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

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) Existing model by which SIRPα suppresses phagocytosis by interacting in trans with CD47 on target cells. See text for details. The 3 Ig-like domains of SIRPα (1 IgV and 2 IgCs) and the single Ig-V domain of CD47 are shown as ellipses. Mβs, macrophages. ( B ) Depiction of SIRPα variants and their functional characteristics. SIRPα FFFF contained substitution of tyrosine (Y)-to-phenylalanine (F) substitution at Y436, 460, 477, and 501; SIRPα ΔIC lacked most of the cytoplasmic domain of SIRPα, ending with arginine 401; SIRPα T96V carried a threonine (T)-to-valine (V) mutation at position 96 (shown by lavender star), which abolishes CD47-binding; SIRPα T96V,FFFF had the T96V and FFFF mutations; SIRPα T96V,ΔIC had the T96V and the ΔIC mutations. KO, knock-out. ITIM, immunoreceptor tyrosine-based inhibitory motif. ( C to G ) SIRPα variants or empty vector were expressed in SIRPα KO BMDMs and tested. Wild-type (WT) BMDMs were used as control. ( C ) Schematic representation of assays performed. Fc, fragment crystallizable. ( D ) Flow cytometry analyses of SIRPα expression and CD47-binding. APC, allophycocyanin. AF647, Alexa fluor 647. ( E and F ) Representative ( E ) and compiled data ( F ) of pHrodo-based phagocytosis assays using L1210 derivatives expressing Tac and opsonized with Tac monoclonal antibody (mAb) 7G7, as targets. Positive cells with percentages are boxed. G , Efficiency of phagocytosis inhibition in SIRPα KO BMDMs expressing or not the indicated SIRPα variants was calculated using the values in ( F ). SIRPα KO expressing WT SIRPα or empty vector displayed 100% and 0% inhibition efficiency, respectively. All data are means ± s.e.m., **** p < 0.0001. Results in ( D and E ) are representative of 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that are representative of 3 experiments. Results in ( F and G ) are pooled from a total of 6 mice studied in 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that involved 3 mice in 3 experiments. Each symbol in ( F ) represents one mouse.

Article Snippet: A histidine (His)-tagged version of mouse CD47 (CD47-His; Cat# 57231-M08H) was from Sino Biological (Beijing, China).

Techniques: Functional Assay, Mutagenesis, Binding Assay, Knock-Out, Plasmid Preparation, Control, Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: A histidine (His)-tagged version of mouse CD47 (CD47-His; Cat# 57231-M08H) was from Sino Biological (Beijing, China).

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: A histidine (His)-tagged version of mouse CD47 (CD47-His; Cat# 57231-M08H) was from Sino Biological (Beijing, China).

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis