Journal: Journal of Translational Medicine

Article Title: Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology

doi: 10.1186/s12967-024-05159-x

Figure Lengend Snippet: Construction of FGFR4 nanoparticles and immunization of alpaca. a Schematic diagram of expression vector encoding FGFR4 antigen’s extracellular domain proteins and ferritin. b Schematic diagram of generation of FGFR4-HPF nanoparticles. c Process of immunizing alpaca with FGFR4-HPF nanoparticles. d Immunized alpaca serum ELISA

Article Snippet: The CM5 censor chip (Cytiva) was activated and then injected with FGFR4-His protein for immobilization using amine coupling kit (GE Healthcare).

Techniques: Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology

doi: 10.1186/s12967-024-05159-x

Figure Lengend Snippet: Construction of electroporation bacteria library and screening of anti-FGFR4 Nbs. a Monoclonal sequencing analysis and genetic evolutionary tree analysis of the electroporation library. Sequencing of 72 monoclonal clones identified 69 positive clones with insertion of single Nb sequences. b Proportion of Nb-displaying phages capable of binding to FGFR4 after screening. Left: output of first round; middle: input of second round; right: output of second round. c Monoclonal phage ELISA with FGFR4. Positive clones: 1, 9, 13, 14, 19. 20, 22, 23. d Identification of monoclonal phages capable of binding to Huh7 cells by flow cytometry. The NC phages uncapable of binding Huh7 cells were used as the control. Clones with positive fluorescence: 1 (anti-FGFR4 Nb1), 14 (anti-FGFR4 Nb2)

Article Snippet: The CM5 censor chip (Cytiva) was activated and then injected with FGFR4-His protein for immobilization using amine coupling kit (GE Healthcare).

Techniques: Electroporation, Bacteria, Sequencing, Clone Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology

doi: 10.1186/s12967-024-05159-x

Figure Lengend Snippet: Functional validation of the screened Nbs in vitro . a Molecular docking models of Nbs with FGFR4. The structure of light blue represents antigen, the structure of dark blue represents Nbs, the interface regions of red and green represent the region of the Nb in contact with antigen, ΔG denotes the free energy of binding. b Schematic diagram of expression vector encoding Nb-Fc. c Detection of the binding ability of Nbs by antibody-antigen binding ELISA. Nc-Linker-Fc: non FGFR4 targeting control nanobody-Linker-Fc antibody. Data were analyzed by the Student’s t-test. d Detection of the binding ability of Nbs by antibody gradient dilution ELISA. Nc-Linker-Fc: non FGFR4 targeting control nanobody-Linker-Fc antibody. Data were analyzed by two-way ANOVA. e Detection of the binding affinity of Nbs by SPR assay. f Evaluation the specificity of Nbs by antibody-antigen binding ELISA. Data were analyzed by one-way ANOVA. The experiments were performed independently in triplicate. Data are expressed as mean ± SEM. **** p < 0.0001

Article Snippet: The CM5 censor chip (Cytiva) was activated and then injected with FGFR4-His protein for immobilization using amine coupling kit (GE Healthcare).

Techniques: Functional Assay, In Vitro, Binding Assay, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, SPR Assay

Journal: Journal of Translational Medicine

Article Title: Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology

doi: 10.1186/s12967-024-05159-x

Figure Lengend Snippet: Construction of Nb-derived CAR-T cells and functional validation of Nb-derived CAR-T cells anti-tumor in vitro . a Schematic diagram of lentivirus plasmid encoding anti-FGFR4 Nb-CAR. b Validation of cytotoxicity of Nb-derived CAR-T cells against Huh7 cells in vitro by LDH assay. NC-T: T cells transduced with empty lentivirus vector. c Validation of cytotoxicity of Nb-derived CAR-T cells against BXPC3 cells in vitro by LDH assay. NC-T: T cells transduced with empty lentivirus vector. d Validation of the cytokine secretion functions of Nb-derived CAR-T cells against Huh7 cells in vitro by ELISA. Mock: T cells transduced with empty lentivirus vector. e Validation of the IFN-γ secretion functions of Nb-derived CAR-T cells against Huh7 cells in vitro by ELISPOT. Mock: T cells transduced with empty lentivirus vector. Data were analyzed by two-way ANOVA. The experiments were performed independent biological replicates (N = 3). Data are expressed as mean ± SEM. Ns: p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The CM5 censor chip (Cytiva) was activated and then injected with FGFR4-His protein for immobilization using amine coupling kit (GE Healthcare).

Techniques: Derivative Assay, Functional Assay, In Vitro, Plasmid Preparation, Lactate Dehydrogenase Assay, Transduction, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Journal of medicinal chemistry

Article Title: Development of Zalfermin, a Long-Acting Proteolytically Stabilized FGF21 Analog.

doi: 10.1021/acs.jmedchem.4c00391

Figure Lengend Snippet: Figure 6. AlphaScreen binding assay data for 1 and 15 toward FGFR1c/KLB (A), FGFR3c/KLB (B), and FGFR4/KLB (C). Data are mean ± SEM (n = 3).

Article Snippet: Biotinylated FGF21 was coupled to streptavidin donor beads (PerkinElmer, cat. no. 6760002), and the ectodomain of human FGFR1c, FGFR2c, FGFR3c, or FGFR4 fused to Fc (R&D Systems, cat. no. 685-FR-050, 712-FR-050, 766-FR-050, and 658-FR050) were coupled to Protein A acceptor beads (PerkinElmer, cat. no. 6760137M).

Techniques: Amplified Luminescent Proximity Homogenous Assay, Binding Assay