Biotin conjugated cetuximab

Apheresis products were obtained from healthy donors and peripheral blood mononuclear cells were isolated using Ficoll-Paque (GE Healthcare). CD8⁺ T cells were obtained by positive selection using immunomagnetic microbeads (Miltenyi Biotec), and activated with anti-CD3/CD28 beads (Life Technologies). On day three, activated CD8⁺ T cells were transduced with the CAR containing lentivirus. The EGFRt⁺ CAR-T cell subset was enriched by immunomagnetic selection with biotin-conjugated cetuximab (Bristol-Myers Squibb) and streptavidin microbeads (Miltenyi Biotec). T cells used as mock negative controls alongside CAR-T cells in experiments were not lentivirally transduced. CAR and mock control T cells were stimulated with irradiated peripheral blood mononuclear cells, irradiated TMLCL, and OKT3 (30 ng/mL, Miltenyi Biotec), expanded according to a rapid expansion protocol [36 (link)] and cryopreserved until further use. Cryopreserved cells were thawed, expanded as described above and functional in vitro assays were conducted between days 11 and 16 of culture.

Apheresis products were obtained from healthy donors (Charité ethics committee approval EA2/216/18) and peripheral blood mononuclear cells were isolated using Ficoll-Paque (GE Healthcare). CD4⁺ and CD8⁺ T cells were obtained by positive selection using immunomagnetic microbeads (Miltenyi Biotec), and activated with anti-CD3/CD28 beads (Life Technologies). On day three, activated T cells were transduced with the CAR-containing lentivirus. EGFRt⁺ CAR T cells were enriched by immunomagnetic selection with biotin-conjugated cetuximab (Bristol-Myers Squibb) and streptavidin microbeads (Miltenyi Biotec). Untransduced T cells were used as negative controls alongside CAR T cells in all experiments. CAR T cells and control T cells were cryopreserved until further use. Cryopreserved cells were thawed, stimulated with irradiated peripheral blood mononuclear cells, irradiated lymphoblastoid TMLCL cells, and OKT3 (30 ng/mL, Miltenyi Biotec), and expanded according to a rapid expansion protocol (26 (link)). CD4⁺ T cells were supplied with IL2 (50 U/μl) and IL7 (10 ng/μl) and CD8⁺ T cells were supplied with IL2 (50 U/μl) and IL15 (10 ng/μl) every other day following expansion. Functional in vitro assays were conducted between days 11 and 16 of culture.