Supplementary Figure 8: BiTE quantification from in vivo specimens.
From: CAR-T cells secreting BiTEs circumvent antigen escape without detectable toxicity
(a) U87KO (EGFR-negative), U87, or U87vIII glioma cells were implanted intracranially in NSG mice. Mice were treated intraventricularly on day 14 post-implantation with CART-EGFRvIII.BiTE-EGFR cells. On day 7 post-treatment, whole brain and blood were isolated from the treated mice. Proteins were extracted from brain tissue and blood. Each lane was loaded with 30 μg total protein and subjeced to SDS-PAGE and blotting with anti-His-tag antibody for BiTE and anti-β-actin antibody. Lanes 1 and 3 included 5 ng of added purified BiTE. (b) Protein samples from blood or brain (c) were analyzed by His Tag ELISA. 10 ng of BiTE was loaded for blood positive controls and 5 ng of BiTE was loaded for brain positive controls. The experiments were repeated independently three times with similar results. Representative data are shown. ELISA was performed in experimental groups as indicated, n = 3 biologically independent animals (mean + SEM is depicted, statistical significance determined by unpaired, two-tailed t-test).
