CN113842467A - 用于抗体与药物偶联体(adc)制备的中间体及其制备方法和应用 - Google Patents
用于抗体与药物偶联体(adc)制备的中间体及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种用于抗体与药物偶联体(ADC)制备的中间体(连接子与载荷组合)及其制备方法和应用。具体地,本发明的连接子与载荷组合具有式I结构,
Description
技术领域
本发明属于医药生物领域,具体地涉及一种用于抗体与药物偶联体(ADC)制备的中间体(连接子与载荷组合)及其制备方法和应用。
背景技术
抗体-药物偶联物(ADC)属于肿瘤靶向治疗的一种,靶向肿瘤抗原的抗体通过化学合成的连接子与具有细胞毒性的有效载荷共价偶联。药物连接子和有效载荷的选择在ADC的治疗效果和安全性中起关键作用。
目前,大多数进入临床开发阶段的ADC分子都利用了可裂解的连接子,包括:酸不稳定连接子、肽连接子和二硫化物的连接子。缬氨酸-瓜氨酸(Val-Cit)与自切除式间隔基苄基羟基(PAB)相连形成的可裂解的二肽连接子被广泛应用在ADC的临床开发中。然而,由于VC连接子具有较强的疏水性,若与超疏水性有效载荷组合在一起,会与抗体中较为亲水的部分一起形成易于聚集的“表面活性剂样”结构,特别是当药物与抗体比值(DARs)高于4时。在体内,ADC的聚集容易导致快速的血浆清除,疗效降低以及毒性增加,特别是对于高DAR值的ADC分子。
紫杉醇(PTX)是紫杉烷类家族的成员,在细胞分裂过程中干扰微管的组装。尽管在许多难治性实体瘤中有效,但因其水溶性较差,阻碍了其临床应用。为了克服PTX溶解度问题,已经开发了一系列亲水性更高的PTX衍生物以及前药,基于PTX的ADC也在研发中。由于PTX在高表达Trop-2的胰腺癌和三阴性乳腺癌(TNBC)的治疗中得以运用,然而,迄今为止,报道的偶联PTX的ADC未能在体内表现出令人满意的肿瘤抑制作用,也未进入临床开发阶段。
综上所述,本领域亟待开发新的稳定的连接子和有效载荷,从而得到有效靶向的ADC,以实现最高的治疗指数。
人滋养层细胞表面抗原2(Trop-2)在多种人类实体瘤中高表达,例如,乳腺癌、肺癌、结肠癌、膀胱癌和胰腺癌。然而,Trop-2在皮肤和粘膜等关键正常组织中不同水平的表达,是它作为ADC靶点是否能够成药的一个很大的挑战。IMMU-132是一种通过带有聚乙二醇的酸不稳定连接子将SN38与靶向Trop2的抗体hRS7连接形成的ADC分子。其中,拓扑异构酶Ⅰ抑制剂SN38是伊立替康的活性代谢产物,具有中等细胞毒作用。在三阴性乳腺癌(TNBC)的III期临床试验(NCT02574455)中显示了积极的结果,目前已经批准上市。另一种靶向Trop-2的ADC分子RN927C使用了毒性更大的澳瑞他汀衍生物Aur0101作为有效负载通过疏水性连接子与抗体相连,然而由于在人体内毒性过大,在I期临床试验(NCT02122146)中被终止,这一结果表明了连接子和有效载荷在以Trop-2为靶点的ADC分子的开发中的重要性。截止目前,其他的靶向Trop-2的ADC分子还在开发中,然而,ADC技术能否成功的运用于Trop-2阳性肿瘤的靶向治疗,其关键问题在于筛选出最佳的连接子和有效载荷的组合,以实现最高的治疗指数。作为优选例之一,我们利用靶向Trop-2的人源化抗体hRS7作为ADC抗体部分,筛选出了式I亲水性ADC连接子与载荷组合。
发明内容
本发明目的是提供一种含有紫杉醇类化合物作为药物分子的ADC,以及可高效地将紫杉醇类化合物与抗体进行偶联的中间体(即式I化合物)。
本发明的第一方面,提供一种式I化合物,
式中,
Z1为用于与抗体进行连接的接头部分(moiety);
m1为0-10的整数;
m2为10-50的整数;
PTX'为紫杉醇类化合物。
在另一优选例中,所述的Z1为取代或未取代的马来酰亚胺基团。
在另一优选例中,所述的Z1通过马来酰亚胺的5元环的1位、3位或4位与抗体连接。
在另一优选例中,所述的连接包括:与抗体上的巯基连接、与抗体上的ε-氨基(如赖氨酸的ε-氨基)连接、与抗体上的硒半胱氨酸进行连接、或其组合。
在另一优选例中,所述的Z1与抗体上天然存在的活性基团或人工引入的非天然活性基团发生反应所生成的化学连接结构。
在另一优选例中,所述的Z1与抗体连接后,形成选自下组的化学连接结构:
(a)与抗体上巯基与马来酰亚胺通过偶联反应,形成的化学连接结构;
(b)与抗体上巯基与二溴马来酰亚胺通过偶联反应,形成的化学连接结构;
(c)抗体上赖氨酸的ε-氨基与连接子上的活性胺基团反应,形成的化学连接结构;
(d)抗体上赖氨酸与含马来酰亚胺的衍生物进行的偶联反应,形成的化学连接结构;
(e)抗体上赖氨酸与碘乙酰胺类的偶联反应,形成的化学连接结构;
(f)抗体上赖氨酸基于亚氨基硫烷的偶联反应,形成的化学连接结构;
(g)抗体上赖氨酸基于吡啶二硫化物的偶联反应,形成的化学连接结构;
(h)与抗体上人工引入的硒半胱氨酸进行的定点偶联,形成的化学连接结构;
(i)上述的任意组合;
在另一优选例中,上式(a)和(b)中,m3为1-8(较佳地2-6)的正整数。
在另一优选例中,上式(d)和(e)中,spacer选自:-(CH2)m4-、-(CH2O)m4-,其中,m4为0-10(较佳地1-6)的整数。
在另一优选例中,所述的式I化合物为式Ia化合物:
式中,
m1为0-10的整数;
m2为10-50的整数;
PTX'为紫杉醇类化合物(即紫杉醇类化合物失去H原子后的基团)。
在另一优选例中,m1为0-8,最佳地为0、4。
在另一优选例中,m2为12-40,更佳地15-35,最佳地为24。
在另一优选例中,PTX'为紫杉醇(PTX),即
在另一优选例中,所述式I化合物的结构如式III所示:
PTX的定义如上所述。
本发明第二方面,提供一种抗体与药物偶联体(ADC),所述的抗体与药物偶联体是第一方面所述的式I化合物与抗体偶联形成的抗体与药物偶联体(ADC)。
在另一优选例中,抗体与药物偶联体具有式II所示的结构:
Ab-(L-PTX')n II
式中,
Ab表示抗体,
PTX'为紫杉醇类化合物;
n为偶联于所述抗体的所述药物的平均偶联数量;
m1和m2的定义如上所述。
在另一优选例中,n是>0整数或非整数,较佳地0.8≤n≤15,最佳为7-8。
