recombinant hfap protein  (R&D Systems)

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Assessment of the binding properties of FAPI TMs to FAP-positive target cells. The surface expression of FAP on HT1080 hFAP (a) and MRC-5 (b) cells was assessed by flow cytometry using commercial anti-hFAP mAb and binding was detected using goat anti-mouse IgG Pacific Blue. Data are shown for one representative experiment out of three independent experiments with similar results ( n = 3). The number of hFAP surface antigens per cell was determined for the HT1080 hFAP and MRC-5 cell lines using QIFIKIT ( n = 1) (c). The FAP-specific binding of 64 Cu-radiolabeled FAPI TMs to HT1080 hFAP cells was evaluated in vitro after 1 h of incubation at 37 °C ( n = 1) (d). The binding of NODA-GA-FAPI-PEG 4/12/24 -E5B9 TMs and NODA-GA-FAPI-PEG 4 to HT1080 hFAP was investigated using flow cytometry. The target cells were incubated with 200 nM of either NODA-GA-FAPI-PEG 4 (black), NODA-GA-FAPI-PEG 4 -E5B9 (red), NODA-GA-FAPI-PEG 12 -E5B9 (blue) or NODA-GA-FAPI-PEG 24 -E5B9 (green), and binding was detected using anti-La mAb 5B9 (e). The data are shown for one representative experiment out of three independent experiments with similar results ( n = 3). The binding affinity of NODA-GA-FAPI-PEG 12 -E5B9 (blue) and NODA-GA-FAPI-PEG 24 -E5B9 (green) TMs to HT1080 hFAP (f, g) and MRC-5 (h) cell lines were further studied by flow cytometry using anti-La mAb 5B9 and the goat anti-mouse IgG Pacific Blue. Titration curves are plotted as the mean fluorescence intensity (MFI) ± SD from three independent experiments ( n = 3). K D : equilibrium dissociation constant; n.a.: not applicable.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: Binding Assay, Expressing, Flow Cytometry, In Vitro, Incubation, Titration, Fluorescence

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Evaluation of cytotoxicity and immunostimulatory cytokine production by UniCAR T-cells redirected by FAPI TMs. UniCAR T-cells were cultivated together with HT1080 hFAP or MRC-5 for 16 h in the absence (dark gray) or presence of NODA-GA-FAPI-PEG 12 -E5B9 (blue) or NODA-GA-FAPI-PEG 24 -E5B9 (green) TM. Target cell lysis was measured using luciferase-based assays. UniCAR T-cells were incubated with target cells at varying E:T ratios in the absence or presence of NODA-GA-FAPI-PEG 12 -E5B9 or NODA-GA-FAPI-PEG 24 -E5B9 TM (a, b). Titration curves were obtained for UniCAR T-cells co-cultured with target cells at an E:T ratio of 5:1. The dotted lines indicate the level of target cell lysis in the absence of FAPI TM (c, d). The statistical significance of the differences between the dose‒response curves was determined by nonlinear regression analysis using the extra sum-of-squares F-test in GraphPad Prism. Cytokines released by UniCAR T-cells cultivated with target cells and FAPI-TMs were quantified by ELISA (e, f). All results are plotted as the mean ± SD from three independent donors and experiments ( n = 3), each performed with three technical replicates. EC 50 : half-maximal effective concentration.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: Lysis, Luciferase, Incubation, Titration, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Assessment of in vivo killing of UniCAR T-cells redirected by NODA-GA-FAPI-PEG 12/24 -E5B9 TMs. Female NXG mice were injected with either HT1080 hFAP cells (black), HT1080 hFAP and UniCAR T-cells (gray), or HT1080 hFAP and UniCAR T-cells at an E:T ratio of 1:1 in combination with 1 nmol of either NODA-GA-FAPI-PEG 12 -E5B9 (blue) or NODA-GA-FAPI-PEG 24 -E5B9 (green). The bioluminescence signal of HT1080 hFAP cells across all groups is shown for days 0 (1 h post-injection), 1, 2, 3 and 6 d post-injection (a). Quantitative analysis of the bioluminescence was conducted, and the results were plotted as mean ± SD from five mice per group ( n = 5) (b). Statistical significance was assessed using two-way ANOVA with Greenhouse–Geisser correction and with Dunett ´ s multiple comparison test with respect to the control group injected with only HT1080 hFAP cells. Stars indicate statistical significance; p -values close to the cutoff (0.05) are shown on the graph.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: In Vivo, Injection, Comparison, Control

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Binding characteristics and pharmacokinetics of 64 Cu-radiolabeled FAPI TMs with different PEG spacer lengths. Maximum intensity projection (MIP) of NRMI nude mice bearing HT1080 (left flank, dashed white circles) and HT1080 hFAP tumor (right flank, white circles) after i.v. injection of 64 Cu-radiolabeled FAPI TMs ( a ). Time–activity curves (TAC, n = 2–3 mice per group) were analyzed for HT1080 hFAP tumor (b) and kidney (c) to determine tumor-to-kidney ratios over time (d). *: free urine.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: Binding Assay, Drug discovery, Injection, Activity Assay

Journal: Communications Biology

Article Title: The characterization of variable new antigen receptors targeting FAP isolated from a novel immunized library

doi: 10.1038/s42003-025-08610-x

Figure Lengend Snippet: BLI sensorgrams of sensors loaded with hFAP ( A ) or mFAP ( B ) and monitored during exposure to serially diluted antibody analytes (300–0.412 nM), followed by dissociation in assay buffer. Data represents raw BLI responses (thin lines) and fitted curves (bold lines) from a representative experiment. C BLI sensorgrams from antibody cross-competition epitope binning experiments, wherein biosensors loaded with hFAP are exposed to a saturating concentration (1 µM) of the indicated primary antibody, followed by exposure to a competing secondary antibody (1 µM). kinetic traces ( D , F ) and cumulative quantification ( E , G ) of proteolytic activity of recombinant hFAP (0.3 nM) in the presence of the indicated VNAR-Fc (1 µM) using 1 µM of either Ac-Gly-Pro-AFC ( D , E ) or MCA-Glu-Arg-Gly-Glu-Thr-Gly-Pro-Ser-Gly-Dnp (“9mer”, F-G) fluorogenic substrates. H Validation of membrane-bound FAP expression in R1-CWR FAP and hPrCSC-44 cell lines by flow cytometry. Cells were stained using a fixed concentration of VNAR-Fc (50 nM) and detected using an anti-IgG1-phycoerythrin secondary (5 µg/mL). Samples were compared to an unstained cell control. I Dose response curves of R1-CWR FAP and hPrCSC-44 cell lines using several staining concentrations of VNAR-Fc antibodies assessed by flow cytometry. p -values, *** p ≤ 0.001 compared to vehicle control using Student’s t test.

Article Snippet: Recombinant human FAP (FAP-H5244, Acro Biosystems), mouse FAP (FAP-M53H3, Acro Biosystems), and human DPP-IV (DP4-H5221, Acro Biosystems) were each biotinylated using EZ-link NHS-PEG4-biotin (Thermo Fisher Scientific) according to the vendor recommendations.

Techniques: Concentration Assay, Activity Assay, Recombinant, Biomarker Discovery, Membrane, Expressing, Flow Cytometry, Staining, Control

Journal: Communications Biology

Article Title: The characterization of variable new antigen receptors targeting FAP isolated from a novel immunized library

doi: 10.1038/s42003-025-08610-x

Figure Lengend Snippet: Confocal microscopy images of hPrCSC-44 cells after incubation with H4-Fc-AF647 ( A ), H15-Fc-AF647 ( C ), H17-Fc-AF647 ( E ), or NGS2405-Fc-AF647 ( G ) for 1 h, using 10 nM of anti-FAP VNAR-Fc-AF647 and 50 µg/ml of fluorescein-dextran. Single-channel images of VNAR-Fc-AF647 localization, fluorescein-labeled endosomes, Hoescht 33342-labeled nuclei, and CellBrite 555-labeled membranes are shown. Merged composite images depicting whole cells and enlarged regions of interest are shown as colored fluorescence overlays. Top right , plots of relative fluorescent signal detected in line scans (teal) in the antibody channel and the endosome channel are shown to illustrate spatial co-localization of punctate structures. Scale bar represents 20 µm in uncropped images, and 10 µm in zoomed insets. Aggregate data from high-content live-cell imaging of anti-FAP VNAR-Fc internalization into CWR-R1 FAP or CWR-R1 cells. Antibodies were directly labeled with pHrodoRed, integrated pHrodoRed fluorescence detected after treatment with the indicated concentration of H4-Fc-pHrodoRed ( B ), H15-Fc-pHrodoRed ( D ), H17-Fc-pHrodoRed ( F ), or NGS2405-Fc-pHrodoRed ( H ) in CWR-R1 cells or CWR-R1 FAP cells that were either treated with DMSO vehicle (0.1%), dynasore (30 µM), or 100 nM of soluble recombinant hFAP. Data represents mean ± s.e.m. from n = 3 independent experiments.

Article Snippet: Recombinant human FAP (FAP-H5244, Acro Biosystems), mouse FAP (FAP-M53H3, Acro Biosystems), and human DPP-IV (DP4-H5221, Acro Biosystems) were each biotinylated using EZ-link NHS-PEG4-biotin (Thermo Fisher Scientific) according to the vendor recommendations.

Techniques: Confocal Microscopy, Incubation, Labeling, Fluorescence, Live Cell Imaging, Concentration Assay, Recombinant