Rapid and robust antibody Fab fragment crystallization utilizing edge-to-edge beta-sheet packing

Abstract

Antibody therapeutics are one of the most important classes of drugs. Antibody structures have become an integral part of predicting the behavior of potential therapeutics, either directly or as the basis of modeling. Structures of Fab:antigen complexes have even greater value. While the crystallization and structure determination of Fabs is easy relative to many other protein classes, especially membrane proteins, broad screening and optimization of crystalline hits is still necessary. Through a comprehensive review of rabbit Fab crystal contacts and their incompatibility with human Fabs, we identified a small secondary structural element from the rabbit light chain constant domain potentially responsible for hindering the crystallization of human Fabs. Upon replacing the human kappa constant domain FG loop (HQGLSSP) with the two residue shorter rabbit loop (QGTTS), we dramatically improved the crystallization of human Fabs and Fab:antigen complexes. Our design, which we call "Crystal Kappa", enables rapid crystallization of human fabs and fab complexes in a broad range of conditions, with less material in smaller screens or from dilute solutions.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants

“…A 10 mg/mL solution of LY-CoV1404 Fab (with CrystalKappa mutations (Lieu et al, 2020b)) in complex with RBD was set up in vapor diffusion sitting drops at a ratio of 1:1 with a well solution of 100 mM Tris HCl pH 6.5, 20% PEG MME 2K and 200 mM Trimethylamine N-oxide. Crystals appeared within two days, grew to their full size and were harvested on the fifth day after the set up.…”

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates

“…For protein crystallography, an isolated RBD (using residues 329 to 527) was fused to a 6× His-tag at the C terminus, expressed in CHO cells, enzymatically deglycosylated using endoglycosidase-H (Endo-Hf, New England Biolabs), and purified by cation exchange chromatography. The Fab portions of selected antibodies, containing mutations in the constant region known to encourage crystallization ( 65 ), were expressed in CHO cells and purified. The Fab-RBD complexes were prepared by mixing the components with a 20% excess of the RBD and then the complex was purified from the excess RBD by size-exclusion chromatography.…”

Structure of the Wnt–Frizzled–LRP6 initiation complex reveals the basis for coreceptor discrimination

“…A fusion protein of disulfide-cyclized haXWnt8 NC-linker with YW210 Fab was designed based upon inspection of our cryo-EM structure and the previously reported structure of YW210 bound to LRP6 E1 ( 25 ), with heterodimerization promoted using AcidP1/BaseP1 zipper fusions ( 56 ) and Crystal Kappa mutations in the light chain to promote crystallization ( 57 ). A synthesized single-chain gene containing the disulfide-cyclized haXWnt8 NC-linker (residues 227 to 243 with S228C and S234C mutations), a (GGGGS) 3 linker, the YW210 Fab heavy chain, and 3C protease site was cloned into the pAcGP67A backbone containing a C-terminal AcidP1 leucine zipper, BAP tag, and hexahistidine tag.…”