在另一优选例中,所述的抗体选自下组:嵌合抗体、二价或双特异性分子、双抗体、三抗体和四抗体。
在另一优选例中,所述抗体可以是任意靶点的抗体,例如选自下组:
Trop-2、PD-L1、CTLA4、OX40、EpCAM、EGFR、EGFRVIII,HER2、PTPN2、VEGF、CD11a、CD19、CD20、CD22、CD25、CD30、CD33、CD40、CD56、CD64、CD70、CD73、CD74、CD79、CD105、CD138、CD174、CD205、CD227、CD326、CD340、MUC16、GPNMB、PSMA、Cripto、ED-B、TMEFF2、EphB2、Apo-2、NMP179、p16INK4A、Nectin-4、B7H3、EMA、CEA、MIB-1、GD2、APRIL受体、叶酸受体、间皮素抑制剂、MET、TM4SF1。
在另一优选例中,所述的抗体与药物偶联体中,m1为0到6的整数。
在另一优选例中,所述的抗体与药物偶联体中,m2为20、21、22、23、24、25、26的整数。
在另一优选例中,所述的抗体与药物偶联体中,m1为4,m2为24。
在另一优选例中,所述的抗体与药物偶联体中,m1为0,m2为24。
在另一优选例中,所述的抗体为hRS7。
在另一优选例中,所述的抗体与药物偶联体为hRS7-VK-PTX。
本发明第三方面,提供一种药物组合物,所述药物组合物包含:(a)如第二方面所述的抗体与药物偶联体,以及(b)药学上可接受的载体。
本发明第四方面,提供一种如第二方面所述的抗体与药物偶联体或如第三方面所述的药物组合物的用途,用于制备治疗肿瘤或免疫疾病的药物。
本发明第五方面,提供一种制备第二方面所述的抗体与药物偶联体的方法,所述方法包括步骤:
(1)提供一反应体系,所述反应体系中包括待偶联的抗体和第一方面所述的式I化合物;
(2)在所述反应体系中,将所述抗体和所述的式I化合物进行偶联反应,从而制得第二方面所述的抗体与药物偶联体。
在另一优选例中,所述反应体系的pH为6.0-8.0;较佳地,为6.0-7.0。
在另一优选例中,所述抗体与式I化合物(或紫杉醇类化合物)的摩尔比为1:1~1:20;较佳地,为1:6~1:15,更佳地,为1:8~1:12。
在另一优选例中,反应时间为1h~48h;较佳地,为3h~36h。
在另一优选例中,所述的偶联包括定点偶联和随机偶联。
在另一优选例中,所述的定点偶联包括K-Lock和C-Lock两种偶联方式。在K-Lock偶联方式中,VK-PTX'偶联于抗体序列中赖氨酸(K)残基,在C-Lock偶联方式中,VK-PTX'偶联于抗体序列中的半胱氨酸(C)残基。
本发明第六方面,提供一种式IV所示的化合物
PTX'、m1、m2、Z1的定义如上所述。
本发明第七方面,提供一种第六方面所述的式IV化合物的制备方法,包括如下步骤:
(S1)在惰性溶剂中,碱存在下,化合物1
PTX'、W1、W2的定义如上所述。
在另一优选例中,步骤(S1中)中,惰性溶剂为DMF。
在另一优选例中,步骤(S1中)中,碱为DIEA。
本发明第八方面,提供一种第一方面所述的式I化合物的用途,用于制备抗体与药物偶联体。
在另一优选例中,所述的抗体与药物偶联体具有式II所示的结构:
Ab-(L-PTX')n II
式中,
Ab表示抗体,
PTX'为紫杉醇类化合物;
n为偶联于所述抗体的所述药物的平均偶联数量;
m1和m2的定义如上所述。
在另一优选例中,n是>0整数或非整数,较佳地0.8≤n≤15。
在另一优选例中,n为4-12的整数或非整数的正数。
在另一优选例中,n为5-10的整数或非整数的正数;较佳地,为6-9的整数或非整数的正数;更佳地,为7-8的整数或非整数的正数。
在另一优选例中,所述抗体为hRS7。
在另一优选例中,所述抗体hRS7具有以下特征:
(a)所述抗体hRS7的轻链可变区的氨基酸序列包含或为如SEQ ID NO.:1所示的序列;和/或
(b)所述抗体hRS7的重链可变区的氨基酸序列包含或为如SEQ ID NO.:2所示的序列。
另一方面,提供一种治疗或预防肿瘤的方法,所述方法包括步骤:给需要的对象施用如上所述的抗体与药物偶联体或如上所述的药物组合物。
在另一优选例中,所述对象为哺乳动物,包括人。
在另一优选例中,所述抗体与药物偶联体的给药剂量为0.3-50毫克/千克;较佳地,为1-15毫克/千克;更佳地,为8-10毫克/千克。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1a显示了VC(Val-Cit-PAB)连接子、VK(Val-Lys-PAB)连接子和hRS7-VK-PTX连接子的分子结构。
图1b显示了RP-HPLC分析不同ADC分子中药物与抗体比(DAR)。
图1c显示了ADC分子抑制移植瘤的生长。
图1d显示了hRS7-VK-PTX单次静脉给药急毒实验。
图1e显示了旁观者效应验证实验。
图1f显示了共聚焦显微镜下检测抗Trop-2ADC的内吞和定位。
图2显示了hRS7-VK-MMAE和hRS7-VK-SN38的分子结构式。
图3显示了抗Trop-2-ADC的相关特性检测。
图4显示了通过流式细胞仪分析Trop-2在不同肿瘤细胞系上的表达水平。
图5显示了通过CCK-8检测了不同有效载荷对不同肿瘤细胞系的抗肿瘤功效。
图6显示了通过CCK-8检测了不同ADC分子在不同肿瘤细胞系中的抗肿瘤功效。
图7显示了ADC分子在小鼠移植瘤模型中抑制了肿瘤的生长。
图8显示了从用不同ADC分子处理的小鼠移植瘤模型中解剖的肿瘤组织的比较。
图9显示了ADC处理小鼠的体重变化。
图10显示了Trop-2-ADC的抗肿瘤活性及其“旁观者效应”。
图11显示了hRS7-VK-MMAE单次给药急毒实验。
具体实施方式
本发明人通过广泛而深入的研究,经过大量筛选,获得一种高效的、稳定性和疏水性经过优化的式I化合物,其可以用于制备靶向抗体与药物偶联体(ADC)。ADC中引入式I化合物可以解决将PTX'作为ADC有效载荷的问题。实验结果表明使用式I化合物和抗体的ADC分子在体内外均显示出较好的药效和安全性。在此基础上完成了本发明。
实验表明,本发明的式I化合物是一类新的稳定的连接子和有效载荷,尤其适合将难以形成ADC的药物分子(如PTX类化合物)与抗体进行高效偶联,从而形成稳定且有效的ADC。基于本发明中间体所制备的ADC具有靶向型,可以实现优异的治疗指数,并可显著降低ADC对正常细胞或组织的毒副作用。
术语
“DAR值”是指药物与抗体的比率(drug antibody ratio)。
本发明中“旁观者效应”是指,ADC释放的小分子使细胞发生裂解后,ADC的有效载荷从目标细胞中释放出来,然后进入周围表达或不表达目标抗原的其他细胞,就会发生“旁观者被杀伤”。当抗原在肿瘤组织中高异质性表达时,“旁观者杀伤”是必要的。
本发明中“紫杉醇类化合物”作为其他化合物的一部分时,其为紫杉醇类化合物的基团,即紫杉醇类化合物失去H原子后的基团。
活性成分
“式I化合物”、“抗体与药物偶联体中间体”、“连接子与载荷组合”“连接子和有效载荷”可互换使用。
本发明提供了式I化合物,其具有如下结构
式中,
Z1、m1、m2、PTX如上定义。
在另一优选例中,m1为0-10的整数;
PTX为紫杉醇类化合物。
在另一优选例中,m1为0-10的整数;如0-8,更佳地0-4,最佳地为0、4。
在另一优选例中,m2为10-50,如m2为12-40,更佳地15-35,最佳地为24。
优选地,m1为1-10(即1、2、3、4、5、6、7、8、9或10)的整数;
优选地,m2为15-30(即15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30)的整数;
优选地,PTX为紫杉醇类化合物(如紫杉醇、多西紫杉醇和天然亲水性紫杉烷类化合物)。
优选地,所述的式I化合物为式Ia化合物:
式中,m1、m2、PTX如上定义。
优选地,式I化合物的结构如式III所示
式中,PTX如上定义。
抗体
如本文所用,术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白,其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连,而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH),其后是多个恒定区。每条轻链的一端有可变区(VL),另一端有恒定区;轻链的恒定区与重链的第一个恒定区相对,轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用,术语“可变”表示抗体中可变区的某些部分在序列上有所不同,它形成了各种特定抗体对其特定抗原的结合和特异性。然而,可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区,它们大致上呈β-折叠构型,由形成连接环的三个CDR相连,在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I,647-669页(1991))。恒定区不直接参与抗体与抗原的结合,但是它们表现出不同的效应功能,例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列,免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白:IgA、IgD、IgE、IgG和IgM,其中一些还可进一步分成亚类(同种型),如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般,抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述,称为可变区域(CDR),将该段间隔成4个框架区域(FR),4个FR的氨基酸序列相对比较保守,不直接参与结合反应。这些CDR形成环状结构,通过其间的FR形成的β折叠在空间结构上相互靠近,重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明不仅包括完整的抗体,还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此,本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中,本发明的抗体还包括其保守性变异体,指与本发明抗体的氨基酸序列相比,有至多10个,较佳地至多8个,更佳地至多5个,最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 | 代表性的取代 | 优选的取代 |
Ala(A) | Val;Leu;Ile | Val |
Arg(R) | Lys;Gln;Asn | Lys |
Asn(N) | Gln;His;Lys;Arg | Gln |
Asp(D) | Glu | Glu |
Cys(C) | Ser | Ser |
Gln(Q) | Asn | Asn |
Glu(E) | Asp | Asp |
Gly(G) | Pro;Ala | Ala |
His(H) | Asn;Gln;Lys;Arg | Arg |
Ile(I) | Leu;Val;Met;Ala;Phe | Leu |
Leu(L) | Ile;Val;Met;Ala;Phe | Ile |
Lys(K) | Arg;Gln;Asn | Arg |
Met(M) | Leu;Phe;Ile | Leu |
Phe(F) | Leu;Val;Ile;Ala;Tyr | Leu |
Pro(P) | Ala | Ala |
Ser(S) | Thr | Thr |
Thr(T) | Ser | Ser |
Trp(W) | Tyr;Phe | Tyr |
Tyr(Y) | Trp;Phe;Thr;Ser | Phe |
Val(V) | Ile;Leu;Met;Phe;Ala | Leu |
本发明抗体或其片段的DNA分子的序列可以用常规技术,比如利用PCR扩增或基因组文库筛选等方法获得。此外,还可将轻链和重链的编码序列融合在一起,形成单链抗体。
一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。
