Production of minicircles

The MC vectors MC-mock and MC-MTA (anti-CD25/IL-10/ CXCR3) were produced as described previously [27]. It is noteworthy that MTA is a combination of anti-CD25 heavy chain (HC)-IL-10-CXCR3 and anti-CD25 light chain (LC), and anti-CD25 is a combination of MC-anti-CD25HC and LC. To construct the MCs, each sequence (Supplementary Tables 1 and 2) was subcloned into the mock plasmid pMC.EF1-MCS-T2A-RFP (or luciferase [LUC])-SV40 PolyA, which was purchased from System Biosciences (Mountain View, CA, USA). The plasmid contained Nhe1 and BamHI restriction sites, and the cloning outcome was confirmed by double digestion at the two sites. The MCs were produced based on the method previously described by Kay et al. [28]. Briefly, Escherichia coli ZYCY10P3S2T cells were transformed with the protein drug-encoding parent plasmids (PPs). E. coli cells were grown overnight at 37°C in Terrific Broth containing 50 μg/mL of kanamycin and then the Luria–Bertani broth containing 0.02% l-(+)-arabinose was added to the E. coli cultures. The mixture was incubated at 30°C for 5 hours, and MC DNA was isolated using Nucleobond Xtra Purification Kits (Macherey-Nagel, Duren, Germany), according to the manufacturer’s instructions.