Harmsen M M, van Solt C B, van Zijderveld-van Bemmel A M, Niewold T A, van Zijderveld F G
Institute for Animal Science and Health (ID-Lelystad) of Wageningen University and Research Centre, Edelhertweg 15, 8219 PH, Lelystad, The Netherlands.
Appl Microbiol Biotechnol. 2006 Sep;72(3):544-51. doi: 10.1007/s00253-005-0300-7. Epub 2006 Feb 1.
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.
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可减轻大肠杆菌诱导的仔猪腹泻，但仅在高VHH剂量下有效。我们现在发现，口服的K609 VHH在胃中大部分被蛋白水解降解。通过严格筛选蛋白水解稳定性，鉴定出七个VHH，它们在胃液中体外孵育后的稳定性提高了7至138倍。通过DNA改组，我们获得了四个克隆，其体外稳定性进一步提高了1.5至3倍。这些VHH彼此之间与K609最多相差十个氨基酸残基，这些残基分散在VHH序列上，并且与预测的蛋白酶切割位点不重叠。最稳定的克隆K922在胃液中孵育后保留41％的活性，在空肠液中保留90％的活性。对仔猪口服K922证实了其改善的蛋白水解稳定性。此外，K922以更高的亲和力与F4菌毛结合，并在较低的VHH浓度下抑制菌毛粘附。因此，K922是预防仔猪腹泻的有希望的候选者。此外，我们的发现可以指导其他口服重组抗体片段的选择和通过基因工程进行改进。
