ARMOZX (Anti-PD-1 mAb ) 阿沐珠(人源化单抗hAB21 or STW204 单抗注射液)

 

Overview

ARMOZX阿沐珠(抗PD-1人源化单抗hAB21 or STW204 单抗注射液)是思坦维公司自主研发的、具全球自主知识产权的(已申请国际PCT专利)、全新的抗PD-1单抗(人源化IgG4/kappa型, 抗体代号:hAB21)。 ARMOZX阿沐珠(hAB21单抗)特异与人PD-1蛋白的胞外区高亲和力结合,并阻断PD-1与其配体(PD-L1,PD-L2)的结合。体外研究表明ARMOZX阿沐珠(hAB21单抗)结合PD-1抗原的位点既不同于Nivolumab (商品名Opdivo,美国Bristol-Myers Squibb公司的抗PD-1 单抗药物),也不同于Pembrolizumab (商品名Keytruda, 美国Merck 公司的抗PD-1 单抗药物)。ARMOZX阿沐珠(hAB21单抗)体外可完全阻断Nivolumab或Pembrolizumab与PD-1的结合;而Nivolumab或Pembrolizumab只可部分阻断hAB21单抗与PD-1的结合。 hAB21 单抗结合PD-1的表位或区域更为广范,其结合位点覆盖过Nivolumab或Pembrolizumab的结合位点。在人源PD-1 knock-in小鼠皮下肿瘤模型测试中,ARMOZX阿沐珠(hAb21单抗)显示出不亚于Nivolumab的抗肿瘤活性。


ARMOZX阿沐珠注射液(STW204 单抗注射液)单药及与化疗药物或抗-VEGF单抗联合治疗晚期恶性实体肿瘤的临床研究批件申请已于2018年初递交至CFDA,目前正在CDE技术审评中。

 

1.1 Background: PD-1/PD-L1 Pathway and Immunotherapy

肿瘤的发生发展与机体免疫功能状态息息相关:体内免疫系统在正常状态下发挥着监控(Immune surveillance)变异的肿瘤细胞增长及抑制肿瘤发生发展的作用;免疫系统如出现功能抑制或低下时,肿瘤则出现免疫逃逸。

 

自2011年以来,免疫疗法(Immunotherapy)在肿瘤临床治疗及研究中取得了一系列具革命性变化(revolutionizing cancer treatment)的重大突破[1-3];其中靶向CTLA-4, PD-1/PD-L1 等checkpoint inhibitors的单抗药物在肿瘤免疫治疗中最为成功及令人鼓舞与注目[4-7]

 

PD-1 基因是由日本科学家Tasuku Honjo及其同事在1992年发现与克隆的,其胞外区有1个IgV样区,与CTLA-4有23%的同源性[8]。 PD-1主要表达于活化的T淋巴细胞、 B淋巴细胞、单核细胞等免疫细胞上[9]。 PD-1的受体或配体有两个:分别为PD-L1[10],又称B7-H1[11]和PD-L2 [12],又称B7-DC [13]。PD-L1及PD-L2主要表达在肿瘤等靶细胞或抗原提呈细胞上[14] 体内如给予抗PD-1/PD-L1 抗体,可激活淋巴细胞, 达到免疫杀伤肿瘤细胞, 甚至彻底消灭与排斥肿瘤的良好效果[15-18]

 

国际药企如Bristol-Myers Squibb /Medarex,Merck,Roche/Genentech等公司早在2003年前后就启动了抗PD-1/PD-L1的抗体新药研制。其中由Bristol-Myers Squibb /Medarex和日本小野制药公司共同开发的抗PD-1单抗Opdivo (通用名Nivolumab)于2014年7月率先在日本获批准上市用于治疗晚期黑色素瘤,成为全球首个批准上市的PD-1抑制剂药物。其后, Opdivo (Nivolumab)于2014年12月首次获美国FDA批准上市用于一线治疗对其它药物没有缓解的手术不可切除的或转移性黑色素瘤患者。目前Opdivo的适应症还包括晚期或转移性非小细胞肺癌(NSCLC)、晚期肾细胞癌(RCC)、头颈部鳞状细胞癌(HNSCC)、霍奇金淋巴瘤等。

 

Opdivo (Nivolumab) 及美国Merck 公司的Keytruda (Pembrolizumab)等抗PD-1单抗药物,在其各自的早期临床试验中均表现有抑制肿瘤生长、甚至彻底消灭与排斥肿瘤及延长患者生存期的良好疗效 [15-18]

 

Opdivo(Nivolumab)及Keytruda (Pembrolizumab)虽已在肿瘤治疗领域带来革命性变化,但其在临床上也还有不少缺陷与不足,这些缺陷与不足至少包括以下几方面:

 

1) Opdivo及Keytruda目前临床上还只获批用于治疗晚期黑素瘤、肾癌、非小细胞肺癌、头颈部鳞状细胞癌、膀胱癌等少数瘤种,对其他常见及多发恶性肿瘤临床治疗上的安全性及有效性还有待研究与确证。

 

2) 对于已获批肿瘤,Opdivo及Keytruda的治疗有效率一般也大多仅在15-30%之间,患者中位PFS也仅有几个月。 这意味着对大多数肿瘤患者来说,即使接受了现有的抗PD-1单抗药物,也并不能确保可以获益或显著延长其生存时间。

 

