Antibody Sequence Analysis and Production

Antibody heavy and light chains were PCR-amplified and sequenced as previously described (61 (link), 62 (link)) Sequence analysis, including the determination of the VH and VL genes and percentage of somatic mutations, was performed using the International Immunogenetics Information System database (63 (link)). Antibody V_H or V_L sequences were cloned into plasmids containing an IgG1 or relevant light chain backbone (GenScript) and used to transfect Expi293 cells (Thermo Fisher Scientific). Recombinant IgG was purified using HiTrap Protein A columns (GE Healthcare Life Sciences). For IgA1 or IgA2 antibodies, V_H sequences were also cloned into plasmids containing the IgA1 or IgA2 constant region (GenScript). Recombinant IgA was expressed without a J chain (to express only monomeric IgA) and purified using columns containing the CaptureSelect IgA Affinity Matrix (Thermo Fisher Scientific). To produce antibody Fabs, heavy chain plasmids encoding only the V_H and C_H1 (domain 1 of constant region of the immunoglobulin heavy chain) were synthesized and used to transfect Expi293 cells along with light chain plasmids. Fab purification (including for the CV503 crystal structure) was performed with the CaptureSelect KappaXP Affinity Matrix or CaptureSelect LC-lambda (Hu) Affinity Matrix (Thermo Fisher Scientific).