Highly sensitive in vitro cytokine release assay incorporating high-density preculture

Abstract

Immunostimulatory effects of monoclonal antibodies (mAb) through binding to F_cγ receptors (F_cγR) on immune cells are a likely cause of cytokine release syndrome. However, it is difficult to detect the potential risk of F_cγR-dependent cytokine release associated with mAb in the current standard cytokine release assays (CRA), including the air-drying solid-phase method using human peripheral blood mononuclear cells (PBMC). To increase the sensitivity to detect F_cγR-dependent cytokine release due to mAb, a high-density preculture (HDC) method was incorporated into the air-drying solid-phase CRA. Here, PBMC were exposed to panitumumab, trastuzumab, rituximab, or alemtuzumab at 0.1, 0.3, 1, and 3 μg/well for 24 or 48 hr under both non-HDC and HDC conditions. T-cell agonists (anti-CD3 mAb, anti-CD28 super-agonist [SA] mAb) were used as reference mAb. Panitumumab, trastuzumab, rituximab, or alemtuzumab induced cytokine release under both non-HDC and HDC conditions, and cytokine release caused by alemtuzumab was more pronounced under HDC conditions. To investigate F_cγR involvement in cytokine release associated with panitumumab, trastuzumab, rituximab, and alemtuzumab, CRA of these four mAb were conducted with anti-F_cγRI, -F_cγRII, or -F_cγRIII F(ab')₂ fragments. The results showed cytokine release caused by trastuzumab, rituximab, and alemtuzumab was significantly suppressed by anti-F_cγRIII F(ab')₂ pretreatment, and slightly reduced by anti-F_cγRI or anti-F_cγRII pretreatment, indicating these mAb induced F_cγR (especially F_cγRIII)-dependent cytokine release from PBMC. Cytokine release caused by panitumumab was slightly suppressed by anti-F_cγRIII F(ab')₂ pretreatment. Anti-CD3 mAb and anti-CD28 SA mAb also induced significant release of cytokines under HDC conditions compared with that under non-HDC conditions. In conclusion, CRA incorporating HDC into the air-drying solid-phase method using human PBMC could sensitively capture the F_cγR-dependent cytokine release potential of mAb.

Keywords: Cytokine release syndrome; FcγR; cytokine release assay; high-density preculture; peripheral blood mononuclear cell; therapeutic monoclonal antibody.