Fig 2: Anti-TEM1 tBs redirect primary human T cell effector functions toward TEM1+ target cells(A and B) Anti-TEM1-tBs specifically activate primary human T cells in the presence of TEM1-expressing A673 and SK-N-AS cells. Upregulation of early activation markers CD69 and CD25 (A) and the inhibitory co-receptor PD1 (B) was measured by flow cytometry after 16 h of co-culture (E:T ratio 1:1, 1 nM tB).(C) Upregulation of CD69 and CD25 by CD8+ T cells co-cultured with TEM1+ A673 WT cells or TEM1-depleted A673 TEM1KO cells in the presence of anti-TEM1-tBs.(D) Anti-TEM1-tBs redirect primary human T cells to kill endogenous TEM1-expressing cells (A673 or SK-N-AS), but spare TEM1-negative AsPC-1 cells. tBs were added at 1 nM, and specific cell killing was calculated as a percentage of complete lysis achieved with 1% Triton X-100. Statistical test was a 2-way ANOVA with multiple comparisons. The significance rating is based on the comparison of the test sample to the “no tB”control for each cell line.(E) Quantification of effector cytokines secreted by polyclonal human T cells stimulated with anti-TEM1 tBs in the presence of TEM1-expressing A673 or SK-N-AS cells or irrelevant AsPC-1 cells for 24 h (E:T ratio 5:1). IFN-? and TNF-a secretion were quantified after stimulation with 1 nM tB, and IL-2 was measured after activation with 10 nM tB.(F ang G) Cytotoxicity exerted by human T cells that were redirected with anti-TEM1 tBs is dependent on TEM1. Filled symbols, TEM1+ A673 WT cells; open symbols, A673 TEM1KO cells. (F) Real-time kinetics of cell killing by pan-T cells redirected by different anti-TEM1 tBs (0.1 nM) over the course of 30 h (E:T ratio 5:1). (G) Cell killing was assessed after 24 h of co-culture (E:T ratio 5:1) as a function of tB concentration. Data are represented as mean ± SD. Representative results of n = 3 experiments (A–C, F, and G) or n = 2 experiments (D and E) using T cells from different donors are shown. See also Figure S5.

Fig 3: Effect of unmodified cNMPs and of membrane-permeant cNMP-AM esters on aCD3-antibody-induced IL-2 production of HuT-78 lymphoma cells. Cells were incubated for 24 h in aCD3-antibody-coated plates with 100 µM of cNMPs (a) or 100 µM of cNMP-AMs (b) The “DMSO” and the “PO4(AM)3” bars represent controls for the DMSO content of the cNMP-AM samples and for the intracellular hydrolysis products of the AM esters. Data shown are means ± SD from n = 3 independent experiments. Statistics: one-way ANOVA and Dunnet’s multiple comparison test with medium (a) or PO4(AM)3 (b) as control columns. Asterisks indicate significance level: * = p < 0.05; ** = p < 0.01

Fig 4: In vitro characterization of convertibleCAR activity.a Ramos (CD20+) target cells were exposed to convertibleCAR-CD8+ cells at an E:T of 5:1 and co-cultured with increasing concentrations of rituximab antibody (ADCC-deficient), Rituximab.LC-U2S3 MicAbody, or Trastuzumab.LC-U2S3 MicAbody. After 24 h, supernatants were harvested and IL-2 (solid bars) or IFN? (hatched bars) quantified by ELISA. b convertibleCAR-CD8+ cells were incubated with increasing concentrations of Alexa Fluor 647 conjugated Rituximab.LC-U2S3 for 30 min, the excess washed away, and the MFI quantified by flow cytometry. Red line at 5 nM indicates inflection point at which receptors are maximally occupied. c convertibleCAR-CD8+ cells were armed with increasing concentrations of Rituximab.LC-U2S3 as described in (b) then co-incubated with calcein-loaded Ramos cells at an E:T of 20:1 for 2 h after which the amount of released calcein was quantified. d iNKG2D.YA-CAR CD8+ cells were pre-armed with 5 nM Rituximab.LC-U2S3, 5 nM Trastuzumab.LC-U2S3, or an equimolar mixture of 2.5 nM of each as described in (b) then exposed to calcein-loaded Ramos or CT26-Her2 cells at two indicated E:T ratios. The amount of calcein released was quantified after two hours. Except for b, data are representative of at least two independent experiments and plotted as an average of technical triplicates.

Fig 5: c-Met CAR Jurkat specifically recognizes the c-Met positive GC cells. (A) The mock CD8sp-c-Met CAR, or CSF2Rsp-c-Met CAR Jurkat cells (1.5 × 105 cells) were co-cultured with GC cell lines (1 × 104 cells) at an E:T ratio of 15:1. After overnight incubation, supernatants were collected and ELISA was performed to measure IL-2 level. The bars show mean ± S.D. of experiments performed in triplicate. Statistical analysis was performed by the paired t-test. *** p = 0.01, compared with the mock Jurkat group. (B) The mock CD8sp-c-Met CAR, CSF2Rsp-c-Met CAR, or FITC CAR Jurkat cells were co-cultured with GC cell lines in the presence or absence of purified soluble c-MET protein. The next day, supernatants were collected and IL-2 ELISA was performed. The bars show mean ± S.D, of experiments performed in triplicate. Statistical analysis was performed by the paired t-test. *** p = 0.01, compared with the c-Met absence group.