Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy
Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy
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DOI：
10.1007/s00253-005-0300-7
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发表时间：
2006-09-01
影响因子：
5
通讯作者：
van Zijderveld, F. G.
中科院分区：
文献类型：
--
作者：
Harmsen, M. M.;van Solt, C. B.;van Zijderveld, F. G.
我们先前证明，口服针对大肠杆菌F4菌毛的重组单域抗体片段（VHH）克隆K609，可减少大肠杆菌引起的仔猪腹泻，但仅在高VHH剂量下。我们现在已经证明，口服K609 VHH的很大一部分在胃中被蛋白水解降解。对蛋白水解稳定性的严格选择鉴定出7种vhs，在胃液中体外培养后稳定性提高了7至138倍。通过DNA洗牌，我们获得了4个克隆，体外稳定性进一步提高了1.5至3倍。这些VHH彼此之间和K609之间最多相差10个氨基酸残基，这些残基分散在VHH序列上，并且与预测的蛋白酶裂解位点不重叠。最稳定的克隆K922在胃液和空肠液中孵育后仍保持41%和90%的活性。仔猪口服K922证实其蛋白水解稳定性提高。此外，在较低的VHH浓度下，K922以较高的亲和力与F4菌毛结合，抑制菌毛粘附。因此，K922是预防仔猪腹泻的有希望的候选药物。此外，我们的研究结果可以指导其他口服重组抗体片段的基因工程选择和改进。
We previously demonstrated that oral application of the recombinant single-domain antibody fragment (VHH) clone K609, directed against Escherichia coli F4 fimbriae, reduced E. coli-induced diarrhoea in piglets, but only at high VHH doses. We have now shown that a large portion of the orally applied K609 VHH is proteolytically degraded in the stomach. Stringent selection for proteolytic stability identified seven VHHs with 7- to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling we obtained four clones with a further 1.5- to 3-fold increased in vitro stability. These VHHs differed by at most ten amino acid residues from each other and K609 that were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Oral application of K922 to piglets confirmed its improved proteolytic stability. In addition, K922 bound to F4 fimbriae with higher affinity and inhibited fimbrial adhesion at lower VHH concentrations. K922 is thus a promising candidate for prevention of piglet diarrhoea. Furthermore, our findings could guide selection and improvement by genetic engineering of other recombinant antibody fragments for oral use.