Protein a hi trap column

The GPC3 and GPC4 mutant proteins without HS chains (GPC3ΔHS and GPC4ΔHS in Figure 4A) were constructed by replacing serine with alanine residues at the HS attachment sites. pVRC8400-GPC3ΔHS-hFc or pVRC8400-GPC4ΔHS-hFc were transiently transfected into HEK-293T cells. The cell supernatant was harvested and the Fc-tagged GPC proteins were purified on a Protein A Hi-Trap column (GE Healthcare Cat. # 29048576) according to the manufacturer’s instructions. The quality and quantity of purified proteins were determined by SDS-PAGE and by measuring absorbance at 280 nm on a NanoDrop instrument (Thermo Fisher Scientific), respectively.

Toxins were conjugated to mAbs by incubating LPETG-tagged monoclonal antibodies [10μM] with Gly₅-modified toxin [200μM] in the presence of 0.62μM sortase A in 50mM Hepes, 150mM NaCl, 5mM CaCl₂, pH 7.5 for 3.5h at 25°C. The reaction was stopped by passing it through a Protein A HiTrap column (GE Healthcare) equilibrated with 25mM sodium phosphate pH 7.5, followed by washing with 5 column volumes (CVs) of buffer. Bound conjugate was eluted with 5 CVs of elution buffer (0.1M succinic acid, pH 2.8) with 1 CV fractions collected into tubes containing 25% v/v 1M Tris-base to neutralize the solution. Protein containing fractions were pooled and formulated in 10mM sodium succinate pH 5.0, 100mg/mL trehalose, 0.1% w/v polysorbate 20 by G25 column chromatography using NAP-25 columns (GE Healthcare) according to the manufacturer’s instructions.

Purified IgG (1 mg) was digested to Fab and Fc fragments by exposure to 40 μg activated papain (Sigma-Aldrich) in a solution containing 10 mM cysteine, 100 mM sodium acetate pH 5.5, 125 μM EDTA for 2–4 h at 37 °C. Fab2 was produced by digestion with pepsin (Sigma-Aldrich) for 2–4 h at 37 °C. Fab and Fab2 fragments were subsequently separated from Fc by affinity chromatography using a protein A Hi-trap column (GE Healthcare) and purified to homogeneity by size exclusion chromatography (SEC)37 (link). SDS–polyacrylamide gel electrophoresis was performed to confirm the sizes of Fab and Fab2. The NativePAGE Novex Bis-Tris gel, SimplyBlue SafeStain staining solution and protein standard were purchased from Invitrogen, Inc.

Full-length antibody versions of 3A3 and 3E11 were cloned as previously described (Nguyen et al., 2015 (link)) as mouse variable region-human IgG1 constant region chimeras. Antibodies hu3A3 and RAY53 were similarly cloned into human IgG1 and IgΚ expression vectors. Antibodies were expressed in ExpiCHO (Thermo Fisher Scientific) cells according to the high titer protocol provided and purified on a Protein A HiTrap column (GE Healthcare) with the ACTA Pure FPLC system (GE Healthcare), and buffer exchanged to PBS.
Human Fab fragments were generated by digestion of full-length antibody with papain and removal of the Fc portion by protein A binding. Mouse Fab fragments of 3A3 were generated by cloning the V_H and V_L regions upstream of heavy chain constant regions with a HRV3C protease site in the hinge (Pallesen et al., 2017 (link)) and a mouse kappa chain, respectively. After expression, protein A purified protein was digested with HRV3C protease, and the flow-through from a Protein A HiTrap column was collected. Excess HRV3C protease was removed by incubation with Ni Sepharose 6Fast Flow beads (GE Healthcare). Fully murine antibodies were produced by cloning the V_H regions into mouse IgG2a and V_L regions in to a mouse IgK expression cassettes in the pAbVec background, co-transfecting, and purifying as described above.

HS20 and YP7 antibodies were purified in-house as previously described32 (link)58 (link). GPC3-hFc, GPC3ΔHS-hFc, GPC1-hFc genes were amplified by adding the IL-2 signal peptide sequence at the 5′ end and were inserted into a pVRC8400 expression vector (provided by Dr. Gary J. Nabel, the National Institute of Allergy and Infectious Diseases). The plasmids were transiently transfected into HEK-293T cells. The media was collected and the proteins were purified using a Protein A Hi-Trap column (GE Healthcare, Pittsburgh, PA) according to the manufacturer’s instructions. The quality and quantity of purified proteins were determined by SDS-PAGE and A280 absorbance on a NanoDrop (Thermo Scientific, Asheville, NC), respectively.