Fab affinity kbp agarose high flow resin

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).

The bsAbs, including Vγ2 x PD-L1 and Vγ2 x Null, were generated similarly to Y111 described previously by Yang et al. (23 (link)). Briefly, the expression plasmids for Vγ2 x PD-L1 and Vγ2 x Null were synthesized and verified by sequencing in AuGCT Biotech (Wuhan, China). Then these expression vectors were transfected into cGMP banked CHO-S cells (Invitrogen, Carlsbad, USA) using the Fecto PRO Reagent (Ployplus, New York, USA) according to the manufacturer’s protocol, respectively. After a week, the cell culture supernatant was collected and serially purified 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). Finally, the purified proteins were analyzed by SDS-PAGE and size-exclusion chromatograms. The Vγ2 x Null served as the control molecule for Vγ2 x PD-L1, with both molecules sharing the same backbone and Vγ2-targeting scFv part. Similarly, its two parental monoclonal antibodies (Vγ2 mAb (Clone 7A5) and PD-L1 mAb (23 (link))) were produced.