Endothelial cells co-stimulate peripheral blood mononuclear cell responses to monoclonal antibody TGN1412 in culture

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

“…TGN1412, Fab and F(ab9) 2 fragments derived from TGN1412, and the panel of recombinant NIB(28SA) mAbs were presented to PBMCs either immobilized by wet-coating onto microtiter plates (11) or in aqueous phase in the presence of an endothelial (HUVEC) cell-derived monolayer (12). These assays were carried out under aseptic conditions using reagents and consumables that were sterile and pyrogen-free.…”

“…Two hundred microliters of PBMCs resuspended in RPMI 1640 culture medium supplemented with 15% (v/v) FCS, 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin) were added to coated plates to give 2 3 10 5 cells/well and incubated in a humidified incubator at 37˚C with 5% (v/v) CO 2 . After 48 h, supernatants were harvested and assayed by ELISA for TNF-a, IL-2, and IFN-g as described previously (12). Proliferation in the remaining cells was assessed by tritiated thymidine incorporation as described previously (12).…”

“…After 48 h, supernatants were harvested and assayed by ELISA for TNF-a, IL-2, and IFN-g as described previously (12). Proliferation in the remaining cells was assessed by tritiated thymidine incorporation as described previously (12).…”

“…Cocultures of PBMCs and primary HUVECs, isolated in-house from umbilical cords, in the presence of aqueous mAbs or fragments were carried out in 96-well flat-bottom microtiter plates (Falcon BD or Nunc, Roskilde, Denmark) as described previously (12). Briefly, mAbs or fragments (0.007-10 mg/well mAb or molar equivalents of fragments) were incubated with PBMCs (12.5 3 10 4 cells/well) in 300 ml RPMI 1640 culture medium supplemented with 2% (v/v) human AB serum, 2 mM L-glutamine, antibiotics, and nonessential amino acids over monolayers of primary HUVECs seeded at 3 3 10 4 cells/well 24 h previously in 100 ml culture medium containing 10% (v/v) human AB serum (supernatants aspirated prior to addition of PBMCs).…”

“…Nevertheless, in the first-in-man phase I trial of TGN1412, all six volunteers receiving the Ab suffered systemic inflammatory response syndrome and life-threatening multiorgan failure (4). The reason(s) for the failure of preclinical testing to predict the severity of the reactions in humans has been the subject of speculation and investigation (5)(6)(7)(8)(9)(10)(11)(12)(13). Suggested contributing factors have included a role for differences between species in Fcmediated interactions with FcgRs in bringing about cross-linking of CD28 and thereby triggering T cell activation and associated cytokine release and proliferation of PBMCs (8,9), although experimental data on the possible influence of Fc on the immunomodulatory activity of TGN1412 has to date been lacking.…”