Transferrin receptor (TfR1) mediated enhanced brain delivery of antibodies have been studied extensively in preclinical settings. However, the brain pharmacokinetics, i.e. brain entry, distribution and elimination are still not fully understood for this class of antibodies. The overall aim of the study was to compare the brain pharmacokinetics of two BBB-penetrating bispecific antibodies of different size (210 vs 58 kDa). Specifically, we wanted to investigate if the faster systemic clearance of the smaller non-IgG antibody di-scFv3D6-8D3, in comparison with the IgG-based bispecific antibody mAb3D6-scFv8D3, was also reflected in the brain. Wild-type (C57/Bl6) mice were injected with 125I-iodinated ([125I]) mAb3D6-scFv8D3 (n = 46) or [125I]di-scFv3D6-8D3 (n = 32) and euthanized 2, 4, 6, 8, 10, 12, 16, or 24 h post injection. Ex vivo radioactivity in whole blood, peripheral organs and brain was measured by γ-counting. Ex vivo autoradiography and nuclear track emulsion were performed on brain sections to investigate brain and parenchymal distribution. Capillary depletion was carried out at 2, 6, and 24 h after injection of [125I]mAb3D6-scFv8D3 (n = 12) or [125I]di-scFv3D6-8D3 (n = 12), to estimate the relative levels of radiolabelled antibody in brain capillaries versus brain parenchyma. In vitro binding kinetics for [125I]mAb3D6-scFv8D3 or [125I]di-scFv3D6-8D3 to murine TfR were determined by LigandTracer. [125I]di-scFv3D6-8D3 showed faster elimination from blood, lower brain Cmax, and Tmax, a larger parenchymal-to-capillary concentration ratio, and a net elimination from brain at an earlier time point after injection compared with the larger [125I]mAb3D6-scFv8D3. However, the elimination rate from brain did not differ between the antibodies. The study also indicated that [125I]di-scFv3D6-8D3 displayed lower avidity than [125I]mAb3D6-scFv8D3 towards TfR1 in vitro and potentially in vivo, at least at the BBB. A smaller size and lower TfR1 avidity are likely important for fast parenchymal delivery, while elimination of brain-associated bispecific antibodies may not be dependent on these characteristics.

中文翻译：

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两种不同大小的 BBB 穿透双特异性抗体的脑药代动力学

转铁蛋白受体 (TfR1) 介导的增强的抗体大脑递送已在临床前环境中得到广泛研究。然而，对这类抗体的脑药代动力学，即脑进入、分布和消除仍未完全了解。该研究的总体目标是比较两种不同大小（210 与 58 kDa）的 BBB 穿透双特异性抗体的脑药代动力学。具体而言，我们想研究与基于 IgG 的双特异性抗体 mAb3D6-scFv8D3 相比，较小的非 IgG 抗体 di-scFv3D6-8D3 的更快全身清除是否也反映在大脑中。向野生型 (C57/Bl6) 小鼠注射 125I 碘化 ([125I]) mAb3D6-scFv8D3（n = 46）或 [125I]di-scFv3D6-8D3（n = 32）并安乐死 2、4、6、注射后 8、10、12、16 或 24 小时。全血、外周器官和大脑中的离体放射性通过 γ 计数测量。对脑切片进行离体放射自显影和核径迹乳液，以研究脑和实质分布。在注射 [125I]mAb3D6-scFv8D3（n = 12）或 [125I]di-scFv3D6-8D3（n = 12）后 2、6 和 24 小时进行毛细管去除，以估计放射性标记抗体的相对水平在脑毛细血管与脑实质中。[125I]mAb3D6-scFv8D3或[125I]di-scFv3D6-8D3与鼠TfR的体外结合动力学由LigandTracer测定。[125I]di-scFv3D6-8D3 与较大的[125I ]mAb3D6-scFv8D3。然而，抗体之间的大脑消除率没有差异。该研究还表明，[125I]di-scFv3D6-8D3 在体外和潜在的体内对 TfR1 的亲和力低于 [125I]mAb3D6-scFv8D3，至少在 BBB。较小的尺寸和较低的 TfR1 亲和力可能对快速实质递送很重要，而消除脑相关双特异性抗体可能不依赖于这些特征。