Fig 1: Effect of IgTT-4E1-S on PD-L1/PD-1 blockade, 4-1BB costimulation, and IFNγ secretion. (a) ADCC reporter bioassay response to rituximab (rtx), IgTT-4E1, and IgTT-4E1-S, using ADCC bioassay effector JurkatNFAT-CD16 cells co-cultured with 3T3 or 3T3EGFR target cells at a 6:1 E/T ratio for 6 h at 37 °C. After incubation, Bio-Glo™ Luciferase Assay Reagent was added for luminescence determination. Data shown represent the mean ± standard deviation of triplicates. Data are presented as the mean ± SD (n = 3). Significance was determined by two-way ANOVA with Tukey’s multiple comparison test. (b) PD-L1/PD-1 blockade bioassay assesses the inhibitory activities of IgTT-4E1-S. Y-axis represents reporter gene fold induction. Cetuximab (ctx) was used as a negative control, and atezolizumab (atz) as a positive control. Fold induction relative to negative control (ctx)-incubated cells. Results are expressed as mean ± SD (n = 3). Significance was measured by one-way ANOVA with Dunnett’s multiple comparison test. (c) Costimulation of JurkatNFkB-4-1BB cells co-cultured with CHO cells, CHOEGFR, or CHOPD-L1 cells in the presence of 10-fold increasing concentrations of IgTT-4E1-S, urelumab (url), and rituximab (rtx) antibodies for 6 h at 37 °C. After incubation, luminescence was determined. Data are presented as fold induction relative to the values obtained from unstimulated JurkatNFkB-4-1BB cells. Representative dose–concentration curves are presented and expressed as mean ± SD (n = 3). Significance was determined by two-way ANOVA with Tukey’s multiple comparison test. (d) Flow cytometry analysis of EGFR and PD-L1 expression in MDA-MB-231 cancer cells. Cells incubated with PE-conjugated and APC-conjugated isotypes are shown as gray-filled histogram. (e) Flow cytometry analysis of PD-1 and 4-1BB expression in PBMCs preactivated with anti-CD3 mAb for 24 h. PBMCs incubated with FITC-conjugated and PE-conjugated isotypes are shown as gray-filled histogram. (f) Anti-CD3 preactivated PBMCs were co-cultured with MDA-MB231 target cells at effector/target (E/T) ratios of 1:1 and 2:1. Control human polyclonal IgG (control hIgG), urelumab (url), atezolizumab (atz), and IgTT-4E1-S were added at 6.67 nM. The IFNγ concentrations in supernatant after 48 h were analyzed and expressed as mean ± SD (n = 3). Significance was determined by two-way ANOVA with Tukey’s multiple comparison test.
Fig 2: Gene construct, molecular model, and binding characteristics of the IgTT-4E1 antibody. (a) Gene layout of the IgTT-4E1 antibody bearing a signal peptide from oncostatin M (white box), one anti-4-1BB scFv (magenta boxes), one anti-EGFR VHH (green box), one anti-PD-L1 VHH (blue box), three collagen-derived trimerization (TIE) domains (yellow boxes) flanked by peptide linkers, and the Fc-encoding element (gray box). N-terminal FLAG-Strep and C-terminal Myc-His tags (orange boxes) were appended for purification and immunodetection purposes. (b) Schematic diagram showing the three-dimensional model of the anti-4-1BB/EGFR/PD-L1 tandem trimerbody (TT). (c) Molecular diagram of the IgTT-4E1 antibody and its three-dimensional modelizations, in front and top views. (d) Human EGFR-Fc (EGFR) was immobilized onto four different biosensors; three biosensors were incubated in 20 nM IgTT-4E1 for 20 min (red trace), while the fourth was kept in HBS as a negative control (gray trace). Then, 50 nM human PD-L1-Fc (PD-L1) was added to two of the IgTT-4E1-loaded biosensors (blue trace) and the control biosensor for 20 min. Also, 50 nM human 4-1BB-hFc (4-1BB) was then added to one PD-L1- and IgTT-4E1-treated biosensor (black trace) and the control biosensor. The cumulative signal increases on the biosensor receiving IgTT-4E1, PD-L1, and 4-1BB demonstrate that all three antigens can be bound by the IgTT-4E1 antibody simultaneously. (e) The binding to human EGFR, PD-L1, and 4-1BB on the cell surface of 3T3, 3T3EGFR, CHO, CHOEGFR, CHOPD-L1, Jurkat, and Jurkat4-1BB cells by rituximab (rtx), cetuximab (ctx), atezolizumab (atz), urelumab (url), and IgTT-4E1 at 6.67 nM was measured by FACS. Cells incubated with PE-conjugated GAH antibody (GAH-PE) are shown as non-filled histogram. The y-axis shows the relative cell number, and the x-axis represents the intensity of fluorescence expressed on a logarithmic scale.
Fig 3: STAb-T19 and STAb-TP4 cells efficiently secrete CD19 × CD3 and PD-L1 × 4-1BB, respectively, and synergize to induce potent specific cytotoxicity. (A, B) Western blot detection of secreted CD19 × CD3 (A) or PD-L1 × 4-1BB (B) in the conditioned media from lentivirally transduced human primary T cells (STAb-T19 or STAb-TP4, respectively). Conditioned media from nontransduced T cells (NT-T) was used as a negative control. (C, D) Detection of soluble functional CD19 x CD3 (C) or PD-L1 x 4-1BB (D) in the conditioned media from STAb-T19 or STAb-TP4 by ELISA against plastic-immobilized human CD19-Fc chimera (hCD19-Fc), human PD-L1-Fc chimera (hPD-L1-Fc), human 4-1BB-Fc chimera (h4-1BB-Fc) or BSA. Conditioned media from NT-T cells was used as a negative control. The data are presented as mean ± SD (n = 3). (E) Representative analysis of intracellular and cell surface-bound (decoration) CD19 × CD3 and PD-L1 × 4-1BB in NT-T, STAb-T19 and STAb-TP4 cells by flow cytometry. One representative experiment out of four independent experiments is shown. The numbers represent the percentage of cells that stained positive for the His tag. (F) Cell cytotoxicity assay. Decreasing numbers of STAb-T19 cells were cocultured with 5 × 104 CD19+PD-L1+ (Nalm6PD-L1/Luc) or CD19-PD-L1-(K562Luc) luciferase-expressing target cells in the presence or absence of a constant number of STAb-TP4 cells. NT-T cells were added as indicated to maintain a constant 1:1 effector:target ratio. Cocultures of target cells with STAb-TP4 cells were used as controls. The percentage of cytotoxicity was calculated after 48 h and normalized to the target cell death observed in cocultures with NT-T cells. The data are shown as mean ± SD (n = 3). Significance was determined by two-way ANOVA with Sidak’s multiple comparisons test. Significance is defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (G) Nalm6PD-L1 cells were cocultured with 1:1 mixtures of STAb-TP4 + NT-T, STAb-T19 + NT-T or STAb-T19 + STAb-TP4 cells at the indicated E:T ratios, and the relative percentages of T cells (CD2+CD10-) and leukemic cells (CD2-CD10+) were analyzed by flow cytometry after 0, 2, 5, and 9 days.
Fig 4: STAb-T19 and STAb-TP4 cells efficiently secrete CD19 × CD3 and PD-L1 × 4-1BB, respectively, and synergize to induce potent specific cytotoxicity. (A, B) Western blot detection of secreted CD19 × CD3 (A) or PD-L1 × 4-1BB (B) in the conditioned media from lentivirally transduced human primary T cells (STAb-T19 or STAb-TP4, respectively). Conditioned media from nontransduced T cells (NT-T) was used as a negative control. (C, D) Detection of soluble functional CD19 x CD3 (C) or PD-L1 x 4-1BB (D) in the conditioned media from STAb-T19 or STAb-TP4 by ELISA against plastic-immobilized human CD19-Fc chimera (hCD19-Fc), human PD-L1-Fc chimera (hPD-L1-Fc), human 4-1BB-Fc chimera (h4-1BB-Fc) or BSA. Conditioned media from NT-T cells was used as a negative control. The data are presented as mean ± SD (n = 3). (E) Representative analysis of intracellular and cell surface-bound (decoration) CD19 × CD3 and PD-L1 × 4-1BB in NT-T, STAb-T19 and STAb-TP4 cells by flow cytometry. One representative experiment out of four independent experiments is shown. The numbers represent the percentage of cells that stained positive for the His tag. (F) Cell cytotoxicity assay. Decreasing numbers of STAb-T19 cells were cocultured with 5 × 104 CD19+PD-L1+ (Nalm6PD-L1/Luc) or CD19-PD-L1-(K562Luc) luciferase-expressing target cells in the presence or absence of a constant number of STAb-TP4 cells. NT-T cells were added as indicated to maintain a constant 1:1 effector:target ratio. Cocultures of target cells with STAb-TP4 cells were used as controls. The percentage of cytotoxicity was calculated after 48 h and normalized to the target cell death observed in cocultures with NT-T cells. The data are shown as mean ± SD (n = 3). Significance was determined by two-way ANOVA with Sidak’s multiple comparisons test. Significance is defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (G) Nalm6PD-L1 cells were cocultured with 1:1 mixtures of STAb-TP4 + NT-T, STAb-T19 + NT-T or STAb-T19 + STAb-TP4 cells at the indicated E:T ratios, and the relative percentages of T cells (CD2+CD10-) and leukemic cells (CD2-CD10+) were analyzed by flow cytometry after 0, 2, 5, and 9 days.
Fig 5: Simultaneous secretion of CD19 × CD3 and PD-L1 × 4-1BB by STAb-T19-P4 cells improves the antitumor efficacy of STAb-T19 cells in vitro. (A) Percentage of reporter protein expression in primary STAb-T19, STAb-TP4, and STAb-T19-P4 cells (GFP or tdTo). (B, C) Percentages of CD4+ and CD8+ T cells (B) and percentages of naïve (TN), central memory (TCM), effector memory (TEM) and effector memory RA+ (TEMRA) T cells (C) among nontransduced (NT-T), STAb-T19-, STAb-TP4-, or STAb-T19-P4-transduced T cells. The transduced populations were further classified into bsAb-secreting (+) or nonsecreting (−) cells on the basis of reporter gene expression. Data are shown as mean ± SD (n = 3). (D) Nalm6PD-L1 target cells were cocultured with STAb-TP4, STAb-T19, or STAb-T19-P4 cells at the indicated E:T ratios, and the relative percentages of CD2+CD10- and CD2-CD10+ cells were analyzed by flow cytometry at days 4, 12, 20, and 24. On day 16, the cocultures were split into two wells, and 5,000 target cells were added to one of them; the relative percentages of T cells and tumor cells were analyzed on days 20 and 24.
Supplier Page from R&D Systems, a Bio-Techne Brand for 4-1BB/TNFRSF9/CD137 Fc Chimera Protein
Available conjugates: Sizes Available: 100 ug