Journal: bioRxiv

Article Title: Retargeted adenoviruses for local IgA and CD47 blocker production as a novel cancer therapy

doi: 10.1101/2025.11.07.686998

Figure Lengend Snippet: (a) ELISA was used to measure the target-binding activity of anti-EGFR and anti-EpCAM IgA antibodies derived from the supernatants of transfected CHO-S cells. Recombinant IgA and IgG antibodies were used as positive controls (ctrl: 0.1 nM), and supernatants from mock-transfected cells served as negative controls. Binding specificity was confirmed by assessing the binding of each IgA antibody to its intended target (EGFR or EpCAM) and to the non-target protein. (b) Western blot analysis of anti-EGFR and anti-EpCAM IgA antibodies in the supernatant of transfected CHO-S cells (lane 1,3). Purified recombinant IgA antibodies were used as controls (lane 2,4). Note that the anti-EGFR IgA control shows incomplete antibody assembly. HC, heavy chain; LC, light chain. (c) Western blot analysis of dimeric IgA antibodies under non-reducing conditions, showing the presence of both the IgA heavy chain (HC), detected by a mouse anti-human kappa chain antibody, and the J-chain (JC), detected by a mouse anti-poly His-tag antibody, in supernatants of CHO-S transiently transfected with the respective expression vectors. 1: anti-EGFR; 2: anti-EpCAM. (d) ELISA was performed to measure SIRPα-Fc protein levels in the supernatant of transfected CHO-S cells, using CD47 as coating. A recombinant anti-CD47 IgG antibody (2.5 nM) was used as a positive control, and supernatant from mock-transfected cells served as a negative control. Wells with no CD47 coating served as additional controls. (e) Western blot analysis of the SIRPα-Fc protein, as detected by a mouse anti-γ heavy chain, in supernatants of CHO-S cells transiently transfected with the respective expression vector, under reducing (red.) and non-reducing (non-red.) conditions. (a-e): representative images from at least three independent experiments are shown.

Article Snippet: The plate was coated with 25 μl/well of 50 nM EGFR ectodomain protein produced and purified as described, biotinylated EpCAM ectodomain protein (Acro Biosystems, Newark, DE, USA) or 0.25 mg/mL biotinylated CD47 protein (Sino Biological, Beijing, China) diluted in PBS, and incubated for 1 h at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Derivative Assay, Transfection, Recombinant, Western Blot, Purification, Control, Expressing, Positive Control, Negative Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Retargeted adenoviruses for local IgA and CD47 blocker production as a novel cancer therapy

doi: 10.1101/2025.11.07.686998

Figure Lengend Snippet: Cells were stained with different CellTrace dyes, proteins were detected with fluorescently labeled antibodies. (a) Anti-EpCAM IgA or anti-EGFR IgA (Payload, magenta) was locally produced in the TME featuring MDA-MB-468 or HCT116 cells (Cancer cells, yellow), respectively, as well as fibroblasts (green) and endothelial cells (cyan). IgA-encoding adenoviruses were retargeted with the respective adapters to EGFR or EpCAM expressed on HCT116 or MDA-MB-468 cells, respectively, at an MOI of 20 i.v.p./cell. (b) Similar to (a), SIRPα-Fc was produced in situ upon AdV retargeting to EGFR (HCT116) or EpCAM (MDA-MB-468) at an MOI of 20 i.v.p./cell. The overlay image shows no specificity of SIRPα-Fc towards either of the cell types, due to ubiquitous expression of CD47 on all cells. (c) Anti-EGFR IgA (magenta) and SIRPα-Fc (green) simultaneously produced in situ upon co-delivery with EpCAM-retargeted adenoviral vectors in MDA-MB-468 (nuclei: cyan, Hoechst) at an MOI of 20 i.v.p./cell (each). Merge depicts SIRPα-Fc and IgA. (d) Adenovirus-mediated co-delivery of IgA and SIRPα-Fc as in (c), represented as orthogonal sections showing 3D distribution of cells with the in situ produced payloads on the cell surface, with the 3D reconstruction shown in (e) (dimensions indicated). Positive controls (recombinant IgA, SIRPα-Fc) and secondary Ab only control images are shown in Figure S9. (f) A schematic image of the previously utilized tumor-on-a-chip (not drawn to scale), consisting of a main chamber with cell co-culture in collagen, and two side channels with medium reservoirs through which retargeted adenoviral vectors or control proteins are administered . Representative images are shown, n=4. Scale bars: 100 μm.

Article Snippet: The plate was coated with 25 μl/well of 50 nM EGFR ectodomain protein produced and purified as described, biotinylated EpCAM ectodomain protein (Acro Biosystems, Newark, DE, USA) or 0.25 mg/mL biotinylated CD47 protein (Sino Biological, Beijing, China) diluted in PBS, and incubated for 1 h at room temperature.

Techniques: Staining, Labeling, Produced, In Situ, Expressing, Recombinant, Control, Co-Culture Assay

Journal: bioRxiv

Article Title: Retargeted adenoviruses for local IgA and CD47 blocker production as a novel cancer therapy

doi: 10.1101/2025.11.07.686998

Figure Lengend Snippet: (a) Set-up of the in vivo study. (b-e) Immunofluorescence (IF) of tumor slices from MDA-MB-468 tumor-bearing SCID mice four days after injection with EpCAM-directed anti-EGFR IgA-encoding and SIRPα-Fc-encoding adenoviral vectors. Nuclei were stained with DAPI, and the produced anti-EGFR IgA was detected with a goat anti-Ckappa light chain. Ly6G and CD45 were detected in the same slice as markers for neutrophils and immune cells, respectively ( b ). EpCAM was detected to identify tumor cells with an anti-EpCAM antibody (c) , SIRPα-Fc with goat anti-human Fc gamma (d) and CD47 with an anti-CD47 antibody (e) . (f) A tilescan of a slice containing a full cross-section of the resected tumor was analyzed by IF for the condition described in (b) (left), or EpCAM (right), showing the distribution of the antibodies and infiltration of immune cells. The scale bar in (b) is applicable to all images from b-e. n = 6, representative images are shown.

Article Snippet: The plate was coated with 25 μl/well of 50 nM EGFR ectodomain protein produced and purified as described, biotinylated EpCAM ectodomain protein (Acro Biosystems, Newark, DE, USA) or 0.25 mg/mL biotinylated CD47 protein (Sino Biological, Beijing, China) diluted in PBS, and incubated for 1 h at room temperature.

Techniques: In Vivo, Immunofluorescence, Injection, Staining, Produced