Computer-based Engineering of Thermostabilized Antibody Fragments

Jiwon Lee; Bryan S Der; Christos S Karamitros; Wenzong Li; Nicholas M Marshall; Oana I Lungu; Aleksandr E Miklos; Jianqing Xu; Tae Hyun Kang; Chang-Han Lee; Bing Tan; Randall A Hughes; Sang Taek Jung; Gregory C Ippolito; Jeffrey J Gray; Yan Zhang; Brian Kuhlman; George Georgiou; Andrew D Ellington

doi:10.1002/aic.16864

Computer-based Engineering of Thermostabilized Antibody Fragments

AIChE J. 2020 Mar;66(3):e16864. doi: 10.1002/aic.16864. Epub 2019 Nov 19.

Authors

Jiwon Lee¹, Bryan S Der², Christos S Karamitros³, Wenzong Li⁴, Nicholas M Marshall³, Oana I Lungu³, Aleksandr E Miklos⁵, Jianqing Xu⁶, Tae Hyun Kang⁷, Chang-Han Lee³, Bing Tan³, Randall A Hughes^{8

9}, Sang Taek Jung¹⁰, Gregory C Ippolito⁴, Jeffrey J Gray⁶, Yan Zhang⁴, Brian Kuhlman², George Georgiou^{3

4

11

12}, Andrew D Ellington^{4

12

13}

Affiliations

¹ Thayer School of Engineering, Dartmouth College, Hanover, NH 03755.
² Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599.
³ Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712.
⁴ Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712.
⁵ U.S. Army Combat Capabilities Development Command Chemical Biological Center, APGEA, MD 21010.
⁶ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MA 21218.
⁷ Biopharmaceutical Chemistry Major, School of Applied Chemistry, Kookmin University, Seongbuk-gu, Seoul 02707, Republic of Korea.
⁸ US Army Research Laboratory, Austin, TX 78712.
⁹ Applied Research Laboratories, The University of Texas at Austin, Austin, TX 78712.
¹⁰ Department of Biomedical Science, Graduate School of Medicine, Korea University, Seoul 02841, Republic of Korea.
¹¹ Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712.
¹² Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712.
¹³ Department of Chemistry, The University of Texas at Austin, Austin, TX 78712.

Abstract

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T_m) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T_m compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.

Keywords: Biochemicals; Bioengineering; Biofuels; Biomolecular Engineering; Food Rosetta; antibody engineering; scAb; scFv; thermostable antibodies.

Abstract

Grants and funding