human cd25 mab 7g7  (Bio X Cell)

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: For opsonization of target cells with IgG, Fc-intact human CD25 mAb 7G7 (mouse IgG2a, BioXCell, Lebanon, NH) and Fc-intact human CD20 mAb rituximab biosimilar (human IgG1, BioXCell) were used.

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 ) 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: For opsonization of target cells with IgG, Fc-intact human CD25 mAb 7G7 (mouse IgG2a, BioXCell, Lebanon, NH) and Fc-intact human CD20 mAb rituximab biosimilar (human IgG1, BioXCell) were used.

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