Fig. 1: CD28 transmembrane in CARs containing a 4-1BB costimulatory domain enhances anti-tumor activity in vitro and in vivo.

a Illustration of a TAG72-CAR T-cell containing the humanized scFv targeting TAG72 with varying five extracellular spacer domains (EQ, dCH2, CD8h, HL, L), three transmembrane domains (CD4tm, CD8tm, CD28tm), and two intracellular costimulatory domains (4-1BB, CD28) followed by a cytolytic domain (CD3z). b Untransduced (UTD) and seven different TAG72-CAR T cells positively enriched for CD19t were evaluated by flow cytometry for Protein L to detect the scFv. c, d In vitro tumor cell killing activity relative to UTD (c) and IFNγ production by ELISA (d), of CAR T cells against tumor targets (TAG72- OVCAR8; TAG72+ OVCAR3, OV90, and OVCAR8-sTn) after 24 hr (for ELISA) or 72 hr of co-culture at an effector:target (E:T) ratio of 1:4. n = 9 from three independent experiments. Data are presented as mean values ±SEM. P values indicate differences between TAG72-dCH2(28tm)28z and TAG72-dCH2(28tm)BBz using a two-tailed Student’s t test. e Western blotting analysis of early downstream signaling mediators following CAR T-cell stimulation of indicated TAG72-CAR T cells. f Quantification of band density of phosphoprotein over their respective total protein levels. n = 2 per timepoint, representative of two independent experiments. Data are presented as mean values ±SD. g TAG72-CAR T-cell killing of OV90 cells measured by xCELLigence over 10 days (E:T = 1:20). h Schema of repetitive tumor cell challenge assay (top). TAG72-CAR T cells were co-cultured with OV90 cells (E:T = 1:2) and rechallenged with OV90 cells every two days. Remaining viable tumor cells and fold change in TAG72-CAR T cells were quantified as described in Methods prior to each tumor cell rechallenge. n = 6–9/group from at least two independent experiments. Data are presented as mean values ±SEM. P values indicate differences between 28tm28z and 28tmBBz using a two-tailed Student’s t test. i Representative bioluminescent flux imaging of intraperitoneal (i.p.) OVCAR3(eGFP/ffluc) tumor-bearing female NSG mice treated i.p. with UTD or indicated TAG72-CAR T cells. j Quantification of flux (individual mice in each group) from treated OVCAR3 tumor-bearing mice. UTD (n = 8/group); TAG72-CAR T cells (n = 10/group). Curative responses: UTD: 0/10, 4tmBBz: 0/10, 28tm28z: 0/10, 28tmBBz: 4/10. P < 0.005 comparing 28tm28z and 28tmBBz using a Multiple Mann–Whitney test.