Use of multicellular human liver models to investigate clearance of therapeutic antibodies.

与单价单抗不同，双特异性抗体可以同时结合两个不同的表位。这些新型的单抗支架在生物制药行业引起了极大的兴趣，它们有可能提供比单一或双重疗法更好的疗效和安全性。然而，双特异性结合的能力可能使它们易于形成免疫复合体，具有加速清除的可能性。有人提出，肝窦内皮细胞(LSECs)可能是清除BsAbs的主要细胞途径(13)，尽管文献中对主要消除途径和涉及的细胞类型存在一些争论(例如(14))。获得和利用原代人类细胞和组织的能力将在抗体清除机制的研究中提供显著的优势。特别令人感兴趣的是全肝灌流技术，因为有证据表明，在持续的组织培养中，肝细胞会发生表型变化，因此不能完全代表体内的药代动力学(15&GSK内部数据)。我们建议使用本提案中描述的模型来研究多种抗体形式的结合、内化和随后的清除，以更好地了解所涉及的分子机制。特别令人感兴趣的是阐明负责内化的细胞类型和细胞表面受体，识别抗体形式依赖的途径，了解抗体与受体的结合，以及评估旨在减轻这种清除途径的影响的可能的工程策略。该项目的具体目标：确定结合的组织定位和所涉及的细胞类型，以及负责结合和内化的关键细胞表面受体。了解不同抗体形式对内化的影响，并确定所涉及的任何形式的特定途径。阐明参与增强清除的关键结构基序以及适当的修饰对结合和内化率的影响适合的方法学包括免疫荧光、流式细胞术、受体的遗传消融(siRNA/CRISPR)、蛋白质工程、亲和力测定。

Bispecific antibodies (BsAbs) can bind two distinct epitopes simultaneously unlike monovalent mAbs. These novel mAb scaffolds are of great interest to the Biopharmaceutical industry with the potential to provide improved efficacy and safety over monospecific or dual therapies. However, the ability to bind bispecifically may predispose them to form immune complexes, with the potential for accelerated clearance.It has been proposed that liver sinusoidal endothelial cells (LSECs) could be the main cellular route by which BsAbs are cleared (13) although there is some debate in the literature as to the main routes of elimination and cell types involved (e.g. (14). The ability to access and utilise primary human cells and tissue would provide a significant advantage in the study of antibody clearance mechanisms. Of particular interest are the whole liver perfusion technology as there is evidence that hepatic cells undergo phenotypic changes upon continued tissue culture thus are not fully representative of in-vivo pharmacokinetics (15 & GSK internal data).We propose using the models described in this proposal to study binding, internalisation and subsequent clearance of multiple antibody formats in order to gain a better understanding of the molecular mechanisms involved. Of particular interest are elucidation of cell-type and cell-surface receptors responsible for internalisation, identification of antibody-format dependant pathways, understanding of antibody - receptor binding and evaluation of possible engineering strategies designed to mitigate the effects of such clearance pathways. Specific aims of this project: Identification of tissue localisation of binding and cell-types involved as well as critical cell surface receptors responsible for binding and internalisation. Understand the impact of differing antibody formats on internalisation and identify any format specific pathways involved. Elucidate key structural motifs involved in enhanced clearance and the effect of suitable modifications on binding and internalisation ratesSuitable methodology includes IHC & immunofluorescence, Flow cytometry, genetic ablation of receptors (siRNA / CRISPR), protein engineering, Affinity determination.