Fab preparation kit

The HM2 antigen-binding fragment (Fab) was prepared using a Fab preparation kit (Thermo Fisher Scientific). GPC1 protein was mixed with HM2 Fab at a 1:1 molar ratio in PBS. In addition, GPC1 protein was mixed with the D4-LR immunotoxin that contains domain III of PE at a 1:1 molar ratio in PBS. The details of data acquisition and 3D model construction were described previously^{40 (link)}. Automatic model building was started with the production of RosettaFold, AlphaFold and YASARA models, and followed by the selection of best templates based on Z-score for all sequences, except for GPC1 in which PDB structures PDBID:4ACR, 4AD7, 4BWE and 4YWT were used as starting models. The models were placed in the maps using several criteria, including Segger (as implemented in Chimera), and the best poses were selected by hand. The models were further refined by Molecular Dynamics (MD) with CC constrains⁵² . We modified the standard procedure to evaluate the ensemble evolution, instead of producing an average model. We extracted the highest probability model that fits the ensemble generated set when the ensemble average converges.

We have developed a human/mouse chimeric anti-fibrin antibody, 102-10 IgG, using antibody engineering technology as previously described44 (link). To prepare the 102-10 Fab fragment, 102-10 IgG was incubated with immobilized papain, a component of the Fab preparation kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The digested antibody was separated from the immobilized papain by centrifugation, and the Fab fragment was separated from the undigested IgG and Fc fragments using Protein A (Thermo Fisher Scientific). Finally, size exclusion chromatography was conducted using Superdex 75 pg (GE Healthcare, Fairfield, CT, USA) to yield the purified Fab fragment. Using the same procedure, an anti-CD20 Fab fragment was prepared from Rituximab (Zenyaku Kogyo, Tokyo, Japan) as an isotype control antibody.