C1-18)烷基链;‘R2’选自经取代的直链或支链(C1-18)烷基链、(C16)烷氧基、(C3-C6)环烷基、芳基和芳烷基基团;其中所述芳基选自苯基、萘基、茚满基、芴基或联苯基基团;‘B’表示R3、-COOR3、-CONHR3或CH2OR3,其中R3选自H、丝氨酸、半胱氨酸、缬氨酸、α-甲基-缬氨酸、Lys(生物素)、Lys(烷基)、Lys(乙酰基),Z1选自丝氨酸、苏氨酸、缬氨酸、丙氨酸、Aib、高丝氨酸、O-甲基-苏氨酸、O-甲基-丝氨酸或O-甲基-高丝氨酸,Z2选自异亮氨酸、亮氨酸、正亮氨酸、甘氨酸、丙氨酸、Aib、N-甲基-异亮氨酸或N-甲基-亮氨酸,Z3选自脯氨酸、羟脯氨酸、硫代脯氨酸、氨基脯氨酸、2-硫代脯氨酸、3-羟脯氨酸、3-氨基脯氨酸、α-甲基-脯氨酸、3-氟脯氨酸、4-氟脯氨酸、1-氨基-环丙烷羧酸、1-氨基-环戊烷羧酸或1-氨基-环己烷羧酸,Z4选自色氨酸、苯丙氨酸、酪氨酸、2-氟苯丙氨酸、α-甲基苯丙氨酸、α-甲基-2-氟苯丙氨酸、α-甲基-2,6-二氟苯丙氨酸、2-氨基-5-苯基-戊酸、α-甲基-2-氨基-5-苯基-戊酸或2’-乙基-4’-甲氧基-联苯丙氨酸,Z5选自谷氨酰胺、组氨酸或天冬酰胺,Z6选自异亮氨酸、亮氨酸、丙氨酸、苏氨酸、天冬氨酸、Aib、正异亮氨酸、N-甲基-亮氨酸、高亮氨酸、β-丙氨酸或α-甲基-天冬氨酸,Z7是谷氨酸、天冬氨酸、α-甲基-天冬氨酸、α-甲基-谷氨酸、2-氨基-4-氰基丁酸或高谷氨酸,Z8选自精氨酸、高精氨酸、瓜氨酸、谷氨酰胺、天冬酰胺或赖氨酸,Z9选自异亮氨酸、亮氨酸、正亮氨酸、甘氨酸、丙氨酸、Aib或N-甲基异亮氨酸,Z10选自丝氨酸、苏氨酸、缬氨酸、Aib或丙氨酸,Z11选自脯氨酸、羟脯氨酸、硫代脯氨酸、氨基脯氨酸、α-甲基-脯氨酸、3-氟脯氨酸、4-氟脯氨酸、1-氨基-环丙烷羧酸、1-氨基-环戊烷羧酸或1-氨基-环己烷羧酸,Z12选自半胱氨酸或高半胱氨酸。
定义
术语‘天然氨基酸’表示自然界中存在的所有那些二十种氨基酸。术语‘非天然氨基酸(unnatural amino acid或non-natural amino acid)’优选表示用相应的D-氨基酸替换L-氨基酸(例如用D-Ala替换L-Ala等)或L或D氨基酸的适当修饰,氨基烷基酸,通过:
-α-烷基化,例如用α-甲基Ala(Aib)替代Ala,用α-甲基Leu替代Leu;
-氨基酸侧链上的取代,例如用卤素、(C1-C3)烷基、芳基取代芳族氨基酸侧链,更具体地用卤素Phe替代Phe;
-β氨基酸,例如β丙氨酸;
在以下段落中描述了说明书中任何地方使用的不同基团、自由基和取代基。
本文中单独或与其他自由基组合使用的术语“烷基”是指包含1至18个碳的直链或支链自由基,例如甲基、乙基、正丙基、异丙基、正丁基、仲丁基、叔丁基、戊基、叔戊基、正戊基、正己基、异己基、庚基、辛基、癸基、十四烷基、十八烷基等。
本文中单独或与其他自由基组合使用的术语“环烷基”是指包含3至7个碳的自由基,例如环丙基、环丁基、环戊基、环己基、环庚基等。
除非另有说明,否则本文中单独或作为另一基团的一部分使用的术语‘氨基酸’包括但不限于连接至相同的碳(称为‘α’碳)的氨基和羧基。
‘α’碳处的绝对‘S’构型通常称为‘L’或天然构型。在‘α’碳处的‘R’构型通常称为‘D’氨基酸。在两个‘α-取代基’都相等(例如氢或甲基)的情况下,氨基酸是Gly或Aib,并且不是手性的。
文件中任何地方提及的术语‘衍生物’表示该特定氨基酸的任何经取代或同源的非天然氨基酸。
尽管已经相对于短链肽首先举例说明了本发明,但是还应理解,残基之间的肽键可被非肽键替代,前提是保留治疗潜力。本领域技术人员将知道合适的修饰,例如硫酰胺键形成、酰胺键的N-甲基化等。
涵盖氨基酸的保守取代的序列也在本发明的范围内,前提是保留生物学活性。
应清楚地理解,本发明的化合物包括肽酰胺和非酰胺以及肽类似物,包括但不限于以下:
a)其中一个或更多个氨基酸被其相应的D-氨基酸替换的化合物。技术人员将知道,可通过标准方法合成逆反(retro-inverso)氨基酸序列;参见例如,Chorev M.,Acc.Chem.Res.,26,1993,266-273;
b)其中肽键被对代谢降解更具抗性的结构替换的化合物。参见例如,Olson G.L.,et al.,J.Med.Chem.,36(21),1993,3039-3049,以及
c)其中单个氨基酸被类似结构替换(例如Ala和Aib;Arg和Cit)的化合物。
在整个说明书中,使用了天然氨基酸的常规一字母和三字母代码,以及使用了其他非天然氨基酸的普遍可接受的三字母代码,例如Hyp(羟脯氨酸)、Thz(硫代脯氨酸)、Aib(α-氨基异丁酸)。
短链肽的制备:
可采用肽合成领域的技术人员熟知的数种合成途径来制备本发明的短链肽。可使用以下描述的方法,与肽合成领域的技术人员已知的常规技术或本领域技术人员所知的其变体一起来合成其中所有符号如先前所限定的式(I)的短链肽。参考的方法包括但不限于以下描述的那些。
可使用溶液相(优选地,使用Boc-chemistry;M.Bodansky,A.Bodansky,“Thepractice of peptide synthesis”,Springer-Verlag,Berlim,1984;E.Gross,J.Meinhofer,“The peptide synthesis,analysis,biology”,Vol.1,Academic Press,London,1979)和或固相技术(例如,G.Barany&R.B.Merrifield,“The peptides:Analysis,synthesis,Biology”;Volume 2-“Special methods in peptide synthesis,Part A”,pp.3-284,E.Gross&J.Meienhofer,Eds.,Academic Press,New York,1980;and inJ.M.Stewart and J.D.Young,“Solid-phase peptide synthesis”2nd Ed.,Piercechemical Co.,Rockford,Il,1984中描述的那些)二者的合适的变体通过化学合成来产生本文中所述的其短链肽。