目前,已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段,或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外,还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞,以使其能够表达蛋白质。
宿主细胞可以是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞;或是高等真核细胞,如哺乳动物细胞。
通常,在适合本发明抗体表达的条件下,培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤,如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如,单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
在另一优选例中,所述抗体为抗体hRS7。
在另一优选例中,所述抗体hRS7的轻链可变区(V-Kappa)氨基酸序列为如SEQIDNO.:1所示的氨基酸序列(DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQ)。
在另一优选例中,所述抗体hRS7的重链可变区(VH)氨基酸序列为如SEQ ID NO.:2所示的氨基酸序列(QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS)。
抗体与药物偶联体及其制备
如本文所用,术语“本发明的抗体与药物偶联体”、“本发明的抗体与药物偶联物”、“本发明的偶联体”或“本发明的ADC”可互换使用,指具有式II结构所示的抗体-药物偶联体。
本发明中,抗体与药物偶联体具有式II所示的结构:
Ab-(L-PTX')n II
其中,
Ab表示抗体,其可以如上所述的任何类的抗体(如嵌合抗体(例如,人源化鼠抗体)、二价或双特异性分子(例如,双特异性抗体)、双抗体、三抗体和四抗体);
PTX'、L、n、m1、m2的定义如上所述。
优选地,式II中,n为4-12的整数或非整数的正数,较佳地,n为5-10的整数或非整数的正数;较佳地,为6-9的整数或非整数的正数;更佳地,为7-8的整数或非整数的正数。
优选地,上述式II中,m1为0、1、2、3、4、5、6的整数;m2为20、21、22、23、24、25的整数。
优选地,上述式II中,m1为4;m2为24。
偶联方法
本发明提供了偶联方法,将式I化合物偶联到抗体上,在不改变抗体亲和性的基础上大幅提高抗体对肿瘤细胞的杀伤力。
典型的适用于本发明的偶联方式,包括C-Lock偶联方式。在C-Lock偶联方式中,药物分子偶联于抗体序列中的半胱氨酸(C)残基。
优选地,本发明的抗体与药物偶联体的制备方法如下:
吸取一定量(如0.5毫克)的抗体,加入一定量(如8倍物质当量)的三(2-羧乙基)膦(Tris(2-carboxyethyl)phosphine,TCEP),在合适的温度(如37℃)条件下反应一段时间(如2.5小时)。然后直接加入式I化合物(如15倍物质当量),进行偶联反应(如4℃过夜反应,如17小时),从而获得本发明的ADC。最后通过Amicon Ultra 4 10K超滤管把缓冲液置换成pH7.0的PBS溶液。
优选地,本发明的抗体与药物偶联体的制备方法如下:
吸取一定量(如0.5毫克)的抗体,加入一定量(如2倍物质当量)三(2-羧乙基)膦(Tris(2-carboxyethyl)phosphine,TCEP),在合适的温度(如37℃)条件下反应一段时间(如2.5小时)。反应完毕以后,通过离心超滤法把反应混合物浓缩到0.5毫升以下,并用偶联溶液(75mM NaAc,pH6.5,1mM DTPA,10%DMSO)加满,再离心浓缩到0.5毫升以下,重复三次。然后直接加入式I化合物(如8倍物质当量),进行偶联反应(如4℃过夜反应,如17小时),从而获得本发明的ADC。
优选地,本发明的抗体与药物偶联体的制备方法如下:
吸取一定量(如0.5毫克)的抗体,加入一定量(如3倍物质当量)的二硫苏糖醇(DTT),在合适的温度(如37℃)条件下反应一段时间(如2小时)。反应完毕以后,通过离心超滤法把反应混合物浓缩到0.5毫升以下,并用偶联溶液(75mM NaAc,pH6.5,1mM DTPA,10%DMSO)加满,再离心浓缩到0.5毫升以下,重复三次。然后直接加入式I化合物(如10倍物质当量),进行偶联反应(如4℃过夜反应,如17小时),从而获得本发明的ADC。
药物组合物和施用方法
本发明还提供了含有本发明ADC的药物组合物,以及使用本发明ADC治疗哺乳动物疾病的方法。优选地,所述的疾病是肿瘤,包括具有特定靶目标的肿瘤,如Trop-2靶向的肿瘤、Her2靶向的肿瘤、EGFR靶向的肿瘤。
本发明还提供了所述抗体与药物偶联体在制备抗肿瘤药物中的应用。
在本发明中,所述药物组合物包括有效量的根据本发明的ADC(作为活性成分),以及至少一种药学上可接受的载体、稀释剂或赋形剂。制备时,通常将活性成分与赋形剂混合,或用赋形剂稀释。当赋形剂起稀释剂作用时,它可采用固体、半固体或液体材料作为赋形剂、载体或活性成分的介质。因此,组合物可以是溶液剂、灭菌注射溶液等。
合适的赋形剂包括:乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水等;制剂还可包括:湿润剂、乳化剂、防腐剂(如羟基苯甲酸甲酯和丙酯)等。所述抗肿瘤药物可制成单元或多元剂型,各剂型包含为了产生所期望的疗效而计算出预定量的本发明的ADC,以及合适的药剂学赋形剂。
所述的抗肿瘤药物可以通过常规途径进行给药,包括(但并不限于):瘤内、肌内、腹膜内、静脉内、皮下、皮内、局部给药等。
使用该药物时,是将安全有效量的所述抗体与药物偶联体施用于人,其中该安全有效量的范围优选为0.5~50毫克/千克体重,更优选为1~10毫克/千克体重。当然,具体剂量还应考虑给药途径、病人健康状况等因素,这些都是在熟练医师技能范围之内的。
此外,本发明的偶联体还可与其他治疗药物联用,其中包括(但并不限于):各种细胞因子,如TNF、IFN、IL-2等;各种肿瘤化疗药物,如5-FU、氨甲喋呤等影响核酸生物合成的药物;氮芥、环磷酰胺等烷化剂类药物;阿霉素、放线菌素D等干扰转录过程阻止RNA合成的药物;长春新碱、喜树碱类等影响蛋白质合成的药物及某些激素类药物,等等。
