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. Author manuscript; available in PMC: 2019 Feb 13.
Published in final edited form as: Nat Biotechnol. 2018 Aug 13;36(9):847–856. doi: 10.1038/nbt.4195

Fig. 2. CAR-T cells secreting RMP1–14 scFv have enhanced anti-tumor function in syngeneic mouse tumor models.

Fig. 2.

(a) Schematic diagram of experimental setup to detect scFv secretion in vivo. C57BL/6 mice were inoculated with ID8 tumor, monitored until development of ascites and subsequently treated i.p. with 4H11m28mZ or 4H11m28mZ/RMP1–14 T cells. In vivo secretion of RMP1–14 scFv was detected by harvesting ascites from tumor-bearing mice 48 hours later. The ascites was immunoprecipitated with an anti-myc-tag antibody and (b) run on a Western blot using an anti-myc tag antibody or (c) run on Luminex utilizing anti-myc-tag beads (*p<0.0004 using an two-tailed unpaired t test, Mean ± SEM of 4H11m28mZ and 4H11m28mZ/RMP1–14 are 2.3 ± 1.9 and 26 ± 1.2, respectively), Data shown is from 2 independent experiments. (d) C57BL/6 mice were injected i.p. with ID8 tumor cells and treated with CAR-T cells, 250 μg RMP1–14 mAb or a combination of both 7 days later. RMP1–14 mAb was given on days 3, 7 and 14 post-tumor inoculation (*p=0.004 by Log-rank Mantel-Cox Test, with a 95% Confidence Interval (CI) of 0.4 to 0.9). Data shown is from 2 independent experiments. (e) PCR of bone marrow from mice surviving >120 days since tumor inoculation in Fig. 2d. CAR-T cells were detected in the bone marrow of long-term surviving mice treated with 4H11m28mZ T cells + RMP1–14 mAb or 4H11m28mZ/RMP1–14 T cells. Data shown is from 2 independent experiments. (f) C57BL/6 mice were inoculated with ID8 tumor and treated 7 days later with 4H11m28mZ or 4H11m28mZ/RMP1–14 T cells. Long term surviving mice in the 4H11m28mZ/RMP1–14 T cell cohort were re-challenged with a second inoculation of ID8 cells and compared to naïve untreated ID8-innoculated mice (*p=0.007 by Log-rank Mantel-Cox Test, with a 95% Confidence Interval of 0.2 to 0.3). (g) Quantification of flow cytometric analysis demonstrating endogenous, CAR- T cells extracted from C57BL/6 mice bearing B16-F10 mouse melanoma and treated with PD-1-blocking scFv CAR-T cells have enhanced activation and cytokines levels as compared to mice treated with second-generation CAR-T cells (*p values indicated on figure, by two-tailed unpaired t test). Data shown is pooled from 6 mice and 2 independent experiments.