In Vitro ELISA and Cell-Based Assays Confirm the Low Immunogenicity of VNAR Therapeutic Constructs in a Mouse Model of Human RA: An Encouraging Milestone to Further Clinical Drug Development.
In Vitro ELISA and Cell-Based Assays Confirm the Low Immunogenicity of VNAR Therapeutic Constructs in a Mouse Model of Human RA: An Encouraging Milestone to Further Clinical Drug Development.
复制标题
体外 ELISA 和基于细胞的测定证实 VNAR 治疗结构在人类 RA 小鼠模型中的低免疫原性：进一步临床药物开发的一个令人鼓舞的里程碑。
DOI：
10.1155/2020/7283239
复制
发表时间：
2020
影响因子：
4.1
通讯作者：
Ubah OC
中科院分区：
文献类型：
--
作者：
Ubah OC
抗药物抗体（ADAs）是生物治疗药物的特异性抗体，与血清药物水平降低和治疗反应受损有关。ADA对畅销抗hTNF α单克隆抗体的生物利用度和临床疗效的影响是公认的，特别是对于阿达木单抗和英夫利昔单抗治疗，经典免疫球蛋白抗体药物的大而复杂的分子结构部分负责患者的免疫原性。本研究的最初目的是开发固相酶联免疫吸附测定法（ELISA）和基于免疫球蛋白的体外细胞法，以准确检测ADA并评估其对两种新型非免疫球蛋白VNAR融合抗hTNF α生物制剂（Quad - X™和D1 - NDure™- C4）和阿达木单抗品牌Humira®的临床前体内疗效结果的影响。当Quad‐X™和Humira®剂量为1mg /kg， D1‐NDure™‐C4剂量为30mg /kg时，在转基因多发性关节炎小鼠模型（Tg197）中测定血清药物水平和ADA的存在。在相同治疗组的所有小鼠中，Quad - X™和D1 - NDure™- C4模式的血清水平一直很高，并且具有可比性。在1 mg/kg和3 mg/kg的Quad - X™和30 mg/kg的D1 - NDure™- C4治疗小鼠中，估计平均谷药血清浓度分别为8μg/mL， 50μg/mL和350μg/mL。与此形成鲜明对比的是，1 mg/kg治疗组的修美乐谷血清浓度范围为<0.008μg/mL至4μg/mL， 8只动物中有7只检测到微量水平。3 mg/kg处理样品的谷血清Humira®和Quad‐X™浓度具有可比性；然而，由于中和ADA，检测到的Humira®血清的功能明显受损。ADA的影响超出了Humira®的简单和快速清除，因为在小鼠纤维肉瘤（L929）细胞试验中，7/8血清样本也显示没有可检测到的中和hTNF - α介导的细胞毒性的能力。所有VNAR结构的中和能力在实验期（10周）结束时保持不变。本文中提供的数据在某种程度上解释了先前发表的临床前体内功效数据的令人兴奋的结果，该数据显示Quad‐X™浓度为0.5 mg/kg，相当于Humira®体内功效的10倍，完全控制了疾病。这一独立的确证也验证了当前手稿中报道的检测技术的稳健性和可靠性，虽然它伴随着小鼠研究的警告，但它似乎表明，至少这些特定的VNAR结构具有较低的固有免疫原性。
Anti‐drug antibodies (ADAs), specific for biotherapeutic drugs, are associated with reduced serum drug levels and compromised therapeutic response. The impact of ADA on the bioavailability and clinical efficacy of blockbuster anti‐hTNF‐αmonoclonal antibodies is well recognised, especially for adalimumab and infliximab treatments, with the large and complex molecular architecture of classical immunoglobulin antibody drugs, in part, responsible for the immunogenicity seen in patients. The initial aim of this study was to develop solid‐phase enzyme‐linked immunosorbent assays (ELISA) and anin vitrocell‐based method to accurately detect ADA and estimate its impact on the preclinicalin vivoefficacy outcomes of two novel, nonimmunoglobulin VNAR fusion anti‐hTNF‐αbiologics (Quad‐X™ and D1‐NDure™‐C4) and Humira®, a brand of adalimumab. Serum drug levels and the presence of ADA were determined in a transgenic mouse model of polyarthritis (Tg197) when Quad‐X™ and Humira® were dosed at 1 mg/kg and D1‐NDure™‐C4 was dosed at 30 mg/kg. The serum levels of the Quad‐X™ and D1‐NDure™‐C4 modalities were consistently high and comparable across all mice within the same treatment groups. In 1 mg/kg and 3 mg/kg Quad‐X™‐ and 30 mg/kg D1‐NDure™‐C4‐treated mice, an average trough drug serum concentration of 8μg/mL, 50μg/mL, and 350μg/mL, respectively, were estimated. In stark contrast, Humira® trough serum concentrations in the 1 mg/kg treatment group ranged from <0.008μg/mL to 4μg/mL with trace levels detected in 7 of the 8 animals treated. Trough serum Humira® and Quad‐X™ concentrations in 3 mg/kg treatment samples were comparable; however, the functionality of the detected Humira® serum was significantly compromised due to neutralising ADA. The impact of ADA went beyond the simple and rapid clearance of Humira®, as 7/8 serum samples also showed no detectable capacity to neutralise hTNF‐α‐mediated cytotoxicity in a murine fibrosarcoma (L929) cell assay. The neutralisation capacity of all the VNAR constructs remained unchanged at the end of the experimental period (10 weeks). The data presented in this manuscript goes some way to explain the exciting outcomes of the previously published preclinicalin vivoefficacy data, which showed complete control of disease at Quad‐X™ concentrations of 0.5 mg/kg, equivalent to 10x thein vivopotency of Humira®. This independent corroboration also validates the robustness and reliability of the assay techniques reported in this current manuscript, and while it comes with the caveat of a mouse study, it does appear to suggest that these particular VNAR constructs, at least, are of low inherent immunogenicity.