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

Journal: Molecular Cancer Therapeutics

Article Title: A B7-H3–Targeted CD28 Bispecific Antibody Enhances the Activity of Anti–PD-1 and CD3 T-cell Engager Immunotherapies

doi: 10.1158/1535-7163.MCT-24-0327

Figure Lengend Snippet: XmAb808 binds avidly to B7-H3 on target cells to costimulate T cells. A, Binding of XmAb808 or its analogue containing a single B7-H3–binding domain (mono–B7-H3×CD28) to target cells with varying B7-H3 densities (left axis) and binding of XmAb808 to human T cells (right axis) are plotted as mean fluorescence intensity (MFI). HEK293 cells express approximately 200,000 B7-H3 antigens; A431, 196,000; 22Rv1, 86,000; LOX-IMVI cells do not express B7-H3. Because mono–B7-H3×CD28 has a lower molecular weight than XmAb808 (145 vs. 192 kDa), the mono–B7-H3×CD28 concentrations in this figure were corrected by a factor of 1.3-fold to adjust for molarity. B, Human PBMCs from 20 different donors were incubated on plates coated with air-dried (immobilized) XmAb808, anti-CD28 (TGN1412), or anti-CD3 (OKT3) antibodies. IFNγ and IL2 were measured after 24 hours. Statistical significance is denoted by **, P < 0.01; ns, not significant. C, IL2 release from PBMCs cocultured with HEK293-αCD3 or HEK293-αCD3-B7-H3 KO cells at an E:T ratio of 25:1 was measured after 24 hours. Data are represented as means ± SEM of cocultures with PBMCs from eight different human donors.

Article Snippet: Binding of XmAb808 to human (Sino Biological; 90182-C08H) and mouse (R&D Systems; 483-CD-200/CF) CD28 was measured using an Octet HTX instrument (Sartorius).

Techniques: Binding Assay, Fluorescence, Molecular Weight, Incubation

Journal: Molecular Cancer Therapeutics

Article Title: A B7-H3–Targeted CD28 Bispecific Antibody Enhances the Activity of Anti–PD-1 and CD3 T-cell Engager Immunotherapies

doi: 10.1158/1535-7163.MCT-24-0327

Figure Lengend Snippet: CD28 costimulation drives activation and survival of T cells, leading to IL2-dependent killing of cancer cells. Target cells were cocultured with human T cells (E:T = 1:1) and treated with XmAb808. A, After 24 hours, IL2 (closed symbols) and IFNγ (open symbols) were measured in culture supernatants. B, The percentages of CD4 + or CD8 + T cells that were CD25 + or Bcl-xL + from cocultures with 22Rv1-αCD3 cells following 24 hours of treatment are plotted. C, Real-time killing of 22Rv1-αCD3 cells in cocultures treated with (closed symbols) or without (open symbols) 1 µg/mL of XmAb808 plus an IL2-neutralizing antibody, an IFNγ-neutralizing antibody, or an isotype control antibody was monitored over time using an xCELLigence Real-Time Cell Analysis System. The arrow indicates the addition of T cells and antibodies at 24 hours after cancer cells were plated. Data are plotted as single points in A and B and as mean ± SEM of three replicates in C .

Article Snippet: Binding of XmAb808 to human (Sino Biological; 90182-C08H) and mouse (R&D Systems; 483-CD-200/CF) CD28 was measured using an Octet HTX instrument (Sartorius).

Techniques: Activation Assay, Control, Cell Analysis

Journal: Molecular Cancer Therapeutics

Article Title: A B7-H3–Targeted CD28 Bispecific Antibody Enhances the Activity of Anti–PD-1 and CD3 T-cell Engager Immunotherapies

doi: 10.1158/1535-7163.MCT-24-0327

Figure Lengend Snippet: XmAb808 enhances T-cell recall responses to endogenous pMHC ligands. A (top graph), Levels of HLA-A2 on A431-β2M-null cells expressing a fusion protein of β2M and HLA-A*0201 fused to CMV pp65 NLV peptide (A431-NLV, dashed black) or melanoma G209 peptide (A431-G209, solid gray) were compared with the signal from unstained cells (solid black). A (two bottom graphs), PBMCs from two unique HLA-A*0201 CMV–seropositive donors were stained with HLA-A*0201-CMV-pp65 (NLV) tetramers; the percentage of tetramer+ CD8 + T cells is shown. B, A431-NLV or A431-G209 cells were cocultured with CD3 + T cells from donor 1 at an E:T ratio of 25:1 and treated with XmAb808. IL2 and IFNγ were measured in the supernatants 24 hours after treatment. C, Parental MDA-MB-231 cells or MDA-MB-231 cells expressing the CMV pp65 protein (MDA-MB-231-pp65) were cocultured with CD3 + T cells from donor 2 at an E:T ratio of 10:1 and treated with XmAb808. IL2 and IFNγ were measured in the supernatants 24 hours after treatment. Data are represented as the mean ± SEM of three replicates.

Article Snippet: Binding of XmAb808 to human (Sino Biological; 90182-C08H) and mouse (R&D Systems; 483-CD-200/CF) CD28 was measured using an Octet HTX instrument (Sartorius).