用于制备本发明的短链肽的优选策略是基于使用基于Fmoc的SPPS方法(其中Fmoc(9-芴基甲氧基羰基)基团用于α-氨基的临时保护),结合用于氨基酸侧链(如果存在的话)的临时保护的酸不稳定保护基团(例如叔丁氧羰基(Boc)、叔丁基(But)、三苯甲基(Trt)基团)(图1)(参见,例如,E.Atherton&R.C.Sheppard,“The Fluorenylmethoxycarbonylamino protecting group”,in“The peptides:Analysis,synthesis,Biology”;Volume 9-“Special methods in peptide synthesis,Part C”,pp.1-38,S.Undenffiend&J.Meienhofer,Eds.,Academic Press,San Diego,1987)。
短链肽可在不溶性聚合物支持物(树脂)上从肽的C端开始以逐步的方式合成。在一个实施方案中,通过形成酰胺、酯或醚键将肽的C端氨基酸附接至树脂来起始合成。这允许所得的肽分别以C端酰胺、羧酸或醇最终释放。
在基于Fmoc的SPPS中,要求在合成中使用的C端氨基酸和所有其他氨基酸具有不同地被保护(正交保护)的其α-氨基和侧链官能团(如果存在的话),使得可在合成期间使用合适的碱(例如20%哌啶溶液)选择性地去除α-氨基保护基团,而没有肽从树脂的任何过早的切割或侧链保护基的去保护,其通常用酸不稳定保护基保护。
氨基酸的偶联是通过激活其羧基作为有活性的酯并将其与附接至树脂的N端氨基酸的未封闭的α-氨基反应来进行。在每次偶联和去保护之后,用过量的溶剂(例如DMF、DCM和乙醚)洗涤肽基树脂。重复α-氨基序列去保护和偶联,直至组装了期望的肽序列为止(方案1)。然后,通常在合适的清除剂的存在下(以限制副反应),使用合适的切割混合物,将肽从树脂切割,伴随着侧链官能团的去保护。最后通过反相HPLC来纯化所得的肽。
最终的肽的前体所需的肽基树脂的合成利用可商购的交联聚苯乙烯聚合物树脂(Novabiochem,San Diego,CA)。优选用于本发明的是C端氨基酸可已附接或可尚未附接的Fmoc-PAL-PEG-PS树脂、4-(2’,4’-二甲氧基苯基-Fmoc-氨基甲基)-苯氧基乙酰基-p-甲基二苯甲基胺树脂(Fmoc-Rink酰胺MBHA树脂)、2-氯-三苯甲基-氯化物树脂或p-苄氧基苄醇树脂(HMP树脂)。如果C端氨基酸是未附接的,则其附接可通过Fmoc保护的氨基酸(通过其与DIPCDI反应形成)的HOBt活性酯来实现。在2-氯三苯甲基树脂的情况下,使用DIPEA实现第一Fmoc保护的氨基酸的偶联。为了组装下一个氨基酸,使用10%至20%的哌啶溶液对肽基树脂的N端保护进行选择性去保护。在每次偶联和去保护之后,通过用DMF、DCM和醚洗涤来除去过量的氨基酸和偶联剂。可分别使用由DIPCDI/HOBt或DIPCDI/HOAT产生的HOBt或HOAT活性酯来完成后续氨基酸的偶联。在一些难以偶联的情况下,特别是疏水的或具有庞大侧链保护的氨基酸的那些氨基酸的偶联;使用高效偶联剂(例如HBTU、PyBOP或TBTU)与添加剂(例如DIPEA)的组合可实现完全偶联。
可通过使用分批或连续流肽合成装置(例如CS-Bio或AAPPTEC肽合成仪)利用Fmoc/叔丁基保护策略来进行本文中所述的短链肽的合成。使用本领域中已知的一种或更多种方法,将存在于不同位置的非天然非商业氨基酸并入肽链。在一种方法中,使用合适的文献方法,在溶液中制备Fmoc保护的非天然氨基酸。例如,使用经修改的文献方法(Betsbrugge J.V.,et al.,Tetrahedron,54,1988,1753-1762)以良好的对映异构体纯度由L-焦谷氨酸制备上述Fmoc保护的APPA类似物。
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图1:用于短链肽的基于Fmoc的固相肽合成(Solid Phase Peptide Synthesis,SPPS)的一些受保护氨基酸的实例。
使用不对称的Strecker合成来制备Fmoc保护的α-甲基化氨基酸(Boesten,W.H.J.,et al.,Org.Lett.,3(8),2001,1121-1124;Cativiela C.,Diaz-de-villegasM.D.,Tetrahedran Asymmetry,9,1988,3517-3599)。然后将所得的衍生物用于肽的逐步合成。或者,使用合成有机化学方法直接在树脂上构建所需的非天然氨基酸,并制备线性肽链。
可使用文献(King D.S.,et al.,Int.J.Peptide Protein Res.,1990,36,255-266)中所述的标准切割方法的任一种的适当变体对其各自短链肽的肽树脂前体进行切割和去保护。用于本发明的一个优选方法是在水和TIPS(作为清除剂)的存在下,使用TFA切割混合物。通常,将肽基树脂在TFA/水/TIPS(95∶2.5∶2.5)中在室温孵育1.5至4小时。然后将切割的树脂过滤出,并将TFA溶液在减压下浓缩或干燥。将所得的粗制肽用Et2O沉淀或洗涤,或直接重新溶解在DMF或50%水性乙酸中用于通过制备型HPLC纯化。
可通过使用制备型HPLC的纯化来获得具有期望纯度的短链肽。将粗制肽溶液进样到尺寸为250×50mm的Semi-Prep柱(Luna 10μ;C18;100A°)中,并用在水中的CAN的线性梯度洗脱,二者均用0.1%TFA缓冲,流速为40ml/分钟,通过PDA检测器在220nm下监测流出物。可通过电喷射质谱(Electrospray Mass Spectroscopy,ES-MS)分析来确认纯化的短链肽的结构。
在制备型HPLC纯化之后,将制备的所有肽分离为具有TFA作为反荷离子的三氟乙酸盐。然而,一些肽通过穿过合适的离子交换树脂床,优选穿过阴离子交换树脂Dowex SBRP(Cl)或等效碱性阴离子交换树脂来进行脱盐。在一些情况下,TFA反荷离子通过穿过合适的离子交换树脂(用稀释的乙酸缓冲液洗脱)而被乙酸根离子替代。为了制备肽的盐酸盐,在制备的最后阶段,将具有乙酸盐的选定的肽用4M HCl处理。所得的溶液通过膜过滤器(0.2μm)过滤,并随后冻干,以产生白色至类白色的HCl盐。按照本领域技术人员已知的类似技术和/或此类合适的改变,制备了本发明的短链肽的其他合适的可药用盐。
方案1:基于Fmoc的SPPS的通用方案
使用SPPS方法制备短链肽的通用方法:
在树脂上组装短链肽:
足量(50至100mg)的Fmoc-PAL-PEG-PS树脂或Fmoc-Rink酰胺MBHA树脂,负载:0.5至0.6mmol/g在DMF(1至10ml/100mg树脂)中溶胀2至10分钟。通过将树脂用10%至30%哌啶在DMF(10至30ml/100mg树脂)中孵育10至30分钟来去除树脂上的Fmoc基团。过滤去保护的树脂,并用过量的DMF、DCM和乙醚(50ml×4)进行洗涤。将洗涤的树脂在新鲜蒸馏的DMF(1ml/100mg树脂)中在氮气氛围下孵育5分钟。将用HOBt(1至3当量)和DIPCDI(1至2当量)在DMF中预激活的第一Fmoc保护的氨基酸的0.5M溶液(1至3当量)添加至树脂,然后将树脂在氮气氛围下摇晃1至3小时。使用定性茚三酮试验监测偶联完成。在第一氨基酸的偶联之后,用DMF、DCM和乙醚(50ml×4)洗涤树脂。对于下一个氨基酸的偶联,首先,使用10%至20%的哌啶溶液对与树脂偶联的第一氨基酸上的Fmoc保护进行去保护,然后使用合适的偶联剂并且如上所述对Fmoc保护的第二氨基酸进行偶联。像根据上述通用方法(方案1)一样,进行去保护、洗涤、偶联和洗涤的重复循环直至期望的肽链在树脂上组装。最后,如上所述,通过20%哌啶处理对上面制备的Fmoc保护的肽基树脂进行去保护,并且用DMF、DCM和乙醚洗涤肽基树脂。将包含期望的肽的树脂在氮气压力下干燥10至15分钟,并进行切割/去保护。
使用上述方案及本领域技术人员已知的其合适变体,使用Fmoc-SPPS方法,制备了本发明中设计的短链肽。此外,使用以下方案对树脂结合的短链肽进行切割和去保护、纯化和表征。
切割与去保护:
如下通过用TFA切割混合物的处理,将期望的短链肽从其各自的肽基树脂上切割并去保护。将TFA/水/三异丙基硅烷(95∶2.5∶2.5)(10ml/100mg肽基树脂)的溶液添加至肽基树脂,并且将混合物在偶尔搅拌的情况下保持在室温下。将树脂过滤,用切割混合物洗涤,并将合并的滤液蒸发至干燥。将获得的剩余物溶解在10ml水中,并用乙醚萃取水层3次,并最终将水层冷冻干燥。冷冻干燥之后获得的粗制肽如下通过制备型HPLC纯化:
粗制短链肽的制备型HPLC纯化:
在Shimadzu LC-8A液相色谱上进行制备型HPLC。将溶解在DMF或水中的粗制肽溶液进样到尺寸为250×50mm的Semi-Prep柱(Luna 10μ;C18;100A°)中,并用在水中的CAN的线性梯度洗脱,二者均用0.1%TFA缓冲,流速为15至50ml/分钟,通过PDA检测器在220nm下监测流出物。在50分钟内以每分钟1%梯度变化,使用用0.1%TFA缓冲的20%至70%水-ACN混合物的典型梯度。以单个10至20ml级分收集洗脱的期望产物,并通过各个HPLC级分的冻干来获得为无定形白色粉末的纯的短链肽。
纯的短链肽的HPLC分析
在如上所述通过制备型HPLC纯化之后,在Shimadzu LC-10AD分析型HPLC系统上通过分析型RP-HPLC分析每种肽。对于短链肽的分析型HPLC分析,以0.1%TFA和ACN缓冲液的线性梯度使用Luna 5μ;C18;100A°、尺寸为250×4.6mm的柱,并使用PDA检测器在220nm下进行色谱图的采集。
通过质谱法表征
通过电喷雾电离质谱(ESI-MS)以流动进样或LC/MS模式表征每种肽。三重四极质谱仪(API-3000(MDS-SCIES,Canada))以正离子和负离子电喷雾模式用于所有分析。在以单位分辨率运行的四极的质量范围内获得全扫描数据。在所有情况下,实验测量的分子量在计算的单一同位素分子量的0.5道尔顿内,使用Analyst 1.4.1软件对质谱图进行定量。
以下表1(i)是如上所述使用SPPS方法合成的短链肽的列表。列表中提及的Seq.ID.No 1被认为是来自WO 2011027257的参考。
表1(i):制备的短链肽的列表
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在另一个优选的实施方案中,可通过本领域中公知的方法化学合成本发明的肽。还可使用重组方法来产生本发明的肽。可在以下中产生肽:微生物,例如细菌(例如大肠杆菌(E.coli)、枯草芽孢杆菌(B.subtilis)、或能够表达这样的肽的任何其他细菌)、酵母菌(例如酿酒酵母(Saccharomyces cerevisiae)、粟酒裂殖酵母(Schizosaccharomycespombe)、假丝酵母(Candida)、巴斯德毕赤酵母(Pichia pastoris)或能够表达肽的任何其他酵母菌)或真菌;真核细胞,例如哺乳动物或昆虫细胞;或重组病毒载体,例如腺病毒、痘病毒、疱疹病毒、塞姆利基森林病毒(Simliki forest virus)、杆状病毒、噬菌体、辛德毕斯病毒(sindbis virus)或仙台病毒(Sendai virus)。用于分离和纯化重组产生的肽的方法也是本领域中公知的,并且包括例如凝胶过滤、亲和色谱法、离子交换色谱法等。
用于DT缀合的方法:
白喉毒素是由535个氨基酸组成的单多肽链,所述氨基酸包含通过二硫键连接的两个亚基。所述亚基中的一个与细胞表面结合,实现更稳定的亚基以穿透宿主细胞。为了缀合,需要等摩尔浓度的白喉毒素和肽。DT和肽的浓度为2至50mg/mL。在第一步中,将白喉毒素通过溶解在磷酸盐缓冲的盐水中制成溶液。接下来,添加EDAC(1-乙基3,3-二甲基氨基丙基碳二亚胺),这提供使羧酸交联的第一步。EDAC激活羧基以通过酰胺键形成与伯胺直接反应,从而使白喉毒素与肽缀合。此外,EDAC介导的交联在酸性pH中更有效。因此,通过在pH6.0的MES缓冲液(40-吗啉代乙烷磺酸)的存在下,将DT用EDAC孵育一分钟来使反应发生。此外,存在MES的该EDAC偶联方法通过形成中间体提高了缀合效率。接下来,添加ADH(己二酸二酰肼),其是同双功能交联剂,导致DT和肽的相对稳定的腙键。通过先氧化然后交联(在pH5.0(由于酰肼的低pKa)下进行),以位置特异性的方式进行连接。这避免了伯胺的竞争。再次添加EDAC,并将混合物在2至8℃下孵育3小时以开始缀合。将上述制备的DT缀合的肽通过10kD柱透析,并进行无菌过滤(0.2μ过滤器)以除去杂质,并且将纯的肽-DT缀合物在2至8℃下储存至少一周。
在另一个实施方案中,通过按照WO 2011027257中给出的通用方法和现有技术,用是白喉毒素的遗传解毒形式的CRM197进行缀合。
在另一个优选的实施方案中,本发明的疫苗可皮下、肌内、皮内、静脉内施用(参见,例如,“Handbook of Pharmaceutical Manufacturing Formulations”,SarfarazNiazi,CRC Press Inc,2004)。根据施用途径,制剂可由分别的载体、佐剂和/或赋形剂组成。
疫苗制剂:
为了设计更具免疫潜能和保护性的疫苗,最终的疫苗制剂可包含佐剂,所述佐剂具有免疫潜能(immunopotentiating)特性,可根据佐剂的类型将免疫应答引导至体液免疫或细胞介导的免疫。传统的佐剂(例如铝盐、乳剂、脂质体和病毒微体)以有效的方式将疫苗抗原呈递给免疫系统,并控制抗原的释放和储存以增强特异性免疫应答。第二类佐剂是免疫刺激剂,其影响免疫系统并提高针对抗原的免疫应答。例如,其通过MHC分子、共刺激信号的激活或通过相关的细胞内信号传导途径来影响细胞因子的产生。
已经发现,许多目前批准的疫苗佐剂并不总是足够有效以诱导针对不同的靶标病原体的有效保护性免疫应答,特别是在免疫功能低下的群体(例如年老和免疫功能受损的群体)中,其中由于受损的T细胞功能,T细胞依赖性抗体应答降低。具有免疫潜能特性的佐剂的使用在大量的疫苗(例如丙型肝炎病毒(hepatitis C virus,HCV)、HIV、HBV、HPV、流行性感冒和癌症)中产生更有效的应答。因此,疫苗设计的更合理的方法是使用免疫刺激剂佐剂,其可通过TLR利用来调节且增强细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)应答,和/或影响树突状细胞(dendritic cell,DC)。T细胞是免疫应答的最有效手段,并且鉴于T细胞在调节针对疫苗的免疫应答中的公认重要性,在疫苗接种策略中使用这些新的佐剂似乎是合适的。
可用于制剂的其他佐剂:为了引起DT缀合的肽的更强的免疫应答,添加至制剂的另外的佐剂如下。
1)明矾(氢氧化铝凝胶,2%湿凝胶混悬液)
明矾是有效的佐剂,因为抗原与不溶性铝盐佐剂结合(adjuvante)在一起,在体内保留很长时间。抗原从不溶性盐颗粒缓慢释放,使得长期且有效地刺激免疫系统(‘贮库效应(depot effect)’)[1]。除了贮库效应之外或与之相反,不溶性铝盐以最终导致T辅助2(Th2)型免疫应答的方式激活固有免疫细胞。明矾通过提高通过抗原呈递细胞(antigen-presenting cell,APC)的抗原的抗原性摄取来诱导Th2应答[2]。
2)明矾与MF-59
AddaVax(也称为MF59)是基于角鲨烯的水包油纳米乳剂。角鲨烯是比弗氏佐剂中使用的石蜡油更容易代谢的油[3]。角鲨烯水包油乳剂可引起细胞(Th1)免疫应答和体液(Th2)免疫应答二者[4,5]。该佐剂类别通过募集和激活抗原呈递细胞以及刺激巨噬细胞和粒细胞产生细胞因子和趋化因子来起作用[3]。
3)明矾与其他佐剂,例如,TLR3激动剂例如聚(I:C)、TLR 4激动剂例如单磷酰脂A或GLA-SE、TLR5激动剂例如鞭毛蛋白、TLR7激动剂例如嘎德莫特和咪喹莫特、TLR7/8激动剂例如R848、NOD2激动剂例如N-乙醇酰-MDP、包含CpG的核酸(其中胞嘧啶是未甲基化的)、QS21(皂苷佐剂)、白介素、β-谷固醇等。
所有实验中使用的佐剂是明矾与单磷酰脂A的稳定制剂的组合。
新肽的亲和力测定:
使用Biacore仪器(Biacore T200,GE Healthcare),通过表面等离激元共振(surface plasmon resonance,SPR)分析重组人PCSK9的新肽的亲和力参数。用BIACORET200仪器(GE Healthcare,Uppsala,Sweden)在25℃下进行SPR实验。
表面处理:(用于蛋白质固定的方法)
通过使用胺偶联化学,在pH 5.0的10mM醋酸盐缓冲液中稀释至100至150μg/mL的纯的重组人PCSK9(His标记,BPS Biosciences)固定在Series S Sensor Chip CM5的四个流通池之一上至约6000至8000RU(共振单位,resonance unit)的水平。用pH 8.5的1M乙醇胺封闭表面。而将一个流通池固定为空白用于参考减除(reference subtraction)(无PCSK9)。将含有150mM NaCl的10mM Hepes(pH 7.4)用作运行缓冲液。保存剩余的两个流通池用于将来使用。
结合和动力学/亲和力实验:
在100%DMSO中制备NCE(新肽)的100×储备溶液,并在10mM Hepes(pH 7.4)+150mM NaCl中稀释至一定浓度范围,使得DMSO的最终浓度保持在1%。通过使不种浓度的NCE经过空白以及固定有配体(蛋白质)的表面来进行结合/动力学研究。每个循环由以下组成:将分析物(稀释的小分子药物)以5至30μL/分钟的流速进行60秒或120秒进样(缔合阶段),然后是60至180秒的解离阶段。如果需要,使用10mM甘氨酸/HCl(pH 1.5)的30秒(15μL)进样和10mM NaOH的20秒或30秒(10或15μL)进样进行再生。使用Biacore T200评估软件分析数据。所有曲线均减去参考。在样品进样结束之前5秒将所有曲线的基线调整为零,并将数据表示为结合RU(5秒窗的平均值)。
对于KD(平衡解离常数)测定,分析物曲线减去了缓冲液空白,并假定简单的1∶1相互作用以产生动力学数据进行建模。
BALB/C小鼠中的免疫原性研究
从动物舍中获得12至15周龄的雌性BALB/c小鼠,并保持2至3次适应(acclimatization)。小鼠可随意获得食物和水,并保持在12小时的昼夜循环下。在第0天(预处理),对动物进行放血并收获血清用于总胆固醇测量。将动物随机化并分组以基于其总胆固醇和体重进行不同的处理。在第0天(预处理)血液收集的第二天,通过皮下或肌内途径用0.3ml如表1所述的疫苗制剂免疫动物。在第一次注射之后的2周和4周给予下一次增强注射,并且在此之后2周直至第7周,然后每四周直至第32周对动物进行放血用于免疫原性测量,详细的血液收集时间表为第0天(预处理)、第一次疫苗施用之后第5、7、20、24、28、32、45和57周。分离血清并测量血清总胆固醇,并使用ELISA进行PCSK9抗体滴度的免疫原性或抗体确认,对于血清抗体与人PCSK9的结合,使用表面等离激元共振(SPR)测定和LDLR-PCSK9相互作用抑制测定。
表1
hApoB100/hCETP双转基因小鼠(dTg)中的免疫原性研究
从动物舍获得超过8周龄的雄性或雌性hApoB100/hCETP dTg小鼠,并保持2至3次适应。小鼠可随意获得食物和水,并保持在12小时的昼夜循环下。在第0天(预处理),对动物进行放血并收获血清用于LDL-胆固醇(LDL-C)、总胆固醇、HDL-C和甘油三酯测量。将动物随机化并分组以基于其LDL-C和体重进行不同的处理。在血液收集的第二天,通过皮下或肌内途径用0.3ml疫苗制剂免疫动物。在第一次注射之后的2周和4周给予下一次增强注射,并且在第3次注射之后2周对动物进行放血用于免疫原性测量。分离血清并测量血清LDL-C、总胆固醇、HDL-C和甘油三酯水平,并使用ELISA进行PCSK9抗体滴度的免疫原性或抗体确认,对于血清抗体与人PCSK9的结合,使用表面等离激元共振(SPR)测定和LDLR-PCSK9相互作用抑制测定。
