Affinity transfer of nanobodies to a CDR3 only single-domain antibody fragment NBL42
10.3760/cma.j.cn121382-20211208-00302
- VernacularTitle:仅具有CDR3的单域抗体NBL42作为纳米抗体亲和力转移的移植框架研究
- Author:
Yafei LIU
1
;
Yaning FENG
;
Xin ZHANG
;
Yuan XIE
;
Jiangwei LI
Author Information
1. 新疆大学生命科学与技术学院，新疆生物资源与基因工程重点实验室，乌鲁木齐 830046
- Keywords:
CDR graft;
Affinity transfer;
Nanobody;
Antigen binding;
Universal framework
- From:
International Journal of Biomedical Engineering
2022;45(3):200-206,219
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To evaluate the potential of a previously identified CDR3 only single-domain antibodies (sdAbs) fragment, NBL42, as a general framework for affinity transfer.Methods:The H3 loops of VHH-A4(A4), VHH-H5(H5), cAb-Lys3(L3) and B6H12 which bind with alliinase, PD-1, lysozyme and CD47, respectively, were grafted into the corresponding loop of NBL42. The genes of the reconstituted CDR3 only sdAbs were synthesized, expressed in E. coliand purified with Ni 2+ column affinity chromatography. The antigen binding and stability of the recombinant CDR3 only sdAbs were assayed by ELISA. Results:The recombinant NBL42-A4CDR3, NBL42-H5CDR3, NBL42-L3CDR3 and NBL42-B6H12CDR3 ran as a single peak at 15, 15, 28 and 16 kDa, respectively, in SDS-PAGE as expected molecular weight. Grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 expressed in a soluble form and specifically bind with alliinase and PD-1, respectively, but lost about 50% of their binding activity. In contrast, the grafted sdAbs NBL42-Lys3CDR3 and NBL42-B6H12CDR3 completely lost their antigen binding capacity. NBL42 sdAbs and grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 retain roughly half of their binding activity after 90 ℃ heat treatment, indicating high stability. The C88Y mutation in NBL42 and the Swiss Mode 3D model predicted that the C88Y residue in FR3 may play a key role in NBL42 stability and CDR3 affinity transfer.Conclusions:The structure of NBL42 has potential as a framework for CDR3 transplantation and affinity transfer.
