Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation

doi: 10.1073/pnas.1909414116

Figure Lengend Snippet: M2 differentially blocks recognition of B7.1 and B7.2 by antibodies and the CD28 and CTLA4 receptors. (A, Left) MEFs transduced to express mouse B7.1 or B7.2 (MEF-mB7.1 or MEF-mB7.2) were preincubated with M2 at the indicated concentration before staining with 1 μg/mL anti-mouse B7.1 (16-10A1) or B7.2 (GL1) blocking antibody. (A, Right) Quantification of mean fluorescence intensity (MFI) of specific antibody staining in the presence of M2 relative to no M2 from 2 independent experiments is shown in the bar chart (mean ± SEM). ns, not significant. (B) Cells used in A were incubated with recombinant M2 or C8 (control PIE) and mouse CD28-Fc or CTLA4-Fc at the indicated concentration for 30 min before CD28-Fc/CTLA4-Fc binding was visualized by fluorescence-labeled anti-human IgG. (Upper) Representative flow cytometric plots of 3 independent analyses are shown. (Lower) Bar chart is the quantification of 3 analyses (mean ± SEM) showing MFI of CD28/CTLA4 in the presence of M2 relative to no M2. (C) Same experiment as in B was conducted with MEF-hB7.1 or MEF-hB7.2 and soluble human CD28-Fc or CTLA4-Fc. *P < 0.05, ***P < 0.001, ****P < 0001.

Article Snippet: The recombinant mouse and human CD28, CTLA4, and PD-L1, all fused with Fc of human IgG1 at the C terminus (except human PD-L1, fused with Fc of mouse IgG1) were from Sino Biological, Inc.

Techniques: Concentration Assay, Staining, Blocking Assay, Fluorescence, Incubation, Recombinant, Binding Assay, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation

doi: 10.1073/pnas.1909414116

Figure Lengend Snippet: ΔM2 CPXV elicits stronger primary T cell responses than WT CPXV. C57BL/6-CR mice were intraperitoneally infected with 50,000 platelet-forming units (PFU) of WT or ΔM2 CPXV. Spleens were analyzed 5 d postinfection. (A) B8R (peptide TSYKFESV) tetramer (TM) staining and total numbers (#) of tetramer-positive CD8 T cells. (B) Percentage of IFN-γ–producing CD8 T cells upon ex vivo stimulation with Δ12Δ203 CPXV-infected DC2.4 cells. (C) Percentage of IFN-γ–producing CD4 T cells upon ex vivo stimulation with peptide-pulsed DC2.4 cells. (D) CPXV titers in the spleen determined by DNA copy number. (E) C57BL/6-J WT and CD28−/− mice were intraperitoneally infected with 50,000 PFU of WT or ΔM2 CPXV and analyzed as in C. *P < 0.05, **P < 0.01, ***P < 0.001. ns. not significant.

Article Snippet: The recombinant mouse and human CD28, CTLA4, and PD-L1, all fused with Fc of human IgG1 at the C terminus (except human PD-L1, fused with Fc of mouse IgG1) were from Sino Biological, Inc.

Techniques: Infection, Staining, Ex Vivo

Journal: bioRxiv

Article Title: Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

doi: 10.1101/2025.10.06.680836

Figure Lengend Snippet: (A) TRIC-based Dianthus screen of 40 catalogue-derived analogues (20 each from 22VS and 8VS chemotypes). Normalized fluorescence (Fnorm) values at 100 μM identified eight compounds exceeding the ±5 SD threshold (shaded region). (B–D) MST validation of three SAR-derived hits. Sigmoidal dose–response binding curves confirmed specific CD28 engagement with dissociation constants of 6.5 ± 2.8 μM for BPU11 (B), 57.8 ± 9.8 μM for BPU16 (C), and 30.5 ± 4.8 μM for BPU18 (D). Data represent mean ± SEM from three independent experiments, fitted using a four-parameter nonlinear regression model.

Article Snippet: His-tagged human CD28 protein (Acro Biosystems) was labeled with RED-tris-NTA 2nd Generation dye (NanoTemper Technologies, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Derivative Assay, Analogues, Fluorescence, Biomarker Discovery, Binding Assay

Journal: bioRxiv

Article Title: Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

doi: 10.1101/2025.10.06.680836

Figure Lengend Snippet: ELISA-based CD28:B7-1 binding assays were performed using recombinant CD28 immobilized on 96-well plates and biotinylated CD80 as ligand. Increasing concentrations of test compounds were added, and binding was detected with streptavidin–HRP. All compounds inhibited CD28– CD80 binding in a concentration-dependent manner. Dose–response curves yielded IC 50 values of 71.61 μM for 8VS (A), 18.89 μM for BPU11 (B), 86.74 μM for BPU16 (C), and 45.16 μM for BPU18 (D). Data represent mean ± SEM from at least three independent experiments, with nonlinear regression fits generated using a four-parameter logistic model.