使用商业试剂盒(Roche Diagnostics,Germany)在Cobas c311自动分析仪(Roche,Germany)上确定血清LDL-C、HDL-C、总胆固醇和甘油三酯水平。
通过抗PCSK-9ELISA评估体液应答
在特定的时间间隔(每次增强免疫之后15天),从所有小鼠组收集血清样品。将96孔ELISA板(#442402)用50ng/孔的PCSK-9蛋白预先涂覆,并将板在2至8℃下孵育过夜。第二天,洗涤孔以除去未结合的蛋白质,并将其在2%脱脂乳-PBST中封闭2小时,并用PBST洗涤。将血清样品用在PBST中制得的0.2%脱脂乳按1∶20稀释。将适当体积的样品和标准品添加至每个孔,并在37℃下孵育1小时。将抗小鼠IgG二级HRP标记的抗体添加至每个孔,并在37℃下孵育1小时,然后洗涤。将OPD底物添加至每个孔,并在黑暗中在室温下孵育20分钟。在Spectramax微板读取仪(Spectramax microplate reader)上在492nm下读取板。在测试的所有DT缀合的PCSK-9肽中,将给出最高响应的肽作为标准品,并使用其安慰剂计算校正的OD。来自用PBS注射的小鼠的血清用作阴性对照。通过使用阴性对照值,计算截止值,并将高于截止值的校正的OD的作为标准。所有测试样品均给出了针对PCSK-9的绝对抗体滴度,其从标准曲线推算并且值以log 10表示。
LDLR-PCSK9相互作用抑制测定:
使用具有较小变化的BPS Bioscience体外PCSK9[生物素化]-LDLR结合测定试剂盒(BPS Bioscience,San Diego,CA,USA),测定了来自疫苗处理的动物的血清中通过抗体(Ab)的LDLR-PCSK9相互作用的抑制。首先,将LDLR胞外域涂覆在96孔板上,并将板孵育过夜。接下来,将PCSK9-生物素在1×PCSK9测定缓冲液中稀释,以获得2.5ng/μl(50ng/20μl)的浓度,并将该主要的混合物、测试抑制剂(来自动物研究的血清样品)、抑制剂缓冲液添加至LDL-R涂覆的板并在室温下孵育2小时。最后,用链霉抗生物素蛋白-HRP处理该板,接着添加HRP底物以产生化学发光,然后根据制造商的说明使用化学发光读取仪对其进行测量。相对抑制表示为来自疫苗处理的动物的血清样品中的PCSK9-LDLR结合强度与安慰剂处理的情况下的PCSK9-LDLR结合强度(其被认为是100%)之间的百分比抑制的差异。
结果:
I)表2:使用Biacore(Biacore T200,GE Healthcare)通过表面等离激元共振(SPR)分析的重组人PCSK9的新肽的亲和力。
表2:重组人PCSK9的新肽的亲和力研究。
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II)通过ELISA测定的处理的动物的血清样品中针对PCSK9的平均抗体滴度在下表3中给出
表3ELISA测定中针对PCSK9的平均抗体滴度
所有肽制剂均示出了非常高水平的针对人PCSK9的抗体滴度,并且与Seq.ID.No.1相比,Seq.ID.No.7、8、12的肽的滴度更高。直至疫苗施用之后第57周,仍保持了非常高的抗体应答,表明少数肽制剂超过一年的免疫原性应答的可能性。
III)使用体外PCSK9[生物素化]-LDLR结合测定试剂盒(作为功能测定的方法),测定了来自疫苗处理的动物的血清中通过抗体(Ab)的LDLR-PCSK9相互作用的抑制。在第5周一次测量的通过抗体(Ab)的百分数抑制LDLR-PCSK9相互作用在表4中给出。
表4通过ELISA测定的LDLR-PCSK9相互作用抑制
LDLR-PCSK-9%相互作用抑制Vs.安慰剂 | 疫苗施用之后的周数 |
测试组 | 第5周 |
佐剂中DT缀合Seq.ID.No.1 | 46 |
佐剂中DT缀合Seq.ID.No.7 | 66 |
佐剂中DT缀合Seq.ID.No.8 | 66 |
佐剂中DT缀合Seq.ID.No.12 | 67 |
在第5周,几乎所有的肽制剂都示出了40%至70%抑制LDLR-PCSK9相互作用。Seq.ID.No.7、8、12示出的相互作用抑制显著高于Seq.ID.No.1示出的相互作用抑制。
IV)使用SPR测定通过针对人PCSK9的亲和力测定,还证实了处理的动物的血清样品中的抗体检测,其在下表5中给出
表5使用SPR测定通过针对人PCSK9的亲和力测定的血清抗体检测。
相比于安慰剂,来自用肽制剂处理的动物的样品血清示出了响应单位的提高。与Seq.ID.No.1相比,在第5周和第57周,序列ID.No.12示出显著更高的抗体应答,并且在第5周,序列ID.No.8示出显著更高的抗体应答。直至疫苗施用之后第57周,保持了非常高水平的抗体应答;其清楚地表明肽制剂超过一年的免疫原性应答的可能性。
本发明的肽以每次免疫接种0.1ng至10mg,优选0.5至500μg的量施用于哺乳动物或个体。在一个优选的实施方案中,这些量是指疫苗中存在的所有肽(如果在疫苗中使用了多于一种的肽)。
可与载体物质组合以产生单剂型的肽的量将根据所治疗的宿主和特定的施用模式而变化。
疫苗的剂量可根据以下因素而变化:例如,哺乳动物或个体的疾病状态、年龄、性别和体重,以及抗体在个体中引起期望响应的能力。可调整剂量方案以提供最佳的治疗响应。例如,可每天施用数个分开的剂量或可如紧急治疗情况所示成比例地减少剂量。疫苗的剂量还可根据情况而变化以提供最佳的预防剂量响应。例如,本发明的肽和疫苗可总是根据针对PCSK9的抗体的水平,以数天、一或两周、或甚至数月或数年的间隔施用于个体。
当与合适的免疫原性载体缀合时,本发明的肽可充当疫苗。这些疫苗在施用之后能够形成与PCSK9结合的抗体。这些疫苗可适合于预防/治疗病症,例如高脂血症、高胆固醇血症或动脉粥样硬化。
参考文献:
1)Glenny AT,Pope CG,Waddington H,Wallace U.Immunological Notes:XVII-XXIV.J Pathol Bacteriol.1926;29:31-40.
2)Grun JL,Maurer PH.Different T helper cell subsets elicited in miceutilizing two different adjuvant vehicles:the role of endogenous interleukinl in proliferative responses.Cell Immunol.1989;121:134-145.
3)Ott G.et al.,2000.The adjuvant MF59:a 10-year perspective.Methodsin Molecular Medicine,Vol42,211-228.