Techniques: Expressing, Staining

Journal: Molecular Cancer Therapeutics

Article Title: A B7-H3–Targeted CD28 Bispecific Antibody Enhances the Activity of Anti–PD-1 and CD3 T-cell Engager Immunotherapies

doi: 10.1158/1535-7163.MCT-24-0327

Figure Lengend Snippet: XmAb808 enhances the activity of an EpCAM×CD3 TCE in vitro . A and B, 22Rv1-NLR cells were cocultured with T cells at an E:T ratio of 10:1 and treated with a dose titration of EpCAM×CD3 with (closed symbols) or without (open symbols) 1 μg/mL of XmAb808. A, After 24 hours, IL2 and IFNγ secretion was measured, and B, CD25 + or Bcl-xL + T cells were counted. CD25 + or Bcl-xL + cell counts are presented as a percentage of total CD4 + or CD8 + T cells following 24 hours of treatment. For cytokine measures, data are represented as the means of two replicates. C, 22Rv1-NLR cells were cocultured with T cells at an E:T ratio of 1:1 and treated with a dose titration of XmAb808 and 1 ng/mL of EpCAM×CD3; 10 μg/mL of an IL2-neutralizing, an IFNγ-neutralizing, or an isotype control antibody was also added. After 5 days, CD4 + and CD8 + T cells in 100 μL were counted with flow cytometry. D, The viability of 22Rv1-NLR cells in coculture with T cells at an E:T ratio of 1:1 was monitored in real time using an Incucyte Live-Cell Analysis System. Cocultures were treated with 10 μg/mL of EpCAM×CD3, 1 μg/mL of XmAb808, and/or 10 μg/mL of an IL2- neutralizing, an IFNγ-neutralizing, or an isotype control antibody, as indicated. The arrow indicates when T cells and antibodies were added. Data are represented as means ± SEM; n = 3.

Article Snippet: Binding of XmAb808 to human (Sino Biological; 90182-C08H) and mouse (R&D Systems; 483-CD-200/CF) CD28 was measured using an Octet HTX instrument (Sartorius).

Techniques: Activity Assay, In Vitro, Titration, Control, Flow Cytometry, Cell Analysis

Journal: Molecular Cancer Therapeutics

Article Title: A B7-H3–Targeted CD28 Bispecific Antibody Enhances the Activity of Anti–PD-1 and CD3 T-cell Engager Immunotherapies

doi: 10.1158/1535-7163.MCT-24-0327

Figure Lengend Snippet: An XmAb808 analogue enhances antitumor activity of an EpCAM×CD3 TCE in human PBMC-engrafted mice. NSG-MHC I/II DKO mice were intradermally inoculated with HPAF-II cells. After 2 weeks, palpable tumors formed, and mice were then engrafted with human PBMCs together with 5 mg/kg of EpCAM×CD3 alone or in combination with 1 mg/kg of B7-H3×Null or 1 mg/kg of XmAb808 s weekly. A, Tumor volumes are shown over time as mean ± SEM with n = 8–10 mice/group. B, Tumor volumes of individual mice at the end of the study. Each horizontal line represents mean values. C and D, Human T-cell counts in peripheral blood after 21 days of treatment. Each horizontal line represents the geometric mean. E, hCD45 + cells counted in the peripheral blood are shown over time as mean ± SEM with n = 8–10 mice/group. For B–D , asterisks denote statistical significance:*, P < 0.05; **, P < 0.01. XmAb808 s is a surrogate B7-H3×CD28 bispecific identical to XmAb808 except it lacks the half-life–extending mutations in its Fc domain.

Article Snippet: Binding of XmAb808 to human (Sino Biological; 90182-C08H) and mouse (R&D Systems; 483-CD-200/CF) CD28 was measured using an Octet HTX instrument (Sartorius).

Techniques: Activity Assay

Journal: Molecular Cancer Therapeutics

Article Title: A B7-H3–Targeted CD28 Bispecific Antibody Enhances the Activity of Anti–PD-1 and CD3 T-cell Engager Immunotherapies

doi: 10.1158/1535-7163.MCT-24-0327

Figure Lengend Snippet: XmAb808 combines with an anti–PD-1 antibody to suppress tumor growth in human PBMC-engrafted mice. NSG-MHC I/II DKO mice were intradermally inoculated with MDA-MB-231-pp65 cells. After 18 days, palpable tumors formed, and mice were engrafted with human PBMCs together with 3 mg/kg of XmAb808 and/or 3 mg/kg of anti–PD-1 weekly. A, Tumor volumes are shown over time as mean ± SEM of 10 mice/group. B, Tumor volumes of individual mice at the end of the study. Each horizontal line represents mean values. C and D, Human CD4 + and CD8 + T-cell counts in the peripheral blood after 14 days of treatment. Each horizontal line represents the geometric mean. For B–D , asterisks denote statistical significance: *, P < 0.05; **, P < 0.01.

Article Snippet: Binding of XmAb808 to human (Sino Biological; 90182-C08H) and mouse (R&D Systems; 483-CD-200/CF) CD28 was measured using an Octet HTX instrument (Sartorius).

Techniques: