Journal: Journal for Immunotherapy of Cancer
Article Title: Nanofilament immunotherapy induces potent antitumor vaccine responses
doi: 10.1136/jitc-2024-011331
Figure Lengend Snippet: Key properties and validation of the TAT003 nanofilament. ( a ) Activation of tumor-associated macrophages (TAMs) after exposure to M13. Flow cytometric analysis of activation markers CD80 and CD86, and antigen presentation markers H2Kb and H2Db (representative data). ( b ) Left side: Schematic representation of TAT003’s key features. Right side: Mechanisms of immunogenicity, a TAT003 nanofilament activating a TAM, and, in the inset, a TAT003 nanofilament interacting simultaneously with a cancer cell, through the binding to PD-L1 via a displayed anti-PD-L1 scFv, and with a T cell, through the binding to IL-2 receptor (IL-2R) via a displayed mIL-2; resulting in the blockade of PD-1/PD-L1 interactions and stimulation of IL-2R. ( c ) Competitive binding flow cytometry assay on A20 mouse cells between an anti-PD-L1-PE and M13, TAT003ΔIL2, TAT003 or atezolizumab (representative data). The dotted line serves as a reference point for the evaluation of PD-L1 signal status. ( d ) Single confocal slices of A20 cells exposed to M13 or TAT003 for 15 min then incubated in Hoechst dye and anti-M13-FITC antibody conjugate. ( e ) Phagocytosis of TAT003 and TAT003-coated A20 cancer cells by bone-marrow-derived macrophages. ( d ) M13 and TAT003 were detected using an anti-M13-FITC antibody (green), and cell nuclei were stained using Hoechst dye (cyan) (representative images). Scale bars: 10 µm. ( e ) PD-1 blockade bioassay using engineered cells that output luminescence on PD-1/PD-L1 axis blockade. Dose response of cells was obtained with various concentrations of either TAT003∆IL2, TAT003 or atezolizumab as a positive control (technical duplicate). ( f ), Dose response of the HEK Blue-IL-2 cell line to various concentrations of human and mouse IL-2 compared with the signal obtained with a TAT003 dose equivalent to 0.1 ng/mL of mIL-2 molecules displayed on the bacteriophage (n=3). ( g ), Indirect ELISA to assess the presence of both anti-PD-L1 scFV and mIL-2 on the same bacteriophage particle (n=3). Measurement of TLR9 dose response activation using HEK Blue TLR9 cell lines for the murine ( h ) and human ( i ) versions of the receptor using commercial ODN as positive controls (n=2). ( j ), Cancer cell phagocytosis frequency evaluation. Bone marrow-derived macrophages were generated and put in contact with CFSE-stained A20 cancer cells and treated with either PBS or TAT003. The proportion of macrophages having phagocytized A20 cells corresponds to the number of CFSE+macrophages (n=4). APC, Allophycocyanin; CTL, cytotoxic T lymphocytes; FITC, Fluorescein isothiocyanate; hIL-2, human IL-2; IL-2, interleukin-2; mIL-2, mouse IL-2; ODN, oligodesoxynucleotides; PBS, phosphate-buffered saline; PD-L1, Programmed Death Ligand 1; PD1, Programmed Death protein 1; PE, phycoetrythrin; scFv, single-chain antibody fragment; TLR, toll-like receptor.
Article Snippet: HEK-Blue STAT5-inducible SEAP reporter cells, which are specifically designed to detect IL-2 (InvivoGen; hkb-il2) or TLR9 (InvivoGen; hkb-hTLR9 and hkb-mTLR9), were cultured under optimal conditions according to the manufacturer’s guidelines.
Techniques: Biomarker Discovery, Activation Assay, Immunopeptidomics, Binding Assay, Flow Cytometry, Incubation, Derivative Assay, Staining, Bioassay, Positive Control, Indirect ELISA, Generated, Saline
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