Supplemental Figure 1. CD69 antigen-response curves of CAR engrafted Jurkat T cells. Supplemental Figure 2.

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1 Supplemental Figure 1. CD69 antigen-response curves of CAR engrafted Jurkat T cells. To evaluate the antigen sensitivity of mutant CARs transduced Jurkat T cells were stimulated with varying concentrations of immobilized CEA for 24 hours before analysis by flow cytometry for expression of the activation marker CD69. (A) Antigen responses of a panel of different CARs. (B) Antigen responses of dimer mutant CARs. (C) Antigen responses of D6 mutant CARs. (D) Antigen responses of combined C2 and D6 mutant CARs Supplemental Figure 2. Antigen sensitivity and TCR-interactions by a mutant F10A CAR. To highlight the specificity of transmembrane targeted mutations an F10A mutant receptor was generated which was hypothesized to have no functional effect on antigen sensitivity or to abrogate TCR interactions. Non-reducing immunoblot analysis of F10A CAR engrafted Jurkat T cell lysate (A). Antigen sensitivity was assessed by measuring upregulation of CD69 in response to 24 hours stimulation by immobilsed CEA (B). MA5.8 cells were transduced with the F10A mutant CAR (C) and analyzed for upregulation of CD3 surface expression (D). FCIP analysis was conducted on lysates from transduced Jurkat T cells with fold increase in anti- TCRV 8 (E) or CEAhFc/anti-hIgG (F) binding analyzed. ** p < 0.01 by 2-way ANOVA with a post-hoc Dunnet test. Data represents mean ± SD from three independent experiments. Supplemental Figure 3. Mutant CAR activity in primary human T-cells. To evaluate CAR activity in primary human T-cells transduced or non-transduced cells were first enriched and then expanded in the presence of irradiated autologous feeder cells to obtain populations with enhanced CAR expression. Cells were equilibrated with mock transduced cells to obtain populations with equivalent CAR surface expression before incubation with varying concentrations of immobilized CEA protein for 24 hours. IFN secretion was analyzed by ELISA from the CAR dimerisation panel (A) or the D6 mutant panel (B). Data represents average ± SD from one donor in triplicate. To assess the effect of transmembrane mutations on tumour killing transduced donor T-cells were incubated with CEA-expressing MKN45k tumour cells before staining for the degranulation marker CD107a. Increase in mean fluorescence intensity values compared to cells incubated without tumour cells is shown for each CAR mutant (C). Surface expression of CAR and CD3 is shown for each cell population expressing the different mutant CARs with CD3 MFI values for CAR + cells indicated above each dotplot (D). Results are from one donor each in triplicate ± SD. Supplemental Figure 4. FCIP negative control and primary human T-cell analysis. To evaluate the specificity of the FCIP approach interactions between MFE.FLAG and three molecules highly expressed in Jurkat T-cells was assessed. (A) Anti-FLAG beads were incubated with lysates from non-transduced or MFE.FLAG expressing Jurkat T-cells and stained with anti-tcrv 8, anti-cd28, anti-cd45ra or anti-cd95 PE antibodies. Analysis was conducted using an LSRII cytometer and FlowJo software. Results are averages ± SD from three experiments. (B) Lysates from non-transduced primary human T-cells or cells expressing MFE.FLAG or MFE.htm.FLAG were incubated with anti-flag beads before staining with anti- 1

2 CD3 PE or CEAhFc/anti-hIgG-PE. Analysis was conducted using an LSRII cytometer and FlowJo software. Results are averages of two experiments. Supplemental Figure 5. Native PAGE analysis of TCR complexes from tranduced and non-transduced Jurkat T-cells. Digitonin lysates from nontransduced Jurkat T-cells or cells expressing MFE or MFE.C2G G-10C were separated on a 4-15 % tris-glycine gel, transferred to PVDF membranes and probed with anti-cd3 or anti-flag antibodies. Results from various exposure times are indicated with arrow indicating the position of the endogenous TCR complex. Approximate molecular weight markers are also shown. 2

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