All protein samples used in this study were expressed using the Expi293 mammalian cell expression system (Thermo Fisher Scientific, MA USA) in HE400 medium (GMEP, Fukuoka, Japan). Mammalian expression vectors for all chains (i.e., the genes for both heavy chains and both light chains) were simultaneously transfected into cells using PEI MAX reagent (Polyscience Inc., PA USA). The mixing ratios of the four expression vectors were set to their respective molar concentrations, 1:1:1:1. After seven days of culturing, supernatants were collected and applied to a Protein A column (UNOsphere SUPrA, Biorad, CA USA). Finally, to remove N-terminus Catcher/Tag units, Thrombin was added at a concentration of 0.2 U/1 mg protein. The final purification was performed by applying the cleaved sample to the Protein A column again.

10% polyacrylamide gels were used for the SDS-PAGE measurements. Samples were concentrated using a TCA-DOC (trichloroacetic acid-deoxycholic acid) precipitation method with 7% TCA and 0.02% DOC to apply 5 µg sample in each well. Samples were prepared in a reduced condition using β-mercaptoethanol.

For chromatography, we used a superdex75 10/300 GL column with PBS to evaluate the IgG-size bispecific molecules after cleavage. All procedures were performed at room temperature. Briefly, 100 µL samples with a concentration of 200 nM were loaded onto the column and detection was performed at 280 nm absorption using a Biorad NGC chromatography system (Biorad Inc.). To minimize the fluctuation of the elution volume of the sample, we measured control experiment in every measurement.

Protein thermal stability measurements were then determined via DSF using a StepOne real-time PCR system (Applied Biosystems). Briefly, protein concentrations were first set to 0.1 mg/mL in PBS buffer. Samples were then mixed with 250X diluted SYPRO Orange dye (Sigma Aldrich). 25 µL aliquots were then loaded onto 48-well sample plates, and samples were then heated from 25 °C to 99 °C.

We performed flow cytometry analyses using 106 cells of HPB-ALL (CD3+), A431 (EGFR+), and SK-BR-3 (Her2+) cells. All cell lines were obtained from the Cell Resource Center for Biomedical Research at Tohoku University. Prior to flow cytometry, cells were incubated for 10 min with 30 nM of bsAb in PBS. After washing cells with PBS twice, PBS-suspended cells were then incubated with anti-human-Fc FITC conjugated goat antibody (SIGMA, F9512, RRID: AB_259808) and washed with PBS twice. PBS-suspended cells were analyzed using an RF-500 flow cytometer (Sysmex, Kobe, Japan). All analyses used 100 µL cell suspension for each measurement. FCSalyzer software (Sourceforge; https://sourceforge.net/projects/fcsalyzer/) was used to analyze all obtained data. Finally, we also obtained measurements for positive controls, anti-CD3 IgG FITC (Proteintech Group, Inc., IL, USA, RRID: AB_2883794), Cetuximab (anti-EGFR), and Herceptin (anti-Her2).

We performed cytotoxicity assays. To do so, T-LAK cells, a type of activated T-cell, were first induced from peripheral blood mononuclear cells. The reason for the use of T-LAK instead of naïve PBMC is to obtain a sufficient number of cells for the experiments. These included Normal Human PBMCs, Purified and Non-Characterized (Precision for Medicine) for Her2/CD3 bsAb measurements and PBMCs from Cellular Technology Limited for EGFR/CD3 bsAb measurements. Briefly, PBMCs were stimulated with IL-2 and Dynabeads Human T-Activator CD3/CD28 beads (Thermo Fisher Scientific) for Her2/CD3 bsAb measurements, or in a culture flask (A/S Nunc) pre-coated with anti-CD3 monoclonal antibody for EGFR/CD3 measurements. In vitro growth inhibition measurements of cancer cells using HER2 positive SK-BR-3 cells or EGFR positive TFK-1 cells were performed using MTS assay kits (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay; Promega). For these assays, the E/T ratio was set to five and the incubation time was 48 h for Her2/CD3 bsAb assays, and the E/T ratio was set to two and the incubation time was 20 h for the EGFR/CD3 bsAb assays. PBS-incubated wells were used for a 100%-cytotoxicity reference because this treatment kills all the cancer cells during the incubation time and medium-incubated wells were used for a 0%-cytotoxicity reference.