Volume 21, Issue 1, Pages 17-26 (October 2017) Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent  Diogo Gomes-Silva, Malini Mukherjee, Madhuwanti Srinivasan, Giedre Krenciute, Olga Dakhova, Yueting Zheng, Joaquim M.S. Cabral, Cliona M. Rooney, Jordan S. Orange, Malcolm K. Brenner, Maksim Mamonkin  Cell Reports  Volume 21, Issue 1, Pages 17-26 (October 2017) DOI: 10.1016/j.celrep.2017.09.015 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 17-26DOI: (10.1016/j.celrep.2017.09.015) Copyright © 2017 The Authors Terms and Conditions

Figure 1 High Expression of BB.z CARs in T Cells Enhances Apoptosis (A) Schematic representation of the expression systems utilized to express BB.z CARs in T cells. (B) Expression of CD19- and GD2-specific CARs on the cell surface of T cells 4 days post-transduction; mean fluorescence intensities are plotted on the bar graphs. (C) Relative magnitude of tonic signaling in BB.z and IRES BB.z CAR T cells 7 days post-transduction measured by western blot analysis. Numbers indicate relative signal intensity normalized to GAPDH. (D) Overall expansion of T cells retrovirally transduced with CD19- and GD2-specific CARs relative to that of control mock-transduced T cells. (E) Representative histograms showing Annexin V staining of CD19 CAR T cells 7 days post-transduction. Bar graphs show summarized data for both CARs from 3 donors. (F) Representative histograms showing expression of CD19-and GD2-specific CARs in expanding T cells 4 and 11 days post-transduction. Data represent two to five independent experiments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 by one-way ANOVA. Cell Reports 2017 21, 17-26DOI: (10.1016/j.celrep.2017.09.015) Copyright © 2017 The Authors Terms and Conditions

Figure 2 High Expression of BB.z CD19 CAR Alters the Phenotype and Undermines the Anti-tumor Activity of CAR T Cells (A) Relative proportion of naive-phenotype (TN), central memory (TCM), effector memory (TEM), and terminally differentiated effector cells (TEMRA) CD19 CAR T cells 14 days post-transduction defined by CCR7 and CD45RA expression. (B) Stacked bar graph showing relative proportion of CD4+ and CD8+ cells among CD19 CAR T cells 14 days post-transduction. (C) CD19 CAR T cells were cocultured with GFP+ Raji cells (CD19+) at a 1:4 ratio for 72 hr. Representative dot plots show the frequency of residual live tumor cells at the end of coculture. Numbers indicate percent of tumor cells among total live cells. (D) Absolute cell counts of Raji and CAR T cells at the end of coculture determined by flow cytometry using counting beads. Data from individual donors are shown on each bar graph. (E) Schematic diagram of the experiment. NSG mice were intravenously engrafted with 1 × 106 GFP+ FFluc+ NALM-6 cells followed by a single intravenous injection of 1.0 × 106 CD19 CAR T cells 3 days later. (F) Representative staining of peripheral blood of tumor-bearing mice 17 days after tumor engraftment with relative frequency of CD19 CAR T cells and GFP+ NALM-6 cells. Relative frequencies of CD19 CAR T cells in peripheral blood 17 days post-engraftment are shown on the bar graph. (G) Kinetics of NALM-6 luminescence in mice measured by IVIS. (H) Kaplan-Meier curves showing advantage in overall survival of mice receiving IRES BB.z CD19 CAR T cells compared to mice receiving BB.z T cells (p = 0.0036 by Mantel-Cox log-rank test). Data represent 2–3 independent experiments. ∗p < 0.05; ∗∗p < 0.01 by Student’s t test. Cell Reports 2017 21, 17-26DOI: (10.1016/j.celrep.2017.09.015) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Enhanced Apoptosis and Increased Expression of BB.z CAR in T Cells Requires 4-1BB-Derived TRAF2 Signaling (A) A schematic drawing of TRAF2 binding motifs in the 4-1BB domain and corresponding alterations in the protein sequence to disrupt either the N-terminal (mut 1) or the C-terminal (mut 2) motif. Histogram on the right shows relative expression of CD19 CARs with mutated TRAF2 motifs. (B) Frequency of apoptotic cells among CD19 CAR T cells with intact or mutated costimulatory domains 7 days post-transduction. (C) Overall