We cloned and expressed canine-ized cytokines IL-2 and IL-12 fused to the collagen-binding domain LAIR1 as detailed in the Supplementary Methods S1 (sequences in Supplementary Table S1 ). To assess IL-2 bioactivity, 10,000 CTLL-2 cells (ATCC Cat# TIB-214, RRID:CVCL_0227) were plated per well in incomplete T-cell media (RPMI 1640, 10% fetal bovine serum, 2mM L-glutamine, 1mM sodium pyruvate) and incubated for 48h at 37°C with dilutions of the IL-2 fusion protein. After incubation, cell viability was assessed using the CellTiter-Glo 2.0 assay (Promega). The HEK Blue IL-12 (Invivogen; RRID:CVCL_UF31) assay was performed according to vendor instructions using dilutions of the collagen-binding IL-12 cytokine. Collagen-binding of both cytokines was evaluated through ELISA with rat collagen I-coated plates and an anti-hexahistidine detection antibody (Abcam Cat# ab1269, RRID:AB_299333) as previously reported(16 (link)). Low endotoxin levels (<5 EU/kg/dose) were confirmed for each cytokine batch using the Endosafe Nexgen-PTS system (Charles River Labs) with typical values <1 EU/mg protein.
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Evaluating Engineered Canine Cytokines
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Other organizations : Massachusetts Institute of Technology, University of Illinois Urbana-Champaign
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Variable analysis
independent variables
- Dilutions of the IL-2 fusion protein
- Dilutions of the collagen-binding IL-12 cytokine
- Cell viability (assessed using the CellTiter-Glo 2.0 assay) for the IL-2 fusion protein
- Response in the HEK Blue IL-12 assay for the collagen-binding IL-12 cytokine
- Collagen-binding of both cytokines (evaluated through ELISA with rat collagen I-coated plates)
- Incomplete T-cell media (RPMI 1640, 10% fetal bovine serum, 2mM L-glutamine, 1mM sodium pyruvate)
- Incubation conditions (37°C, 48h)
- None specified
- Low endotoxin levels (<5 EU/kg/dose) confirmed for each cytokine batch using the Endosafe Nexgen-PTS system
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