Bonvicini Gillian, Singh Sunitha, Sandersjöö Lisa, Sehlin Dag, Syvänen Stina, Andersson Ken G
BioArctic AB, Warfvinges Väg 35, 112 51, Stockholm, Sweden.
Department of Public Health and Caring Sciences/Geriatrics, Rudbeck Laboratory, Uppsala University, Dag Hammarskjölds Väg 20, 751 85, Uppsala, Sweden.
Fluids Barriers CNS. 2025 Apr 8;22(1):36. doi: 10.1186/s12987-025-00643-y.
Monovalent binding to the transferrin receptor (TfR) is considered the most efficient mode for high delivery of protein constructs across the blood-brain barrier via TfR-mediated transcytosis at therapeutic doses. However, growing evidence suggests this is not the case at lower, diagnostic doses. There is also a lack of data on how valency and affinity to TfR affect brain uptake independently since previous studies have not compared monovalent and bivalent antibodies with similar affinities regardless of valency (i.e. apparent affinity). Therefore, the aim was to evaluate the independent effects of valency and affinity on TfR-mediated brain delivery at different doses.
Affinity variants of antibody 8D3 were produced by introducing alanine point mutations into the complementarity-determining regions. Eleven Fab fragments and 29 IgGs were affinity screened against mouse TfR (mTfR). Six of each were chosen for production with a knob-into-hole design to have monovalent and bivalent TfR binders in full-length antibody format. The apparent affinity of these 12 antibodies were tested in an Sp2/0-Ag14 cell assay. The 10 nM apparent affinity set and the bivalent wild-type antibody were radiolabelled and injected into wild-type mice at a low (0.22 ± 0.03 mg/kg) or high (7.5 ± 0.43 mg/kg) dose. The biodistribution was measured in brain, blood and peripheral organs 4 h post-injection.
Two sets of monovalent and bivalent 8D3 formats with similar mTfR apparent affinities were identified. Brain and tissue uptake was higher at the low dose than the high dose for all antibodies. At the low dose, the higher apparent affinity, bivalent antibody had higher brain uptake than either of the two lower apparent affinity antibodies. At the high dose, the monovalent antibody had higher brain uptake than the two bivalent antibodies. The peripheral distribution of the three antibodies were similar to the brain distribution at both doses.
Valency and apparent affinity affect brain uptake in a dose-dependent manner such that: brain uptake was affected more by apparent affinity at the low dose and by valency at the high dose. Thus, when designing constructs for TfR-mediated brain delivery, the application, and consequently the dose, are critical to consider.
单价结合转铁蛋白受体（TfR）被认为是通过TfR介导的转胞吞作用在治疗剂量下高效递送蛋白质构建体穿过血脑屏障的最有效方式。然而，越来越多的证据表明，在较低的诊断剂量下情况并非如此。由于先前的研究没有比较具有相似亲和力（即表观亲和力）的单价和二价抗体，因此也缺乏关于TfR的价态和亲和力如何独立影响脑摄取的数据。因此，本研究旨在评估价态和亲和力在不同剂量下对TfR介导的脑递送的独立影响。
通过在互补决定区引入丙氨酸点突变来产生抗体8D3的亲和力变体。针对小鼠TfR（mTfR）对11个Fab片段和29个IgG进行亲和力筛选。每种选择6个用于采用引入式设计生产，以获得全长抗体形式的单价和二价TfR结合物。在Sp2/0-Ag14细胞试验中测试这12种抗体的表观亲和力。将10 nM表观亲和力组和二价野生型抗体进行放射性标记，并以低剂量（0.22±0.03 mg/kg）或高剂量（7.5±0.43 mg/kg）注射到野生型小鼠体内。在注射后4小时测量脑、血液和外周器官中的生物分布。
鉴定出两组具有相似mTfR表观亲和力的单价和二价8D3形式。所有抗体在低剂量时的脑和组织摄取均高于高剂量。在低剂量时，表观亲和力较高的二价抗体的脑摄取高于两种表观亲和力较低的抗体中的任何一种。在高剂量时，单价抗体的脑摄取高于两种二价抗体。三种抗体在两种剂量下的外周分布与脑分布相似。
价态和表观亲和力以剂量依赖性方式影响脑摄取，具体如下：在低剂量时脑摄取受表观亲和力影响更大，而在高剂量时受价态影响更大。因此，在设计用于TfR介导的脑递送的构建体时，应用以及相应的剂量是需要考虑的关键因素。
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