expansion of CD19 CAR T cells on day 7 post-transduction. (D) Tonic CD19 CAR signaling and total CAR expression in T cells 7 days post-transduction was measured with anti-pTyr (CD3ζ) and total anti-CD3ζ antibodies by western blot. Numbers indicate relative signal intensity normalized to GAPDH. (E and F) Relative phosphorylation of IKKα/β in normal and TRAF-mutant (E) and IRES-reduced (F) BB.z CD19 CAR T cells 7 days post-transduction by a western blot analysis. (G) Relative abundance of CD19 CAR transcripts was measured by calculating relative expression of retroviral mRNA normalized to the copy number of CD19 CAR-containing proviruses per 100 ng of genomic DNA by qPCR in T cells 7 days post-transduction. Data represent 2–3 independent experiments with n = 3 donors in each. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 by one-way ANOVA. Cell Reports 2017 21, 17-26DOI: (10.1016/j.celrep.2017.09.015) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Tonic 4-1BB Signaling Promotes FAS-Mediated Apoptosis in CAR T Cells (A) Representative staining for surface FAS, FASL, and TRAIL by flow cytometry in CD19 CAR T cells 8 days post-transduction. (B) MFI of FAS and FASL on the surface of CD19 CAR on day 8 post-transduction. ∗∗p < 0.01; ∗∗∗p < 0.001 by one-way ANOVA. (C) Representative images showing cellular localization of CD19 CAR fused with Emerald fluorescent protein, FAS and FASL. Bottom panel shows overlay of all three signals. Scale bar, 5 um. Bar graphs show quantification of FAS (top) and FASL (bottom) total and surface signal by confocal microscopy. ∗p < 0.05; by Student’s t test. (D) Staining for activated caspase 8 in CD19 CAR T cells using a specific fluorescent inhibitor FAM-LETD-FMK. Numbers indicate mean ± SD percentage of cells with activated caspase 8. (E) BB.z CD19 CAR T cells were electroporated with Cas9 protein and a FAS-specific sgRNA 24 hr post-transduction and allowed to expand for 7 days. Histograms show expression of CD19 CAR (left) and surface expression of FAS (center) on electroporated T cells. Corresponding changes in the frequency of Annexin V cells gated on FAS+ and FAS− populations are shown on the right with lines indicating cells from the same donor. ∗∗p < 0.01 by paired two-tailed Student’s t test. Experiments were replicated twice with n = 3 donors in each. Cell Reports 2017 21, 17-26DOI: (10.1016/j.celrep.2017.09.015) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Expression of BB.z CD19 CAR from a Lentiviral Vector Reduces Toxicity and Improves the Anti-tumor Function of CAR T Cells (A) Schematic representation of the gammaretroviral (BB.z) and self-inactivating lentiviral (LV BB.z) vectors. Arrows indicate promoter activity in each expression system. (B) Representative histograms showing CD19 CAR expression in T cells 7 days after transduction BB.z or LV BB.z vectors. (C) Representative histograms showing surface expression of FAS and FASL in CD19 CAR T cells. (D) Frequency of apoptotic cells in CD19 CAR T cells 7 days post-transduction measured by Annexin V staining. (E) Expansion of CD19 CAR T cells over 7 days. (F) CD19 CAR T cells were cocultured with GFP+ CD19+ Raji cells at a 1:4 ratio for 72 hr. Representative dot plots show the frequency of residual live tumor cells at the end of coculture. Numbers indicate percent of tumor cells among total live cells. (G) Expansion of LV BB.z CD19 CAR T cells in peripheral blood of NSG mice engrafted with 1 × 106 GFP+ FFluc+ NALM-6 cells 15 days prior followed by a single intravenous injection of 8 × 105 CD19 CAR T cells 3 days later. (H) Frequency of GFP+ tumor cells in peripheral blood of mice 15 days post-engraftment. (I) Tumor burden in mice measured by IVIS imaging at indicated time points. (J) Survival of tumor-bearing mice receiving either BB.z or LV BB.z CD19 CAR T cells (p = 0.0062 by Mantel-Cox log-rank test). Experiments were replicated three times for (A)–(F) and twice for (G)–(J). ∗p < 0.05; ∗∗p < 0.01 by Student’s t test or one-way ANOVA. Cell Reports 2017 21, 17-26DOI: (10.1016/j.celrep.2017.09.015) Copyright © 2017 The Authors Terms and Conditions