3)Opdivo及Keytruda的长期使用可导致患者发生包括如免疫介导性肺炎(Immune-mediated pneumonitis)等免疫相关不良事件(immune-related adverse events,irAE);这些免疫相关不良事件还涉及皮肤、胃肠道、肝脏、内分泌等其他多个组织器官[19]

 

要克服上述这些缺陷与不足,除需加强对已有的抗PD-1单抗药物如Opdivo及Keytruda药物的具体靶向位点(epitope)、作用机制、药物不良反应产生的原因等作进一步的基础及临床研究之外,临床上还需推出具独特抗原结合区域或结合位点(epitop)且体内外生物活性/抗肿瘤疗效更优良的、全新的抗PD-1的单抗药物,以克服的现有PD-1单抗药物的缺陷与不足,并不断满足国内外众多肿瘤患者日益增长的需求。

 

这些新的抗PD-1单抗,一方面有望开发成为单独使用的新型免疫功能增强剂或抗肿瘤药物制剂;另一方面也可与目前已上市的PD-1/PD-L1单抗或其他类型药物如抗血管增生(抗-VEGF单抗)药物联合或序贯使用,达到进一步增强机体免疫功能及提高抗肿瘤临床疗效的效果。如最近,Roche/Genentech公司的一项在肾癌临床研究中就证明了其抗PD-L1单抗药物Atezolizumab (商品名Tecentriq)与抗-VEGF单抗药物Bevacizumab(商品名Avastin)上联合治疗,肿瘤区域的 CD8+ T 淋巴细胞数量显著增多,the combination exhibited a response rate of 40%, which is greater than either single agent alone.[20]


1.2 鼠源抗PD-1单抗Ab21的获得及其基因工程人源化改造 

hAb21单抗是思坦维公司自主研发的针对人PD-1的、全新的人源化单克隆抗体(IgG4/kappa 型)。 该人源化单抗的前身为鼠源抗PD-1单抗Ab21。鼠源Ab21单抗则来源于由人PD-1蛋白免疫后小鼠脾细胞与骨髓瘤细胞经PEG融合与HAT药物筛选获得的一代号为Ab21的小鼠杂交瘤细胞株。

 

体外包括ELISA,flow-cytometry等研究结果显示:与Nivolumab, Keytruda(MK3475)一样,鼠源Ab21单抗及其人源化hAB21单抗均可特异与PD-1抗原结合(Fig.1)。


Fig.1  Anti-PD1 mAbs(hAB21, Nivolumab, Keytruda-MK3475) or anti-VEGF mAb (hPV19) were added into 96-well ELISA plates pre-coated with PD-1-Fc or other related protein as indicated; the bound mAbs were detected by HRP-goat anti-human IgG-Fab antibodies and OPD.

 

体外竞争ELISA等研究结果显示:鼠源Ab21单抗及其人源化hAB21单抗不但可竞争阻断PD-1受体(PD-L1及PD-L2)与PD-1的结合(Fig.2A);而且也可竞争阻断Nivolumab及Keytruda (MK3475) 单抗与PD-1的的结合(Fig.2B


Fig. 2  Various amount of anti-PD-1 mAbs(mAB21, Keytruda-MK3475)or anti-VEGF mAb (hPV19) were co-incubated with fixed amount of biotin-PDL1-Fc protein (A) or biotin-MK3475(B) and then transferred into 96-well ELISA plates pre-coated with PD-1 protein; bound biotin-protein or mAb were then detected by adding Avidin-HRP and OPD.


1.3  The heavy-chain and light-chain variable region of ARMOZX (hAb21has novel amino acid sequences, and hAb21 recognizes and binds to PD-1 antigen at a unique site, which is different from that of Nivolumab (Opdivo) or PembrolizumabKeytruda

Fig. 3  Various amount of anti-PD-1 mAbs(hAB21, Nivolumab, Keytruda-MK3475)or anti-VEGF mAb (hPV19) were co-incubated with fixed amount of biotin-MK3475 (A), biotin-Nivolumab(B) or biotin-hAB21(C) and then transferred into 96-well ELISA plates pre-coated with PD-1 protein; bound biotin-mAbs were then detected by adding Avidin-HRP and OPD.

根据体外研究结果及文献报道(Tan S et al. Nature Comm. 2017, 8:14369), ARMOZX(hAb21)结合PD-1 抗原的表位位于Nivolumab or Pembrolizumab之间(如下图)


Original figure: from Tan S et al. Nature Comm. 2017, 8:14369


1.4  In-vitro IHC analysis data demonstrated that ARMOZXhAb21specifically binding to normal human lymphoid organs and tissues such as spleen, lymph nodes and thymus

Fig. 4  In-vitro IHC analysis of normal human spleen tissue


如上图所示:正常人体冰冻组织经生物素标记的hAB21抗体染色后,免疫相关淋巴细胞/组织(包括脾脏、淋巴结和胸腺等)等可见阳性染色。而同型抗体对照组和阴性对照组均未见阳性染色。该研究结果也与文献报道一致,如Agata等的研究表明PD-1抗原主要表达于激活的T细胞和B淋巴细胞表面[9]


1.5      In-vivo anti-tumor activity: 在人源PD-1 knock-in小鼠皮下肿瘤模型测试中,ARMOZX阿沐珠(hAb21单抗)显示出不亚于Opdivo的抗肿瘤活性 (Fig.5)

Fig.5   MC38 tumor cells were inoculated in human PD-1 knock-in mice, when tumors reached at 60mm3,

mice were separated into different groups and received hAB21 or Nivolumab (i.p) twice/week for two weeks

 

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