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 VH and VL 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 VH regions into mouse IgG2a and VL regions in to a mouse IgK expression cassettes in the pAbVec background, co-transfecting, and purifying as described above.
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 VH and VL 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 VH regions into mouse IgG2a and VL regions in to a mouse IgK expression cassettes in the pAbVec background, co-transfecting, and purifying as described above.
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