一种三特异性抗体设计以及功能研究

Structure Design and Function Research of the Trispecific Antibody

收稿日期:2021-11-23修回日期:2022-01-06作者简介:张瑞(1988-),男,安徽蚌埠人,硕士研究生在读,研究方向:生物工程:E-mail:634947112@qq:com。通信作者:李大力(1965-),男,江苏徐州人,副教授,研究方向:生物工程:E-mail:lidali@njust.edu.cn。

To design the trispecific antibody Tris1 that targets are ROR1 and CMET on the tumor with CD3 on the T cell and verify the biology activity, the trispecific antibody Tri1 was obtained by Fab exchange through IgG1 cFAE(controlled Fab-arm-exchange) technology, based existing anti-cMET monoclonal antibodies and FIT-Ig(Fabs-in-tandem immunoglobulin) bispecific antibodies that target ROR1 and CD3.For the construction of the FIT-Ig expression vector, the point mutation K409 R was added to the CHregion of the Fc segmentfor cFAE;While the expression vector for the cMET monoclonal antibody was added to the CHregion on the Fc for the FAE point mutation F405 L.Then, using SEC-HPLC analysis method and OctetRed system to identify the purity and binding affinity.Monoclonal antibody and FIT-Ig antibody were transiently expressed in Expi293 F mammalian cell expression system, and protein A gravity column affinity purification was used for protein purification.The purified monoclonal antibody and FIT-Ig antibody were mixed in 25 mmol/L 2-MEA buffer at a molar ratio of 1∶1 and incubated at 37 °C for 90 min to form a stable trispecific antibody structure.At the same time, the molecular weight was detected by LC-MS to confirm the structural correctness and further investigation experiment at 37 °C test its stability. The purity of the trispecific antibody was 94% and retained good binding activity for the three target antigens.It can be reserved for a long time due to the structure is stable. Trispecific antibody target to ROR1,CD3 and CMET was produced successfully, which retain the Fc and Fab.Moreover, it has the perfect industrial production prospect with the compatibility of production technical and standard mass antibody manufacture procedure.By exploring the multi-target antibodies in cancer therapy around the world, the development and preparation of trispecific antibody can lay a new macromolecular structure foundation for the treatment of various diseases.