Journal: Scientific Reports

Article Title: Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients

doi: 10.1038/s41598-021-00325-3

Figure Lengend Snippet: Development of Nb-based anti-CTLA4 heavy chain only antibody (HcAb). ( A ) Predicted structure of cHcAb6. The DNA sequence coding for the cNb6 was genetically fused to the hinge and Fc domains of canine IgG (subclass B). ( B ) The cHcAb6 form dimers as demonstrated by western blotting. The cHcAb6 was expected to form dimers via hinge and Fc domains of canine IgG. The cHcAb6 protein, expressed in ExpiCHO-S cells, was resolved under reducing and non-reducing conditions and detected by anti-IgG Fc antibody. The cHcAb6, as expected, forms dimers of ~ 83 kDa under non-reducing conditions. (R-reducing condition, NR-non reducing). ( C ) Purity of cHcAb6 assessed by SDS-PAGE. The cHcAb6 was expressed and purified from the ExpiCHO-S cells by affinity (Protein A) and size-exclusion chromatography. The purified cHcAb6 was resolved under reducing (R) and non-reducing (NR) condition and stained with GelCode Blue Stain. ( D ) Binding of cHcAb6 to cells expressing canine CTLA4 demonstrated by flow cytometry. MDCK cells transiently expressing CTLA4 were stained with cHcAb6, washed, and bound cHcAb6 was detected using anti-Fc-750 antibody by flow cytometry. cHcAb6 does not bind to untransfected cells.

Article Snippet: The cHcAb6 band was detected with anti-Strep Tag II and rabbit anti-canine IgG Fc antibodies (Novus Biologicals).

Techniques: Sequencing, Western Blot, SDS Page, Purification, Size-exclusion Chromatography, Staining, Binding Assay, Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients

doi: 10.1038/s41598-021-00325-3

Figure Lengend Snippet: Canine Tregs constitutively express CTLA4. Activated cPBMCs were first stained with cHcAb6. The cHcAb6 stained PBMCs were fixed, permeabilized, and treated with anti-CD3, CD4, CD8, and FoxP3 antibodies. The bound cHcAb6 was detected using anti-canine IgG Fc-750 Ab. Canine IgG was used as an isotype control. ( A ) The CTLA4 was predominantly expressed on helper T cells and a small subset of cytotoxic T cells. ( B ) CTLA4 was constitutively expressed on Tregs. MFI Mean fluorescence intensity.

Article Snippet: The cHcAb6 band was detected with anti-Strep Tag II and rabbit anti-canine IgG Fc antibodies (Novus Biologicals).

Techniques: Staining, Control, Fluorescence

Journal: Scientific Reports

Article Title: Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients

doi: 10.1038/s41598-021-00325-3

Figure Lengend Snippet: cHcAb6 binds to native CTLA4 on cPBMCs. CPBMCs were stimulated with PMA and Ionomycin for 8 h. Activated and control PBMCs were stained with cHcAb6 and analyzed by flow cytometry using anti-canine IgG Fc-750 Ab. Canine IgG was used as an isotype control. The CTLA4 expression was markedly increased after PMA/Ionomycin stimulation.

Article Snippet: The cHcAb6 band was detected with anti-Strep Tag II and rabbit anti-canine IgG Fc antibodies (Novus Biologicals).

Techniques: Control, Staining, Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients

doi: 10.1038/s41598-021-00325-3

Figure Lengend Snippet: ( A ) cHcAb6 binds to cells expressing the canine FcγRI receptor. MDCK cells transiently expressing FcγRI were stained with cNb6, and bound cHcAb6 was detected using anti-Fc-750 antibody by flow cytometry. Canine IgG and cells stained with secondary Ab only were used as controls. ( B ) cHcAb6 induces IFN-γ expression from PBMCs after CD3 stimulation. CPBMCs were stimulated with 1 μg/mL of anti-CD3 antibody in the presence or absence of 100 nM of cHcAb6. After 72 h, IFN-γ expression was quantified using TaqMan assays. HRPT1 was used as endogenous control, and expression levels of IFN-γ were normalized to CD3 stimulated PBMCs. All experiments were performed in triplicates.

Article Snippet: The cHcAb6 band was detected with anti-Strep Tag II and rabbit anti-canine IgG Fc antibodies (Novus Biologicals).

Techniques: Expressing, Staining, Flow Cytometry, Control