4)Calabro,S.et al.,2013.The adjuvant effect of MF59 is due to theoil-in-water emulsion formulation,none of the individual components induce acomparable adjuvant effect.Vaccine 31:3363-9.
5)Ott G.et al.,1995.MF59.Design and evaluation of a safe and potentadjuvant for human vaccines.Pharm Biotechnol 6:277-96.
序列表
<110> Cadila Healthcare Limited
<120> 基于新的肽的PCSK9疫苗
<130> CHL-PCT0769
<140> PCT/IB2018/052555
<141> 2018-04-12
<150> 201721013281
<151> 2017-04-13
<160> 58
<170> PatentIn version 3.5
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<221> Xaa
<222> (11)..(11)
<223> Xaa是4-羟脯氨酸
<400> 27
Ser Ile Pro Trp Asn Leu Glu Xaa Ile Thr Xaa Cys
1 5 10
<210> 28
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (6)..(6)
<223> Xaa是α-氨基异丁酸
<220>
<221> Xaa
<222> (11)..(11)
<223> Xaa是4-羟脯氨酸
<400> 28
Ser Ile Pro Trp Asn Xaa Glu Arg Ile Thr Xaa Cys
1 5 10
<210> 29
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (6)..(6)
<223> Xaa是α-氨基异丁酸
<220>
<221> Xaa
<222> (11)..(11)
<223> Xaa是4-羟脯氨酸
<400> 29
Ser Ile Xaa Trp Asn Xaa Glu Arg Ile Thr Xaa Cys
1 5 10
<210> 30
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (6)..(6)
<223> Xaa是α-氨基异丁酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<220>
<221> Xaa
<222> (11)..(11)
<223> Xaa是4-羟脯氨酸
<400> 30
Ser Ile Pro Trp Asn Xaa Glu Xaa Ile Thr Xaa Cys
1 5 10
<210> 31
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<220>
<221> Xaa
<222> (11)..(11)
<223> Xaa是4-羟脯氨酸
<400> 31
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Xaa Cys
1 5 10
<210> 32
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是α-甲基-脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 32
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 33
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是1-氨基-环丙烷羧酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 33
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 34
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是1-氨基-环戊烷羧酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 34
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 35
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是1-氨基-环己烷羧酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 35
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 36
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (6)..(6)
<223> Xaa是β丙氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 36
Ser Ile Xaa Trp Asn Xaa Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 37
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (7)..(7)
<223> Xaa是α-甲基-谷氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 37
Ser Ile Xaa Trp Asn Leu Xaa Xaa Ile Thr Pro Cys
1 5 10
<210> 38
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (7)..(7)
<223> Xaa是高谷氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 38