与现有技术相比,本发明的主要有益效果包括:
(1)本发明式I化合物能有效地将紫杉醇类化合物偶联到抗体上,而常规的val-cit-PAB连接子不能。
(2)本发明式I化合物具有较好的亲水性。
(3)由本发明式I化合物得到的抗体与药物偶联体具有经优化的DAR值和更优的载荷,使得本发明ADC的治疗窗口更大。
(4)本发明提供的抗体与药物偶联体对肿瘤细胞具有显著杀灭肿瘤细胞的的活性。
(5)本发明首次开发了可与抗体(如hRS7抗体)高效偶联并稳定的含PTX'分子的ADC,所述的ADC具有协同的治疗作用,可以显著地扩大PTX'对肿瘤细胞的杀伤活性。
(6)本发明式I化合物可与各种不同抗体偶联,所形成的抗体与药物偶联体具有经优化的DAR值(如2、4、6或8或以上),从而使得本发明ADC更高的亲水性和热稳定性。
(7)本发明的抗体偶联体具有更高的药效。
(8)本发明提供的抗体与药物偶联体具有更好的药代性能以及更低的毒副作用。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
材料与方法
(a)细胞系和试剂
MDA-MB-468、BXPC-3、SKBR3、CFPAC-1、SCLC-21H、HCC1806、Capan-1,NCIH2452、MDA-MB-231、COLO205、HT29、NCI-H1688和SK-MES-1由中国上海细胞系库提供。
DMEM中添加了10%新生小牛血清(Thermo Fisher Scientific,美国马萨诸塞州沃尔瑟姆)。细胞在5%CO2的潮湿气氛中于37℃孵育。PE标记的山羊抗人IgG标记购自Biolegend(美国圣地亚哥)。人源化的Trop-2抗体hRS7是根据美国专利9931417B2制备的。Cell Counting Kit-8(CCK8)购自Dojindo Laboratories(日本东京)。Matrigel(#354234)购自BD Biosciences(美国圣何塞)。紫杉醇(PTX),MMAE和SN38由Levena Biopharma(中国苏州)合成。Anti-LAMP-1(#9091),抗Clathrin(#4796),抗GM130(#12480)和555-抗兔IgGF(ab')2片段购自Cell Signaling Technology(Trask美国莱恩·丹佛斯(Lane Danvers)。IgG H&L购自Abcam Trading(Shanghai)Company Ltd.人Trop-2重组蛋白(6xHisTag)购自Sino Biological(中国北京)。甲醛购自Polysciences,Inc.(目录号18814)。正常山羊血清#5425购自Cell Signaling Technology(美国,Trask LaneDanvers)。ProLongTMLive防褪色试剂(#P36974)和TMB底物购自Thermo Fisher ScientificInc.。TCEP(BondBreakerTM)购自Pierce(美国伊利诺伊州罗克福德)。二甲基亚砜(DMSO)和其他化学品是(Merck KGaA)的产品。
(b)ADC的制备与DAR值的检测
通过在37℃下将6摩尔当量的TCEP(1mg/mL)加入8mg/mL的含有9%蔗糖,10mM乙酸盐,0.01%Tween-20,pH 5.0的抗体溶液中进行2小时还原反应。还原后,每个抗体巯基加入20摩尔当量的接头有效载荷,并在4℃下孵育16小时以上。通过用AmiconUltracontainer(50,000MWCO,Millipore Corporation)离心(4000rpm,15分钟,Thermo Fisher ST40R TX-1)浓缩溶液混合物。ADC的浓度通过UV检测器(Nanodrop 100,Thermo Fisher ScientificInc.)在280nm吸收下测量。通过反相(RP)-HPLC和QTOF质谱仪确认最终产物的DAR值。
(c)RP-HPLC分析ADC的DAR值
RP-HPLC分析是在1260Infinity UHPLC(Agilent Technologies)上,在以下条件下进行的:1)Agilent8μm,4.6*250mm;2)流动相A:0.01%三氟乙酸水溶液(TFA);3)流动相B:含0.01%TFA的乙腈溶液;4)渐变程序:0-3min(100%A),3-25min(100%A-100%B),25-30min(100%B);5)柱温:70℃;6)进样量:15ul(1mg/mL)。ADC分子的疏水程度与偶联的有效载荷的数量成正比,并且可以通过其保留时间来反映。由于药物接头具有紫外线吸收性,因此可以根据以下表达式计算ADC中每个抗体分子的共轭药物分子的平均数:偶联的药物分子的平均数=(L0峰面积比×0+L1峰面积比×1+H0峰比×0+H1峰比×1+H2峰比×2+H3峰比×3)/100×2。
(d)SEC-HPLC分析ADC的聚集体
SEC-HPLC方法用于监测ADC的聚集和降解。在60℃孵育1小时后,使用1260HPLC系统(Agilent)在5μm,(7.8×300mm)P/N 088460的Thermo MAbPac SEC-1上分析样品。流动相:磷酸盐缓冲盐水(PBS),50mM磷酸钠和300mM氯化钠,pH 6.8。分离期间将柱保持在26℃。检测器设置在280nm。流速:0.7mL/min。样品进样量:15μl。
(e)SDS-PAGE
将样品在1x上样缓冲液中煮沸10分钟,将含有5μg的hRS7或ADC的样品通过SDS-PAGE(10%Bio-Rad Criterion Tris-HCl凝胶)分离,然后用考马斯亮蓝染色5分钟,并用BioRad ChemiDoc MP检测。
(f)检测肿瘤细胞Trop-2表达水平
将细胞与5μg/mL的hRS7在冰冷的染色培养基中在冰上孵育30分钟,并用冰冷的染色培养基洗涤两次以去除未结合的抗体。然后将细胞在冰冷的染色培养基中用PE山羊抗人IgG(5μg/mL)染色30分钟,并洗涤两次。通过流式细胞术检查细胞。
(g)ELISA检测抗原与抗体的结合
