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

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journal contribution
posted on 13.10.2021, 20:40 by Shiho Ito, Kyoko Miwa, Chiharu Hattori, Tetsuo Aida, Yoshimi Tsuchiya, Kazuhiko Mori

Immunostimulatory effects of monoclonal antibodies (mAb) through binding to F receptors (FR) on immune cells are a likely cause of cytokine release syndrome. However, it is difficult to detect the potential risk of FR-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 FR-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 FR involvement in cytokine release associated with panitumumab, trastuzumab, rituximab, and alemtuzumab, CRA of these four mAb were conducted with anti-FRI, -FRII, or -FRIII F(ab’)2 fragments. The results showed cytokine release caused by trastuzumab, rituximab, and alemtuzumab was significantly suppressed by anti-FRIII F(ab’)2 pretreatment, and slightly reduced by anti-FRI or anti-FRII pretreatment, indicating these mAb induced FR (especially FRIII)-dependent cytokine release from PBMC. Cytokine release caused by panitumumab was slightly suppressed by anti-FRIII 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 FR-dependent cytokine release potential of mAb.