Ser Ile Xaa Trp Asn Leu Xaa Xaa Ile Thr Pro Cys
1 5 10
<210> 39
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (7)..(7)
<223> Xaa是α-甲基-天冬氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 39
Ser Ile Xaa Trp Asn Leu Xaa Xaa Ile Thr Pro Cys
1 5 10
<210> 40
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (6)..(6)
<223> Xaa是高亮氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 40
Ser Ile Xaa Trp Asn Xaa Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 41
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (6)..(6)
<223> Xaa是α-甲基-天冬氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 41
Ser Ile Xaa Trp Asn Xaa Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 42
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-硫代脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 42
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 43
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是2-硫代脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 43
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 44
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是3-羟脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 44
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 45
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-氨基脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 45
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 46
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是3-氨基脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 46
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 47
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是3-氟脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 47
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 48
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-氟脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 48
Ser Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 49
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (1)..(1)
<223> Xaa是O-甲基-丝氨酸
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 49
Xaa Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 50
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (1)..(1)
<223> Xaa是高丝氨酸
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 50
Xaa Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 51
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (1)..(1)
<223> Xaa是O-甲基-苏氨酸
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 51
Xaa Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 52
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (1)..(1)
<223> Xaa是O-甲基-高丝氨酸
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 52
Xaa Ile Xaa Trp Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 53
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (4)..(4)
<223> Xaa是2-氟苯丙氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 53