96孔免疫板在4℃过夜涂2μg/mL带有组氨酸标签的抗原蛋白(Sino BiologicalInc.)。洗涤板,用1%酪蛋白封闭,然后在室温下与10nM抗体或ADC一起孵育1小时。洗涤后,加入抗人κ-HRP(Biolegend San Diego,美国)并孵育1小时。洗涤后,加入TMB底物,并用SpectraMax M5e(Molecular Devices)酶标仪在450nm处检测。
(h)ADC内吞速率的检测
为了评估ADC的内吞速率,将MDA-MB-231或CFPAC-1细胞(1×106)与5μg/mLADC样品在染色培养基中在冰上孵育30分钟,并洗涤两次,然后将其放置在新鲜的完全培养基中。37℃加湿室初始化内在化。孵育结束时,将细胞用冰冷的染色培养基洗涤两次,然后用山羊抗人IgG-PE染色。通过流式细胞仪分析细胞以检测剩余表面ADC的水平。
(i)共聚焦显微镜检测ADC的内吞和胞内定位
将SKBR3细胞与5μg/mL hRS7-VK-PTX或hRS7-VK-SN38在24孔板(1×104细胞/孔)中,于5%CO2,37℃孵箱中孵育45或90min。将细胞在PBS中洗涤一次,固定并透化。洗涤后,将细胞用488山羊抗人IgG(H&L)或555抗兔IgG F(ab')2片段染色。核DNA用4',6-二mid基-2-苯基吲哚(DAPI)染色。用Olympus BK71共聚焦显微镜记录免疫荧光。
(j)细胞活力测定
使用细胞计数试剂盒8(CCK-8)测定细胞活力。将细胞以3000-10000细胞/孔接种到96孔板中。孵育过夜后,以不同浓度添加药物。将板在37℃,5%CO2的潮湿箱中孵育96小时。加入CCK8试剂并放回培养箱中,直到未处理的对照细胞在450nm处的吸光度大于1.0。以相对于未处理细胞的生长百分比来测量生长抑制。由两次或三次测定的平均值产生剂量反应曲线,并使用Prism GraphPad软件计算IC50值。
(k)小鼠体内药效的检测
五周龄的雌性无胸腺BALB/c nu/nu小鼠购自Charles River Company(中国上海),每组随机分为5-6只小鼠。按照中国科学院上海药物研究所机构动物护理和使用委员会(IACUC)的要求,批准并执行了动物处理和程序。所有模型均采用在小鼠侧面皮下接种。BXPC-3,HCC1806和NCIH1688细胞是通过注射混匀在Matrigel基质中的5×106个细胞而建立的。通过注射悬浮在Matrigel基质中的1×107细胞来建立COLO205细胞。肿瘤体积达到约200mm3后,将荷瘤小鼠根据肿瘤体积随机分为治疗组和对照组。每种药物经腹膜内给药。以3或10mg/kg(10mL/kg)的剂量注射到小鼠身上。肿瘤体积定义为长×1/2×宽×宽。通过下式计算抑制百分比:(1-治疗组平均肿瘤体积/对照组平均肿瘤体积)×100%。通过监测体重减轻来评估与不同治疗组相关的毒性。
(l)剂量耐受性测定
剂量耐受性测定以BALB/cmice(每剂3只小鼠)进行。药物以单次静脉内给药。
hRS7-VK-PTX的注射剂量分别为60、80和100mg/kg,hRS7-VK-MMAE的注射剂量为60和80mg/kg。通过观察小鼠行为,体重减轻和存活来评估毒性。体重变化率(%)计算为:(体重-治疗前体重)/(治疗前体重)×100%。
(m)统计分析
Prism GraphPad版本6用于统计分析。结果显示为平均值±SD。对照组和实验组之间的统计学差异通过学生t检验计算,P<0.05被认为有显著差异。
实施例1
抗体的制备
在本实施例中,表达与纯化hRS7抗体,从而获得hRS7抗体。方法如下:
hRS7轻链和重链的氨基酸序列来自美国专利9931417B2,然后通过Vector NTI软件逆转录为cDNA序列。合成DNA序列,并将其亚克隆到pcDNA3.1载体中,并在大肠杆菌中扩增。通过PEI将纯化的质粒转染到HEK293细胞中。然后将细胞悬浮在FreeStyleTM293表达培养基中进行培养。培养5天后,收集细胞培养上清液,并通过protein A亲和纯化抗体。
实施例2
化合物VK-PTX的制备
化合物VK-PTX的结构如下所示:
将紫杉醇与VK连接子进行反应,从而形成化合物VK-PTX,即
Mal-peg4-Val-Lys(peg24)-PAB-紫杉醇(VK-PTX),方法如下:
步骤1:化合物3(Fmoc-Val-Lys(Trt)-PAB-PTX)的合成
将Fmoc-Val-Lys(Trt)-PAB-PNP(购买自Levena Biopharma,美国)和PTX(购买自Levena Biopharma,美国)溶解于无水DMF,并在溶液中加入DIEA,混合物在室温22℃下搅拌反应2小时,并通过反向HPLC直接纯化出目的产物Fmoc-Val-Lys(Trt)-PAB-PTX,冷冻干燥后形成白色粉末化合物3。
步骤2:化合物4(Fmoc-Val-Lys-PAB-PTX TFA盐)的合成
将步骤1得到的化合物3溶于10%TFA/DCM溶液并在室温下搅拌20分钟,混合物反应后通过减压浓缩和反相HPLC纯化,得到化合物4,Fmoc-Val-Lys-PAB-PTX TFA盐。
步骤3:化合物5的合成
将步骤2得到的化合物4,m-PEG24酸和HATU溶于DMF,加入DIEA,并于室温下搅拌30分钟,获得产物5a。
将二异丙胺加入到反应混合物中,室温搅拌3小时,混合物反应后进行浓缩和反向HPLC纯化,在冻干后获得蜡状的化合物5。
步骤4:化合物6(Mal-peg4-val-Lys(m-PEG24)-PAB-PTX)的合成
将步骤3得到的化合物5、m-PEG4-酸和HATU溶于DMF,加入DIEA,并搅拌20分钟。之后通过反向HPLC纯化及冷冻干燥,最终获得所需化合物6,Mal-peg4-val-Lys(m-PEG24)-PAB-PTX(VK-PTX)。
实施例3 VK-MMAE、VK-SN38的制备
采用类似方法,制得化合物VK-SN38、VK-MMAE。
(1)VK-SN38的制备
步骤1:化合物3的合成
将Fmoc-Val-Lys(Trt)-PAB-PNP(购买自Levena Biopharma,美国)和SN38(购买自Levena Biopharma,美国)溶解于无水DMF,并在溶液中加入DIEA。混合物在室温22℃下搅拌反应2小时,并通过反向HPLC直接纯化出目的产物Fmoc-Val-Lys(Trt)-PAB-SN38,冷冻干燥后形成白色粉末化合物3。
步骤2:化合物4的合成
将化合物3溶于10%TFA/DCM溶液并在室温下搅拌20分钟,混合物反应后通过减压浓缩和反相HPLC纯化,得到化合物4,Fmoc-Val-Lys-PAB-SN38TFA盐。
步骤3:化合物5的合成
将化合物4、m-PEG24酸和HATU溶于DMF,加入DIEA,并于室温下搅拌30分钟,获得产物5a。
将二异丙胺加入到反应混合物中,室温搅拌3小时,混合物反应后进行浓缩和反向HPLC纯化,在冻干后获得蜡状的化合物5。
步骤4:化合物6的合成
将化合物4、m-PEG24酸和HAT
将化合物5,m-PEG4-酸和HATU溶于DMF,加入DIEA,并搅拌20分钟。之后通过反向HPLC纯化及冷冻干燥,最终获得所需化合物6:Mal-peg4-val-Lys(m-PEG24)-PAB-SN38(VK-SN38)。
(2)VK-MMAE的合成
步骤1:化合物3的制备
将Fmoc-Val-Lys(Trt)-PAB-PNP(购买自Levena Biopharma,美国)和MMAE(购买自Levena Biopharma,美国)溶解于无水DMF,并在溶液中加入DIEA。混合物在室温22℃下搅拌反应2小时,并通过反向HPLC直接纯化出目的产物Fmoc-Val-Lys(Trt)-PAB-MMAE,冷冻干燥后形成白色粉末化合物3。
步骤2:化合物4的制备
将化合物3溶于10%TFA/DCM溶液并在室温下搅拌20分钟,混合物反应后通过减压浓缩和反相HPLC纯化,得到化合物4,Fmoc-Val-Lys-PAB-MMAE TFA盐。
步骤3:化合物5的制备
将化合物4,m-PEG24酸和HATU溶于DMF,加入DIEA,并于室温下搅拌30分钟,获得产物5a。
将二异丙胺加入到反应混合物中,室温搅拌3小时,混合物反应后进行浓缩和反向HPLC纯化,在冻干后获得蜡状的化合物5。
步骤4:化合物6的制备
将化合物5、m-PEG4-酸和HATU溶于DMF,加入DIEA,并搅拌20分钟。之后通过反向HPLC纯化及冷冻干燥,最终获得所需化合物6:Mal-peg4-val-Lys(m-PEG24)-PAB-MMAE。
实施例4
ADC的制备
通过偶联反应,分别将实施例2和3中制备的VK-PTX、VK-MMAE、VK-SN38与实施例1中制备的抗体进行偶联,形成相应的ADC。
通过VK连接子,成功制备了稳定的DAR值为8的以PTX为有效载荷的ADC分子,命名为hRS7-VK-PTX(图1a和b)。
为了进行比较,同时制备了有效载荷为MMAE和SN38的DAR值为8的Trop-2-ADC,即hRS7-VK-MMAE和hRS7-VK-SN38(图1b,图2)。
在还原条件下,DAR值为0至8的hRS7-VK-MMAE的SDS-PAGE如图3a所示。其中,MMAE由于毒性太强,不能单独作为药物用于肿瘤治疗,但已作为ADC的有效载荷得到广泛应用;SN38是上述IMMU-132中使用的中等毒性的有效载荷。MMAE和SN38都具有一定的疏水性,但不如PTX疏水。用VK连接子制备出的三种Trop2-ADC即使在60℃下孵育1小时(图3c、d、e),都没有显示出严重的聚集和降解,表明连接子的聚乙二醇化可以显著提高ADC分子的疏水性和稳定性。
DAR值为8的不同ADC分子与人Trop-2蛋白(6xHis标签)的亲和力如图3b所示。
对比例1
对照ADC分子的制备
通过疏水性VC连接子(图1a),制备一个靶向Trop-2的hRS7-VC-PTX分子。然而,该分子在制备过程中完全从溶液中沉淀出来,因此无法评估其治疗能力和安全性。
因此,可以看出将疏水性连接子与超疏水性有效载荷结合会损害ADC分子的稳定性,从而导致聚集体的增加和沉淀。
实施例5
hRS7-VK-PTX的药效研究
在本实施例中PBS中的癌细胞(1×106细胞/mL)在4℃与5μg/mL hRS7孵育30分钟,人IgG1用作同种型对照,结果如图4所示。
通过CCK-8检测了不同有效载荷对不同肿瘤细胞系的抗肿瘤功效,结果如图5所示。
通过CCK-8检测了不同ADC分子在不同肿瘤细胞系中的抗肿瘤功效,如图6所示。
在Trop-2不同表达水平的肿瘤细胞系中,单独的PTX分子的细胞毒性弱于MMAE,但与SN38相近(图4、图5、图6)。然而,制备成ADC后,在Trop-2中低表达的肿瘤细胞系中,hRS7-VK-PTX比hRS7-VK-MMAE和hRS7-VK-SN38更有效,包括COLO205、MDA-MB-231、SK-MES-1、Capan-1、NCIH2452(见表1和表2)。
此外,hRS7-VK-PTX在PTX耐药的肿瘤细胞系中也显示出较好的抑制效果,包括SKBR3、CFPAC-1、MDA-MB-231和COLO205(见表1和表2)。
表1:比较hRS7-VK-PTX、hRS7-VK-MMAE和hRS7-VK-SN38在Trop-2不同表达水平的肿瘤细胞系中的药效
表2有效载荷PTX、MMAE和SN38对具有不同Trop-2表达水平的癌细胞的抑制效力
这些结果证明,在以PTX为有效载荷的ADC中使用VK连接子显著增强了PTX在体外实验中对肿瘤细胞的抑制。因此,以PTX为有效载荷的Trop-2-ADC在Trop-2中低表达的细胞系中显示出了较好的体外药效,且在一定程度上克服了PTX单药的耐药性。
实施例6 hRS7-VK-PTX的内化速率研究
ADC的内化速率可能会影响其对肿瘤细胞的抑制效率。有趣的是,hRS7-VK-PTX在Trop-2表达的细胞中的的内化速率比hRS7-VK-MMAE和hRS7-VK-SN38快(图1g)。
将SKBR3细胞与ADC分子在37℃下孵育45或90分钟。通过在培养基中洗涤细胞去除未结合的ADC,然后固定并通透。用各自的抗体和与结合的二级抗体(红色)检测到标记蛋白LAMP-1、GM130和网格蛋白。用标记的二抗(绿色)检测到Anti-Trop-2ADC。DAPI用于染色核DNA(蓝色)。共聚焦结果显示,hRS7-VK-PTX分子与溶酶体和网格蛋白重链共定位,表明其通过网格蛋白介导的内吞作用而被内化并运入溶酶体区室。相比之下,hRS7-VK-SN38没有发生明显的内化(图1f)。
这些结果表明,hRS7-VK-PTX可以触发更快的内化速率,这为较高的细胞杀伤效力提高支持。
