Li Xinyang, Wang Meiniang, Zhang Xinhua, Liu Chuxin, Xiang Haitao, Huang Mi, Ma Yingying, Gao Xiaoyan, Jiang Lin, Liu Xiaopan, Li Bo, Hou Yong, Zhang Xiuqing, Yang Shuang, Yang Naibo
BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, 518083, China.
BGI-Shenzhen, Shenzhen, 518083, China.
Clin Transl Med. 2020 Feb 13;9(1):16. doi: 10.1186/s40169-020-0265-2.
The advent of proprotein convertase subtilisin/kexin type 9 (PCSK9)-inhibiting drugs have provided an effective, but extremely expensive treatment for the management of low density lipoprotein (LDL). Our aim was to explore a cost-effective application of camelid anti-PCSK9 single domain antibodies (sdAbs), which are high variable regions of the camelid heavy chain antibodies (VHHs), as a human PCSK9 (hPCSK9) inhibitor. One female llama was immunized with hPCSK9. Screening of high affinity anti-PCSK9 VHHs was carried out based on surface plasmon resonance (SPR) technology. We reported a lysate kinetic analysis method improving the screening efficiency. To increase the serum half-life and targeting properties, the constant region fragment of the human immunoglobulin gamma sub-type 4 (IgG4 Fc) was incorporated to form a novel llama-human chimeric molecule (VHH-hFc).
The PCSK9 inhibiting effects of the VHH proteins were analyzed in two human liver hepatocellular cells (HepG2 and Huh7) and in the hPCSK9 transgenic Sprague-Dawley (SD) rat model. The hPCSK9 antagonistic potency of the bivalent VHH-hFc exceeded the monovalent VHH (P < 0.001) in hepatocarcinoma cells. Furthermore, the llama-human chimeric VHH-Fc protein had a similar reduction (~ 40%) of the LDL-c and total cholesterol when compared to the approved evolocumab in transgenic SD rat model, but with low cost. More surprisingly, the chimeric heavy chain antibodies could be persevered for 3 months at room temperature with little loss of the affinity.
Due to the high yield and low cost of Pichia pastoris, lipid-lowering effect and strong stability, the llama-human chimeric antibody (VHH-Fc) offers a potent therapeutic candidate for the control of the serum lipid level.
前蛋白转化酶枯草溶菌素/kexin 9型（PCSK9）抑制药物的出现为低密度脂蛋白（LDL）的管理提供了一种有效但极其昂贵的治疗方法。我们的目的是探索骆驼科动物抗PCSK9单域抗体（sdAbs）作为人PCSK9（hPCSK9）抑制剂的经济有效应用，骆驼科动物抗PCSK9单域抗体是骆驼科动物重链抗体（VHHs）的高变区。用hPCSK9免疫一只雌性美洲驼。基于表面等离子体共振（SPR）技术进行高亲和力抗PCSK9 VHHs的筛选。我们报道了一种提高筛选效率的裂解物动力学分析方法。为了延长血清半衰期并改善靶向特性，将人免疫球蛋白γ亚类4（IgG4 Fc）的恒定区片段掺入以形成一种新型的骆驼科动物-人嵌合分子（VHH-hFc）。
在两个人类肝肝细胞（HepG2和Huh7）以及hPCSK9转基因Sprague-Dawley（SD）大鼠模型中分析了VHH蛋白的PCSK9抑制作用。在肝癌细胞中，二价VHH-hFc的hPCSK9拮抗效力超过单价VHH（P < 0.001）。此外，与转基因SD大鼠模型中已获批的依洛尤单抗相比，骆驼科动物-人嵌合VHH-Fc蛋白使LDL-c和总胆固醇降低程度相似（约40%），但成本较低。更令人惊讶的是，嵌合重链抗体在室温下可保存3个月，亲和力几乎没有损失。
由于毕赤酵母产量高、成本低，具有降脂作用且稳定性强，骆驼科动物-人嵌合抗体（VHH-Fc）为控制血脂水平提供了一种有效的治疗候选药物。
