Does anyone think we can't provide Occupancy? Or was this just a delay tactic by Gilead - ugh I mean the FDA?
Leronlimab CCR5 RO. To measure the percentage of CCR5 RO on the surface ofCD4+T cells, we developed the RO equation shown in Supplementary Fig. 5.%RO¼%IgG4%IgG4þ%LeronlimabPB ́100%The equation measures unoccupied CCR5 receptors by using Pacific Blue-conjugated Leronlimab (termed Leronlimab-PB). CCR5 RO is defined as thepercentage of cells CCR5+(measured by clone 3A9) and Leronlimab+(measuredby anti-human IgG4) divided by the percentage of cells CCR5+and Leronlimab+(measured by the sum of anti-human IgG4 and Leronlimab-PB) cells followingincubation with a saturating concentration of Leronlimab-PB. This method isbased on RO assays for anti-PD-1 antibodies in clinical trials38.PBMC or single cells from tissue homogenates (0.3–1×106) were stained withanti-human IgG4 for 30 min at RT in the dark. Next, cells were washed once withFACS buffer and three times with PBS and then stained with CD45, CD3, CD4,CD8, CD16, CD14, amine-reactive dye, CCR5 (via antibody clone 3A9), andLeronlimab-PB for 30 min at RT in the dark. Finally, cells were washed twice withPBS andfixed with 2% PFA for more than 30 min before collecting on LSR-IIinstrument and FACsDIVA version 6.1. Using FlowJo v10, cells were gated onCD45+, singlet, live, CD3+, CD4+, and CCR5+(via 3A9 staining) events. TheCD4+CCR5+population was further gated on human IgG4+or Leronlimab-PB+events.