Cho cells

Chinese hamster ovary (CHO) cells with a single integrated Flp-In recombination site were obtained from Invitrogen (Carlsbad, CA, USA). Preparation of cell lines that stably expressed hMATE1 (NM_018242.3) and hOCT2 (BC039899.1) (described in [64 (link),65 (link)]) used the pcDNA5/FRT/V5-His TOPO mammalian expression vector, which added a 23 amino acid extension to the C-terminus of these proteins that included the V5 (GKPIPNPLLGLDST) and 6xHis epitope tags. Cells were passaged every 3–4 days and maintained at 37 °C in a humidified environment with 5% CO₂. Continued expression of MATE1 and OCT2 in these cell lines was maintained using hygromycin (200 µg/mL; Invitrogen, Carlsbad, CA) selection pressure; continued expression of the Flp-In recombination site in wild-type (WT; non-transporter expressing) CHO cells was maintained using Zeocin (100 µg/mL; Invitrogen) selection pressure. When seeded into 96-well plates (Greiner; VWR Intl., Arlington Heights, IL, USA) for transport assays, cells were grown to confluence in antibiotic-free medium.

The cell fusion assay was performed as previously described [13 (link)]. Briefly, Chinese hamster ovary (CHO) cells (Invitrogen) that overexpress HA protein from A/Brisbane/10/2007 strain were plated at approximately 90% confluence in six-well plates. After incubation at 37°C for 24 hr in DMEM with 10% (v/v) fetal bovine serum (FBS), cells were washed with serum-free DMEM and incubated for 30 min. Cells were washed with serum-free DMEM again and incubated with 5 μg/ml tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin in serum-free DMEM for 5 min. Subsequently, trypsin was neutralized by adding FBS to a final concentration of 10%. Cells were incubated for 30 min with 10 μg/ml CT302 IgG₁, washed with PBS, and incubated with low-pH fusion-inducing buffer (150 mM NaCl buffered to pH 5.0 with 10 mM HEPES). Cells were returned to DMEM with 10% FBS and incubated 2–3 hr at 37°C. Finally, cells were fixed with ice-cold methanol and stained with trypan blue.

The Y111 is a recombinant anti-PD-L1 and anti-CD3 (PD-L1 x CD3) bispecific antibody (Figure 1A) generated from the CHO cell expression system. The anti-PD-L1 monovalent unit was from the drug bank website (https://go.drugbank.com/drugs/DB11595). The anti-PD-L1 sequence was reversely translated into the DNA sequence, and the anti-CD3 single-chain DNA sequence was reversely translated from the protein sequences of anti-CD3 monoclonal antibody 2A5 (27 ). These coding gene sequences were synthesized, inserted into the pEASY-T1 vector (Transgene, Beijing, China), and verified by sequencing the entire vectors by Huada Gene (Wuhan, China). The control molecule, CD3 Isotype, targeting both CD3 and fluorescein [derived from Clone 4-4-20 (28 (link))] was similarly constructed (Supplementary Figure 1). Subsequently, these expression vectors were transfected into the CHO cells (Invitrogen, Carsbad, USA) using Fecto PRO Reagent (Ployplus, New York, USA) according to the manufacturer’s protocols. After culturing for 7-days, the supernatant was collected and purified serially by Sepharose Fast Flow protein A affinity chromatography column (GE, Milwaukee, USA), Fab Affinity KBP Agarose High Flow Resin (ACROBio systems, Newark, USA), and SP cation exchanged chromatography column (GE, Milwaukee, USA).