Cytokines and chemokines from EDTA plasma, plasma from whole-blood IFN-γ release assay incubations, and medium from in vitro cell stimulations were measured at ImmusanT Inc. using a magnetic bead–based assay according the manufacturer’s protocol (MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel; EMD Millipore Corp., Billerica, MA, USA; MAGPIX, Luminex Corporation, Austin, TX, USA). Final concentrations were the average of triplicate measurements. Olink Proteomics (Uppsala, Sweden) was provided frozen EDTA plasma collected at baseline 2, 4, and 6 hours after the first dose from three patients in the first cohort and three in the last cohort of the 16-dose study. Olink performed a 92-plex PEA with the Proseek Multiplex Inflammation I 96 × 96 panel. Cytokine data were expressed as relative fold change from predose levels. IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α, eotaxin, eotaxin-3, IL-8, IL-8 (HA), IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, TARC (thymus and activation regulated chemokine) in EDTA plasma, and sera were measured at ImmusanT Inc. using ECL assay kits (V-PLEX Proinflammatory Panel 1 Human Kit, V-PLEX Chemokine Panel 1 Human Kit, or V-PLEX IL-2, IL-8, and IL-10 as a three-plex; Meso Scale Discovery, Rockville, MD). Data were analyzed by DISCOVERY WORKBENCH 4.0. Calculated values lower than LLOQ (lower limit of quantification) or values on the low end of the scale that were not achieved using the standard curves, were reported as equal to the LLOQ concentration analyzed for each cytokine on each plate.

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