Ser Ile Xaa Xaa Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 54
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (4)..(4)
<223> Xaa是α-甲基-苯丙氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 54
Ser Ile Xaa Xaa Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 55
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (4)..(4)
<223> Xaa是α-甲基-2-氟苯丙氨酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 55
Ser Ile Xaa Xaa Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 56
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (4)..(4)
<223> Xaa是α-甲基-2,6-二氟丙基苯胺
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 56
Ser Ile Xaa Xaa Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 57
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (4)..(4)
<223> Xaa是α-甲基-2-氨基苯基戊酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 57
Ser Ile Xaa Xaa Asn Leu Glu Xaa Ile Thr Pro Cys
1 5 10
<210> 58
<211> 12
<212> PRT
<213> 人工
<220>
<223> PCSK9
<220>
<221> Xaa
<222> (3)..(3)
<223> Xaa是4-羟脯氨酸
<220>
<221> Xaa
<222> (7)..(7)
<223> Xaa是2-氨基-4-氰基丁酸
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa是瓜氨酸
<400> 58
Ser Ile Xaa Trp Asn Leu Xaa Xaa Ile Thr Pro Cys
1 5 10
Claims (7)
1.由氨基酸序列组成的肽,所述氨基酸序列选自以下序列:
SIPWN-Nle-ERITPC;
SI-Hyp-WNLERITPC;
SIPWNLE-Cit-ITPC。
2.与免疫原性载体缀合的权利要求1所述的由氨基酸序列组成的肽,其中所述免疫原性载体选自白喉毒素(DT)、经修饰的DT、匙孔血蓝蛋白(KLH)、破伤风类毒素(TT)、蛋白D或包含辅助性T细胞表位的任何其他蛋白质。
3.如权利要求2中所述的肽,其中所述免疫原性载体是白喉毒素(DT)或CRM。
4.与一种或更多种可药用佐剂和其他药用赋形剂组合的如权利要求2中所述的肽。
5.如权利要求4中所述的肽,其中所述可药用佐剂选自:明矾、TLR3激动剂聚(I:C)、TLR4激动剂单磷酰脂A或GLA、TLR5激动剂鞭毛蛋白、TLR7激动剂嘎德莫特和咪喹莫特、TLR7/8激动剂R848、NOD2激动剂N-乙醇酰-MDP、其中胞嘧啶是未甲基化的包含CpG的核酸、QS21、白介素、β-谷固醇。
6.如权利要求5中所述的肽,其中一种或更多种可药用佐剂选自明矾和/或GLA和/或单磷酰脂A。
7.权利要求1至6中任一项所述的肽在制备适合于治疗选自高脂血症、高胆固醇血症或动脉粥样硬化的病症的药物中的用途。
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PCT/IB2018/052555 WO2018189705A1 (en) | 2017-04-13 | 2018-04-12 | Novel peptide based pcsk9 vaccine |
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EP (1) | EP3609532A1 (zh) |
JP (1) | JP7050807B2 (zh) |
KR (1) | KR102401796B1 (zh) |
CN (1) | CN110536696B (zh) |
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- 2018-04-12 CA CA3058567A patent/CA3058567A1/en not_active Abandoned
- 2018-04-12 CN CN201880023715.5A patent/CN110536696B/zh active Active
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- 2018-04-12 US US16/498,084 patent/US11325945B2/en active Active
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US11325945B2 (en) | 2022-05-10 |
US20200399311A1 (en) | 2020-12-24 |
EP3609532A1 (en) | 2020-02-19 |
MX2019012083A (es) | 2019-11-21 |
KR102401796B1 (ko) | 2022-05-25 |
CA3058567A1 (en) | 2018-10-18 |
JP2020512995A (ja) | 2020-04-30 |
CN110536696A (zh) | 2019-12-03 |
WO2018189705A1 (en) | 2018-10-18 |
BR112019020148A2 (pt) | 2020-05-05 |
JP7050807B2 (ja) | 2022-04-08 |
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