Article Snippet: His-tagged human CD28 protein (Acro Biosystems) was labeled with RED-tris-NTA 2nd Generation dye (NanoTemper Technologies, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Concentration Assay, Generated

Journal: bioRxiv

Article Title: Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

doi: 10.1101/2025.10.06.680836

Figure Lengend Snippet: (A) Three-dimensional representation of BPU11 (stick representation) docked within the CD28 binding site (ribbon representation, colored by secondary structure). The binding site is located within the conserved region comprising residues 99-104. (B) Two-dimensional interaction diagram showing detailed molecular interactions between BPU11 and CD28 residues, including hydrogen bonds (dashed lines) with key residues LYS95, PHE93, HIS38, SER110, LYS109, and ASN111. (C-E) Surface representations of the CD28 binding site showing conformational changes during molecular dynamics simulation.

Article Snippet: His-tagged human CD28 protein (Acro Biosystems) was labeled with RED-tris-NTA 2nd Generation dye (NanoTemper Technologies, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

doi: 10.1101/2025.10.06.680836

Figure Lengend Snippet: (A) RMSD trajectories over 50 ns simulation time comparing the BPU11/CD28 complex (blue) with unbound CD28 protein (green). The complex shows initial equilibration followed by stable dynamics with RMSD values around 0.5 nm. (B) Per-residue RMSF analysis showing differential flexibility patterns between bound (blue) and unbound (green) states, with notable changes in the 80-100 residue region indicating allosteric effects of ligand binding.

Article Snippet: His-tagged human CD28 protein (Acro Biosystems) was labeled with RED-tris-NTA 2nd Generation dye (NanoTemper Technologies, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Residue, Ligand Binding Assay

Journal: bioRxiv

Article Title: Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

doi: 10.1101/2025.10.06.680836

Figure Lengend Snippet: (A) Dose-dependent inhibition of CD28–CD80 interactions by BPU11 measured in CHO-K1 cells co-expressing CD28-SmBiT and CD80-LgBiT. (B) Dose-dependent inhibition of CD28–CD86 interactions by BPU11 in CHO-K1 cells co-expressing CD28-SmBiT and CD86-LgBiT. Cells were treated with BPU11 (0.1–500 μM) for 2 h at 37 °C, and luminescence was normalized to DMSO-treated controls. (C) Jurkat T-cell viability following exposure to BPU11 (10–500 μM) for 24 h, assessed by MTS assay. Viability remained >85% at concentrations up to 300 μM, with modest reductions (∼60% of control) at 500 μM. BPU16 and BPU18 were excluded from NanoBit functional assays due to cytotoxicity observed at ≥30 μM in preliminary viability screens. Data represent mean ± SEM from three independent experiments.

Article Snippet: His-tagged human CD28 protein (Acro Biosystems) was labeled with RED-tris-NTA 2nd Generation dye (NanoTemper Technologies, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Inhibition, Expressing, MTS Assay, Control, Functional Assay

Journal: bioRxiv

Article Title: Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

doi: 10.1101/2025.10.06.680836

Figure Lengend Snippet: Jurkat effector cells were co-cultured with Raji antigen-presenting cells in the CD28 Blockade Bioassay to measure inhibition of CD28-mediated costimulation. Cells were treated with increasing concentrations of compounds for 5 h, and luminescence was quantified using the Bio-Glo™ Luciferase Assay System. (A) 8VS inhibited CD28 signaling with an IC 50 of 22.77 ± 6.8 μM. (B) BPU11 displayed stronger inhibition with an IC 50 of 7.9 ± 2.3 μM. Data represent mean ± SEM from at least three independent experiments, and curves were fitted using a four-parameter logistic regression model.

Article Snippet: His-tagged human CD28 protein (Acro Biosystems) was labeled with RED-tris-NTA 2nd Generation dye (NanoTemper Technologies, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Cell Culture, Bioassay, Inhibition, Luciferase

Journal: bioRxiv

Article Title: Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

doi: 10.1101/2025.10.06.680836

Figure Lengend Snippet: Quantification of IFN-γ (A) , IL-2 (B) , and TNF-α (C) secretion (pg/mL) in apical supernatants by ELISA. BPU11 suppressed CD28-induced cytokine production in a dose-dependent manner. Statistical comparisons to the “CD3/CD28 + Vehicle” group were performed using one-way ANOVA followed by Dunnett’s post-hoc test. * p < 0.05 and *** p < 0.001 relative to “CD3/CD28 + Vehicle” group. Error bars represent the standard error mean from n=5 replicates.

Article Snippet: His-tagged human CD28 protein (Acro Biosystems) was labeled with RED-tris-NTA 2nd Generation dye (NanoTemper Technologies, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay