Capto l

The CD3 and EpCAM fragments were purified by Capto L chromatography (Cytiva, Uppsala, Sweden) through the Akta150 system (Cytiva, Uppsala, Sweden). The supernatant was centrifuged and flirted through a 0.45 μm filter membrane. The column was loaded with binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4), supernatants, wash buffer (100 mM sodium citrate, pH 5.0), and eluted with elution buffer (100 mM sodium citrate, pH 2.8). The elution was neutralized to pH 7.0 with Tris-HCl buffer (1 M, pH 8.0). Fragment A and B samples were dialyzed into phosphate buffer (PBS, pH 7.4) followed by splicing reaction catalyzed mediated by split intein.
After dialyzing all of the fragments in PBS buffer and adding 2 mM dithiothreitol (DTT, Sigma Aldrich, Shanghai, China), 100 mM fragment A was combined with 375 mM fragment B and incubated at 37°C for 2 h. The mixture was dialyzed to PBS to remove DTT, sterilized using a 0.22 μm filter (Millipore, Shanghai, China), and the oxidation reaction was carried out at room temperature for 2–3 days. The CD3×EpCAM BsAb was isolated through Protein A affinity chromatography (Cytiva, Uppsala, Sweden). The eluates were adjusted to pH 7.0 using Tris-HCl buffer, dialyzed to PBS, and sterilized through a 0.22 μm filter (Millipore, Shanghai, China).

The rmAbs were generated as previously reported (Nihei et al, 2023a (link)). Briefly, cDNA was synthesized using total RNA extracted from single IgA⁺ PBs sorted from the kidneys of gddY mice. The amplified variable regions of the IgA heavy chain and Igκ or Igλ light chain genes were ligated with the pCAGGS expression vector (Hitoshi et al, 1991 (link)) into which cDNA encoding human Cγ1, Cκ, or Cλ was inserted. An rmAb specific for 4-hydroxy-3-nitrophenyl acetyl (NP) was generated by inserting a V_HB1-8hi heavy chain (Shih et al, 2002 (link)) and λ light chain gene into the same vectors. For injection into mice, variable region genes of rmAb#66 or rmAb#N9 were recombined with mouse Cα, Cκ, or Cλ, and the resultant vectors were transfected into HEK293T cells expressing the J-chain (Nihei et al, 2023a (link)). As a control for in vivo injection, an IgA rmAb derived from a randomly selected single IgM⁺ IgD⁺ naïve B cell from a gddY mouse spleen was generated. The rmAbs with human Fcγ and polymerized rIgA were purified with Protein A (Cytiva) and CaptoL (Cytiva), respectively, according to the manufacturer’s instructions.