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: Molecular Cancer Therapeutics

Article Title: Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFRpos Mouse Xenograft Model

doi: 10.1158/1535-7163.mct-22-0805

Figure Lengend Snippet: Figure 1. Intratumoral infiltration 90 minutres after systemic administration. A, Intratumoral infiltration of antiEGFR Nanofitins or Cetuximab, revealed by anti-HA and anti-IgG IHC, respectively. Host vasculature is revealed by anti-CD31 staining of consecutive slice sections. Zoom of selected regions illustrates EGFR labeling at the vessel proximity. B, Labeling index, on the basis of cells positively labeled, in the whole tumor. C, Labeling index relative to the distance from the closest blood vessel. , P < 0.0001; , P < 0.0005.

Article Snippet: Affinities were also determined for cysteine-free and HAtagged (35) Nanofitins (500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 nmol/L), either on human EGFR as described above, or on murine EGFR by using Recombinant Mouse EGFR Fc chimera protein (1280- ER, R&D Systems) for the loading step.

Techniques: Staining, Labeling

Journal: Molecular Cancer Therapeutics

Article Title: Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFRpos Mouse Xenograft Model

doi: 10.1158/1535-7163.mct-22-0805

Figure Lengend Snippet: Figure 2. Biochemical profiles of Nanofitin-drug conjugates. A, Schematic representation of a Nanofitin-drug conjugate. The single chain of the Nanofitin scaffold (rainbow cartoon) is engineered to target EGFR by randomizing up to 14 amino acids (spheres in lieu of carbon alpha). Each Nanofitin is genetically fused to a C-terminal cysteine (gray/yellow stick) to allow the regioselective chemistry on the only thiol group. The vc-MMAE payload (structural formula) is coupled via its maleimide- based moiety (black) and releases the MMAE toxin (red) after proteolytic cleavage of the valine-citrulline linker (orange). B, UPLC-RP/MS profiles. Peaks were identified by ESI-MS spectral deconvolution to determine their mass. Percentages of corresponding species were determined from the area under the absorbance curves. C, Determination of the binding characteristics of the antiEGFR Nanofitin-drug conjugates D8-vc-MMAE (left) and B10-vc-MMAE (right) by biolayer interferometry on human EGFR, using the antiEGFR Nanofitin at concentrations of 500, 125, 31.25, and 7.81 nmol/L. Fittings are represented as solid red lines.

Article Snippet: Affinities were also determined for cysteine-free and HAtagged (35) Nanofitins (500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 nmol/L), either on human EGFR as described above, or on murine EGFR by using Recombinant Mouse EGFR Fc chimera protein (1280- ER, R&D Systems) for the loading step.

Techniques: Binding Assay

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