HT1376, NCI-H292, PC3, and
CT26 cells were obtained from ATCC (American Type Culture Collection).
MC38 cells were obtained from the National Cancer Institute (L-159-2018/1).
CT26 and MC38 cells were engineered to express mouse Nectin-4 (NM_027893.3)
as described.21 (link) Human peripheral blood
mononuclear cell (PBMC) isolation was described.21 (link)Recombinant proteins: human CD137 (92 204B, R&D
Systems) and human CD137L (2295-4L-025, R&D Systems) were purchased.
Human Nectin-4 (residues 32–349) and rat Nectin-4 (residue
31–347) with a gp67 signal sequence and C-terminal FLAG tag
were cloned into pFastbac-1 and baculovirus using standard Bac-to-Bac
protocols (Life Technologies). Sf21 cells at 1 × 106 mL–1 in Excell-420 medium (Sigma) at 27 °C
were infected at a multiplicity of infection (MOI) of 2 with a P1
virus stock for protein expression. The supernatant was harvested
at 72 h and incubated for 1 h at 4 °C with anti-FLAG M2 affinity
agarose resin (Sigma) followed by a phosphate-buffered saline (PBS)
wash. Resin was subsequently transferred to a column and washed extensively
with phosphate-buffered saline (PBS). Protein was eluted with 100
μg/mL FLAG peptide concentrated to a volume of 2 mL and loaded
onto an S-200 Superdex column (GE Healthcare) in PBS at 1 mL/min.
2 mL fractions were collected and fractions containing Nectin-4 protein
were concentrated.
CT26 cells were obtained from ATCC (American Type Culture Collection).
MC38 cells were obtained from the National Cancer Institute (L-159-2018/1).
CT26 and MC38 cells were engineered to express mouse Nectin-4 (NM_027893.3)
as described.21 (link) Human peripheral blood
mononuclear cell (PBMC) isolation was described.21 (link)Recombinant proteins: human CD137 (92 204B, R&D
Systems) and human CD137L (2295-4L-025, R&D Systems) were purchased.
Human Nectin-4 (residues 32–349) and rat Nectin-4 (residue
31–347) with a gp67 signal sequence and C-terminal FLAG tag
were cloned into pFastbac-1 and baculovirus using standard Bac-to-Bac
protocols (Life Technologies). Sf21 cells at 1 × 106 mL–1 in Excell-420 medium (Sigma) at 27 °C
were infected at a multiplicity of infection (MOI) of 2 with a P1
virus stock for protein expression. The supernatant was harvested
at 72 h and incubated for 1 h at 4 °C with anti-FLAG M2 affinity
agarose resin (Sigma) followed by a phosphate-buffered saline (PBS)
wash. Resin was subsequently transferred to a column and washed extensively
with phosphate-buffered saline (PBS). Protein was eluted with 100
μg/mL FLAG peptide concentrated to a volume of 2 mL and loaded
onto an S-200 Superdex column (GE Healthcare) in PBS at 1 mL/min.
2 mL fractions were collected and fractions containing Nectin-4 protein
were concentrated.
