Journal: Journal for Immunotherapy of Cancer

Article Title: Metabolic reprogramming via an engineered PGC-1α improves human chimeric antigen receptor T-cell therapy against solid tumors

doi: 10.1136/jitc-2022-006522

Figure Lengend Snippet: hEGFR-targeted CARs with 4-1BB co-stimulatory domains exhibit suppressed mitochondrial mass and exhaustion in the tumor microenvironment. (A) Structure of the anti-EGFR CAR that was used for the experiments, (B) in vitro killing assay of EGFR-specific CAR-T cells incubated overnight with A549 cells at the reported concentrations, (C) 10 7 hEGFR-targeted CARs were injected in NSG mice carrying 100 mm 3 tumors, (D) MitoTracker FM staining of CAR-T cells in tumor infiltrating lymphocyte (TIL) preparations from A549 tumors and respective spleens, (E) PD-1, Tim-3, and Lag-3 staining in CAR-T cells infiltrating A549 tumors and the respective spleens. (F) Cytokine production of TIL after 6 hours of ex vivo restimulation with phorbol 12-myristate 13-acetate and ionomycin. All experiments were repeated with three different donors. Plot B is representative of three different experiments and plots C–E contain all data from all donors. Each dot represents one mouse. Statistics are Wilcoxon matched-pairs signed-rank test. *p<0.05, **p<0.01. CAR, chimeric antigen receptors; IFN, interferon; PD-1, programmed cell death protein-1; TNF, tumor necrosis factor; ACT, adoptive cell therapy

Article Snippet: EGFR protein conjugated to Allophycocyanin was obtained by CREATIVE BIOMART (EGFR-692HA).

Techniques: In Vitro, Incubation, Injection, Staining, Ex Vivo

Journal: Journal for Immunotherapy of Cancer

Article Title: Metabolic reprogramming via an engineered PGC-1α improves human chimeric antigen receptor T-cell therapy against solid tumors

doi: 10.1136/jitc-2022-006522

Figure Lengend Snippet: Anti-EGFR PGC-1α S571A CAR-T cells, but not NT-PGC1α CAR-T cells, exhibit significant antitumor efficacy in vivo driven by increased cytokine production and memory formation. (A, B) Tumor growth curve and survival of A549-bearing NSG mice treated with 10 7 anti-EGFR PGC-1α S571A CAR-T cells when tumors reached 100 mm 3 (arrow). (C) Tumor growth curve as in A but using 10 7 anti-EGFR NT-PGC-1α CAR-T cells. (D) MitoTracker FM staining of TIL from tumors treated with unmodified or PGC-1α S571A CAR-T cells. (E) PD-1 and Tim-3 staining from TIL, (F) Cytokine from TIL after 6 hours ex vivo restimulation with phorbol 12-myristate 13-acetate and ionomycin, (G) Memory markers of TIL from day 10. The PGC-1α S571A growth curve was repeated with two donors while the TIL analysis was performed once across multiple mice. The NT-PGC-1α growth curve was repeated with three donors. Statistics are repeated-measures analysis of variance (A, C), log-rank test (B), and Wilcoxon rank-sum test (D–G). *p<0.05, **p<0.01 ***p<0.001. CAR, chimeric antigen receptors; IFN, interferon; PD-1, programmed cell death protein-1; PGC-1α, PPAR gamma coactivator 1α; TIL, tumor-infiltrating lymphocyte; TNF, tumor necrosis factor.

Article Snippet: EGFR protein conjugated to Allophycocyanin was obtained by CREATIVE BIOMART (EGFR-692HA).

Techniques: In Vivo, Staining, Ex Vivo

Journal: Biomedicines

Article Title: A Rational Designed Novel Bispecific Antibody for the Treatment of GBM

doi: 10.3390/biomedicines9060640

Figure Lengend Snippet: ( a ) EGFRvIII antigen expression level on mutant GBM cell line U87MG.ΔEGFR and wild-type GBM cell line U87MG. ( b ) A binding activity comparison of EGFRvIII-BsAb and CD3 mAb with Jurkat cells (CD3-positive) (upper), as well as a binding activity comparison of the EGFRvIII-BsAb and the EGFRvIII mAb with U87MG.ΔEGFR cells (EGFRvIII-positive) (lower). ( c ) Photographs of the redirection of T cells to cancer cells by 0.01 ng/mL EGFRvIII-BsAb or EGFRvIII mAb. ( d ) FACS analysis of the redirection of CD3+ Jurkat cells to cancer cells by EGFRvIII-BsAb. Jurkat (CD3+) cells labeled by PKH26 (PE-A), as well as U87MG.ΔEGFR cells labeled by CFSE (FITC-A).

Article Snippet: The EGFRvIII antigen (AVI10494; R&D System, Minneapolis, MN, USA) and extracellular domain of human CD3D/CD3E heterodimer (CT038-H2508H; Sino Biological, Beijing, China) were immobilized to a CM5 chip (29149603; GE Healthcare, Chicago, IL, USA) surface using standard protocols with 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) amine.

Techniques: Expressing, Mutagenesis, Binding Assay, Activity Assay, Comparison, Labeling