Nanolipoprotein Particles as a Delivery Platform for Fab Based Therapeutics
Nanolipoprotein Particles as a Delivery Platform for Fab Based Therapeutics
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DOI：
10.1021/acs.bioconjchem.0c00349
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发表时间：
2020-08-01
影响因子：
4.7
通讯作者：
Blanchette, Craig
中科院分区：
文献类型：
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作者：
Darwish, Martine;Shatz, Whitney;Blanchette, Craig
纳米脂蛋白颗粒（NLP）是一种基于脂质双层的纳米颗粒平台，最近被开发用于体内递送多种具有治疗意义的分子，但其递送价数超过当前多价形式的 Fab 的潜力尚未得到评估。在这里，我们描述了 Fab-NLP 缀合物的开发、优化和表征。 NLP 是用马来酰亚胺反应性脂质生成的，用于与带有 C 末端半胱氨酸的 Fab 缀合。值得注意的是，当 NLP 在 pH 7.4 下组装时，马来酰亚胺反应性脂质可与载脂蛋白结合。然而，在 pH 6 下组装时，并未观察到这种不良反应。随后优化了位点特异性 Fab 缀合条件，并证明每个 NLP 最多可缀合 30 个 Fab。有趣的是，虽然大量 Fab 的缀合对 NLP 分子量有显着影响，但仅观察到对 NLP 流体动力学半径的影响很小，这表明颗粒尺寸很大程度上取决于 NLP 的盘状形状。还评估了 Fab-NLP 粘度及其冻干后的稳定性，作为 Fab-NLP 可制造性的评估。相对于另一种多价形式（Fab-PEG 缀合物），Fab-NLP 缀合物获得了显着更高的 Fab 浓度。在抑制和激动剂环境中，也未发现 Fab 与 NLP 缀合对 Fab 活性有影响。最后，在 50% 血清中评估 FabNLP 缀合物的稳定性，Fab-NLP 表现出稳定性增加，在 Fab/颗粒比率为 7 或更大的情况下，24 小时后，>63% 的 Fab-NLP 保持完整。我们的研究结果表明，Fab-NLP 是一个有前途的平台，用于以多价格式定向交付 Fab，并且与现有的制造工艺兼容。
Nanolipoprotein particles (NLPs), a lipid bilayer-based nanoparticle platform, have recently been developed for in vivo delivery of a variety of molecules of therapeutic interest, but their potential to deliver Fabs with valencies that exceed those of current multivalent formats has not yet been evaluated. Here we describe the development, optimization, and characterization of Fab-NLP conjugates. NLPs were generated with maleimide reactive lipids for conjugation to a Fab with a C-terminal cysteine. Of note, maleimide reactive lipids were shown to conjugate to the apolipoprotein when the NLPs were assembled at pH 7.4. However, this undesirable reaction was not observed when assembled at pH 6. Site-specific Fab conjugation conditions were then optimized, and conjugation of up to 30 Fab per NLP was demonstrated. Interestingly, although conjugation of higher numbers of Fabs had a significant impact on NLP molecular weight, only a minimal impact on NLP hydrodynamic radius was observed, indicating that particle size is largely dictated by the discoidal shape of the NLP. Fab-NLP viscosity and its stability upon lyophilization were also evaluated as an assessment of the manufacturability of the Fab-NLP. Significantly higher Fab concentrations were achieved with the Fab-NLP conjugates relative to another multivalent format (Fab-PEG conjugates). Fab conjugation to the NLP was also not found to have an impact on Fab activity in both an inhibitory and agonist setting. Finally, the stability of the FabNLP conjugates was evaluated in 50% serum and Fab-NLPs demonstrated increased stability, with >63% of Fab-NLP remaining intact after 24 h at Fab per particle ratios of 7 or greater. Our findings suggest Fab-NLPs are a promising platform for the targeted delivery of Fabs in a multivalent format and are compatible with established manufacturing processes.