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Supplemental Figure 1
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C D 3+ C D 4+ C D 3+ C D 8+ C D 3-C D 56+ C D 3+ C D 45R A+ C D 3+ C D 45R O + C D 3+ C D 62L-C C R 7- C D 3+ C D 62L+ 0 20 40 60 80 100 NT IL13K-C AR IL13KR -C AR IL13Y-C AR IL13YR -C AR Positive (% ) Supplemental Figure 1 Supplemental Figure 1: IL13 mutein-CAR T cells show no difference in T-cell phenotype. The phenotype of IL13 mutein-CAR T cells and NT T cells was evaluated using a panel of antibodies. CAR and NT T-cell products consisted of CD4-positive and CD8-positive subpopulations (mean 34.8±3.73%; mean 65.9±2.4%), and contained naïve (CD3+/45RA+) and memory (CD3+/45RO+) T cells. Central (CD3+/CD62L+: 47.1±8.6%) as well as effector memory T cells (CD3+/CD62L-/CCR7-: 33.0±8.5%) were present.
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Transcript of Supplemental Figure 1
CD
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Supplemental Figure 1
Supplemental Figure 1: IL13 mutein-CAR T cells show no difference in T-cell phenotype. The phenotype of IL13 mutein-CAR T cells and NT T cells was evaluated using a panel of antibodies. CAR and NT T-cell products consisted of CD4-positive and CD8-positive subpopulations (mean 34.8±3.73%; mean 65.9±2.4%), and contained naïve (CD3+/45RA+) and memory (CD3+/45RO+) T cells. Central (CD3+/CD62L+: 47.1±8.6%) as well as effector memory T cells (CD3+/CD62L-/CCR7-: 33.0±8.5%) were present.
Supplemental Figure 2: IL13 mutein-CAR T cells secret IFNγ in an antigen-dependent manner. IL13K-CAR, IL13Y-CAR or non-transduced (NT) T cells were stimulated with increasing amounts of recombinant IL13Rα1 (A) or IL13Rα2 (B) proteins. After 24h IFNγ was measured by ELISA (n=2, assay performed in duplicate for each donor; mean and SD is shown); ≥250mg of IL13Rα1 and ≥ 125mg of IL13Rα2 protein induced significant IFNγ production (p<0.05) of IL13K-CAR or IL13Y-CAR T cells in comparison to unstimulated T cells.
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Supplemental Figure 2
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Supplemental Figure 3: IL13 mutein-CAR T cells kill IL13Ra1+ and IL13Ra2+ cells. All four IL13 mutein-CAR constructs (K, KR, Y, YR) killed IL13Ra1+/a2+ cells (U373), IL13Ra1+/a2- cells (A431 or 293T). In contrast IL13Ra1-/a2- cells (Raji or T cells) were not killed. Non-transduced (NT) T cells had no cytolytic activity.
Supplemental Figure 3
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Supplemental Figure 4: IL13 mutein-CAR T cells recognize and kill murine IL13R 1a - and IL13Ra2-positive cells. (A) qRT-PCR analysis of murine glioma (GL261), melanoma (B16F10), and murine T cells. (B) Human IL13-mutein CAR T cells (IL13K, IL13KR) killed IL13R 1a -positive (GL261) and IL13R 2a -positive (B16F10) cells. In contrast murine IL13Ra1- and IL13Ra2-negative cells (T cells) were not killed. NT T cells had no cytolytic activity.
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Supplemental Figure 5: Limited persistence of 2nd generation IL13 mutein-CAR T cells in vivo. IL13KR-CAR T cells were transduced to express eGFP.ffLuc. (A) FACS analysis confirmed the expression of CAR and eGFP.ffLuc transgenes. (B) IL13KR-CAR and IL13KR.eGFP.ffLuc T cells killed IL13Ra1+/IL13Ra2+ cells (U373) in contrast to IL13Ra1-/IL13Ra2- cells (Raji cells) in a standard 4h chromium release assay. (C) 1 x 105 unmodified U373 cells were injected intracranially into mice. On day 7 mice received 2 x 106 IL13KR.eGFP.ffLuc CAR T cells intracranially using the same tumor coordinates. Bioluminescence imaging was used to monitor T-cell persistence.
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Supplemental Figure 5