实施例7 hRS7-VK-PTX在体内对Trop-2特异性表达的肿瘤细胞的抑制效果以及“旁观者效应”
I.hRS7-VK-PTX在体内对Trop-2特异性表达的肿瘤细胞的抑制效果
在第7、11、15和19天,用ADC或对照处理接种COLO205细胞的小鼠(每组n=6),每周两次或三次测量肿瘤体积。两尾t检验用于评估治疗组和对照组之间的统计学显着性。***P<0.001,将hRS7-VK-PTX与hRS7进行比较;p<0.001,将hRS7-VK-PTX与hRS7-VK-SN38进行比较,数据=平均值±SD,结果如图1c。
在第5、13和21天,用ADC或裸抗体处理接种BXPC-3细胞的小鼠(每组n=5),每周两次或三次测量肿瘤。在第25天,使用两尾t检验来评估治疗组和对照组之间的统计学显着性。***P<0.001,与hRS7治疗组相比;*P<0.05,将hRS7-VK-PTX与PTX治疗组进行比较;与hRS7治疗组相比,P<0.001,数据=平均值±SD,结果如图7a所示。
在第7、10和14天治疗接种了HCC1806细胞的小鼠(每组n=6)。每周两次或三次测量肿瘤。在20天时,使用两尾t检验来评估治疗组和对照组之间的统计学显着性。*P<0.05,将hRS7-VK-PTX与hRS7-VK-SN38治疗组进行比较,数据=平均值±SD,结果如图7b所示。
从用不同ADC分子处理的小鼠移植瘤模型中解剖的肿瘤组织的比较。ADC处理抑制了BXPC3(a)或COLO205细胞衍生(b)小鼠异种移植模型中的肿瘤生长。在第25天(图8a)或第22天(图8b)收集肿瘤组织,“—”表示完全抑制。
通过检测hRS7-VK-PTX在小鼠异种移植瘤模型中的治疗潜力,发现hRS7-VK-PTX在抑制BXPC-3、COLO205和HCC1806肿瘤细胞的移植瘤模型中比hRS7-VK-SN38更有效(图1c和图7、图8)。
此外,在抑制BxPC-3移植瘤模型中,3mg/kg的hRS7-VK-PTX比10mg/kg的PTX更有效(P=0.0248)(图7a)。
在HCC1806细胞移植瘤模型中,hRS7-VK-PTX在30mg/kg(1.3mg/kg PTX当量)剂量下的抑瘤效果与10mg/kg的PTX相当(图7b)。
这些结果表明当PTX利用亲水性连接子制备成ADC的形式后,能够显著改善PTX的体内抗肿瘤作用。
II.hRS7-VK-PTX在小鼠移植瘤模型中的“旁观者效应”
将Trop-2阳性肿瘤细胞BXPC-3与Trop-2阴性肿瘤细胞NCIH1688的混合物或单独的NCIH1688细胞接种到BALB/c-nu/nu小鼠中(图1e,右图),检测hRS7-VK-PTX在小鼠移植瘤模型中的“旁观者效应”。
免疫组织化学染色表明了混合细胞来源的移植瘤中Trop-2表达的异质性和NCI-H1688细胞单独来源的移植瘤中Trop-2表达的缺失(图10b和c)。尽管PTX作为小分子的药效远不如MMAE,使用hRS7-VK-PTX(10mg/kg)和hRS7-VK-MMAE(3mg/kg)进行治疗均显著抑制了异质表达Trop-2的移植瘤生长,且肿瘤生长抑制指数(TGI)分别为99.5%和91.7%(图1e和图10a)。
此外,hRS7-VK-MMAE对不表达Trop-2的单独接种NCIH1688细胞移植瘤也产生了杀伤作用,而hRS7-VK-PTX没有(图1e和图10a)。
上述结果表明,hRS7-VK-PTX具有“旁观者效应”,并且还表明hRS7-VK-PTX的细胞杀伤作用比hRS7-VK-MMAE更具有Trop-2特异性,这将为临床应用上的安全性带来可靠的证据。此外,hRS7-VK-PTX(3或10mg/kg)不会引起经治疗的小鼠的不良反应或体重减轻(图9),与hRS7-VK-MMAE(3mg/kg),hRS7-VK-SN38(3或10mg/kg)或单独使用PTX的治疗组形成鲜明对比。
实施例8 hRS7-VK-PTX的耐受性研究
通过在小鼠单次静脉给药的急毒实验研究了相对较高的剂量的hRS7-VK-PTX在BALB/c小鼠中的安全性。尽管由于有限的试剂供应量,未能达到hRS7-VK-PTX的最大耐受剂量(MTD),但与安慰剂相比,在单次100mg/kg剂量下,其没有显示出明显的毒性或体重减轻的迹象(图1d)。但是与hRS7-VK-MMAE相比,仅60mg/kg的hRS7-VK-MMAE即会在小鼠中引起严重不良反应以及超过15%的体重减轻(图11)。
因此,hRS7-VK-PTX具有比hRS7-VK-MMAE更好的安全性基及耐受性。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
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<110> 达石药业(广东)有限公司
<120> 用于抗体与药物偶联体(ADC)制备的中间体及其制备方法和应用
<130> P2020-1184
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Claims (10)
3.一种抗体与药物偶联体(ADC),其特征在于,所述的抗体与药物偶联体是权利要求1所述的式I化合物与抗体偶联形成的抗体与药物偶联体(ADC)。
5.如权利要求3所述的抗体与药物偶联体,其特征在于,所述的抗体选自下组:嵌合抗体、二价或双特异性分子、双抗体、三抗体和四抗体。
6.一种药物组合物,其特征在于,所述药物组合物包含:(a)如权利要求3所述的抗体与药物偶联体,以及(b)药学上可接受的载体。
7.如权利要求3所述的抗体与药物偶联体或如权利要求6所述的药物组合物的用途,其特征在于,用于制备治疗肿瘤或免疫疾病的药物。
8.一种制备权利要求3所述的抗体与药物偶联体的方法,其特征在于,所述方法包括步骤:
(1)提供一反应体系,所述反应体系中包括待偶联的抗体和权利要求1所述的式I化合物;
(2)在所述反应体系中,将所述抗体和所述的式I化合物进行偶联反应,从而制得权利要求3所述的抗体与药物偶联体。
10.如权利要求1所述的式I化合物的用途,其特征在于,用于制备抗体与药物偶联体。
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