αpd l1 10f 9g2

PEG-PCL copolymer production was based on a previous report [40 (link)]. Then, αPDL1 (10F.9G2; BioXCell, Lebanon, NH, USA) conjugates were developed as previously proposed [19 (link)]. In brief, dry PEG-PCL NPs underwent incubation with αPDL1 in borate buffer containing EDAc (N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride) and Sulfo-NHS (N-Hydroxysulfosuccinimide, Shanghai, China), overnight at ambient. The samples were centrifuged, and the resulting pellet was resuspended with ultrapure water after washing, for subsequent assays. The supernatant was assessed for antibody amounts at 595 nm on a microplate plate Reader (Synergy HT, BioTek, Winooski, VT, USA). The antibody amounts on NPs were obtained as initial levels minus supernatant amounts.

C57BL/6 mice were given 5 × 10⁵ B16-OVA cells s.c. in the dorsal flank; 3, 8, and 18 days later 20 μg MS-OVA was injected s.c. at the base of the tail away from the tumor site. A total of 100 μg α-CTLA-4, Clone 9D9 (BioXcell, West Lebanon, NH, USA) was given s.c. alongside the archaeosome treatment (days 3, 8, and 18). A total of 250 μg each of α-PD-1, RMP1-14 (BioXcell, West Lebanon, NH, USA) and α-PD-L1, 10F.9G2 (BioXcell, West Lebanon, NH, USA) was given i.p. on days 9, 12, 15, and 18. Rationale for timing and injection site choice for checkpoint inhibitors: α-CTLA-4 was given alongside the archaeosome vaccine s.c. so that it would drain to the same lymph nodes and act on the same CD8⁺ T cells that were being activated by the vaccine. α-PD-1 and α-PD-L1 were given in parallel i.p. at the time when tumors were thought to be growing so they could systemically act on augmenting CD8⁺ T cells responses to the tumor.