Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 1. Expression of TIM3 and TGFβRII and their ligands in prostate cancer: (A) Expression of GAL9 in prostate cancer and normal prostate tissues in the GEPIA 2 database; (B) Expression of TGF-β in prostate cancer and normal prostate tissues in the GEPIA 2 database; (C) Correlation between the expression of TIM3 and TGFβRII, GAL9, and TGF-β in the GEPIA 2 database; (D) IHC of GAL9 in prostate cancer in the HPA database; (E) IHC of TGF-β in prostate cancer in the HPA database; (F) The left panel shows unsupervised graph-based clustering of all samples visualized by UMAP delineated by cell type, and the right panel shows the expression of TIM3 and TGFβRII in different cell populations in the single-cell sequencing results; (G) Expression of TIM3 and TGFβRII in T cells in the single-cell sequencing results (the left panel is a scatter plot, and the right panel is a violin plot); (H) Coexpression of TIM3 and TGFβRII in T cells in the single-cell sequencing results, where 1 (blue) represents T cells that coexpress TIM3 and TGFβRII, and 0 (gray) represents other T cells.

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Expressing, Sequencing

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 2. Preparation and phenotypic characterization of DT-PSMA-CAR-T cells: (A) Gene structure of PSMA-CAR and DT-PSMA-CAR; (B) Expansion of the UTD group, PSMA-CAR-T group, and DT-PSMA-CAR-T group T cells 14 days after electroporation; (C) The positivity rate of CAR in the PSMA-CAR-T group and DT-PSMA-CAR-T group, after magnetic bead sorting, assessed by flow cytometry; (D) Expression of TIM3 and TGFβRII on the three groups of T cells assessed by flow cytometry before performing in vitro killing assays; (E) Expression of CD4 and CD8 on the three groups of T cells assessed by flow cytometry before performing in vitro killing assays; (F) Expression of CD45RO on the three groups of T cells assessed by flow cytometry before performing in vitro killing assays.

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Electroporation, Flow Cytometry, Expressing, In Vitro

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 4. Analysis of the resistance of DT-PSMA-CAR-T cells to immune suppression caused by TIM3 activation in vitro was performed by tumor lysis assays: (A and B) UTD group, PSMA-CAR-T group, and DT-PSMA-CAR-T group T cells were cocultured with LNCAP cells in the presence of 10 μg/ml TIM3 activating monoclonal antibody; (C and D) Under an effector-to-target ratio of 5:1, the three groups of T cells were cocultured with LNCAP cells in the presence of different concentrations of TIM3 activating monoclonal antibody; (E) Cytokine secretion was detected in the supernatant of all experimental groups in (C).

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Activation Assay, In Vitro, Lysis

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 5. Analysis of the resistance of DT-PSMA-CAR-T cells to immune suppression caused by TIM3 and TGFβRII activation in vitro was performed by tumor lysis assays: (A and B) UTD group, PSMA-CAR-T group, and DT-PSMA-CAR-T group T cells were cocultured with LNCAP cells in the presence of 20 ng/ml TGF-β and 10 μg/ml TIM3 activating monoclonal antibody; (C and D) Under an effector-to-target ratio of 5:1, the three groups of T cells were cocultured with LNCAP cells in the presence of different combinations of TGF-β and TIM3 activating monoclonal antibody; (E) Cytokine secretion was detected in the supernatant of all experimental groups in (C).

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Activation Assay, In Vitro, Lysis

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 6. DT-PSMA-CAR-T cells can inhibit the growth of NSG mouse xe nografts in an environment activated by TIM3 and TGFβRII: (A) The timeline of animal experiments: 1 × 106 GAL9- PSMA-PC3 cells were subcutaneously injected; two weeks later, 5 × 106 UTD T cells, PSMA-CAR-T cells, or DT- PSMA-CAR-T cells were infused per mouse via the tail vein; and biolumi nescence imaging was performed weekly; (B) The location and tumor burden of the xenografts displayed by bioluminescence imaging; (C) Quanti tative results of tumor burden obtained by Aniview100.

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Injection, Imaging

Journal: Frontiers in immunology

Article Title: AI-enhanced profiling of phage-display-identified anti-TIM3 and anti-TIGIT novel antibodies.

doi: 10.3389/fimmu.2025.1499810

Figure Lengend Snippet: FIGURE 2 Binding validation and specificity assessment of the 5 final candidates. The 5 final candidates and the reference antibodies were produced as human IgG1 in HEK293 supernatant, concentrated and dosed. (A, B): Binding validation. For HTRF assays, the recombinant extracellular domains of TIM3 (A) or TIGIT (B) fused to a biotinylated Avitag were incubated with increasing concentrations of the antibodies. Antibodies and targets were detected with fluorophore-coupled sensors: d2 acceptor coupled to an anti-IgG and terbium donor coupled to the streptavidin. The binding was assessed as the energy transfer between the donor and acceptor and computed as the HTRF ratio: 665nm acceptor emission/620 nm donor emission x 10,000. Curves were fitted mathematically with GraphPad Prism software. (C) Specificity evaluation of the candidates. HEK293 cells were transiently transfected with the Flag-tagged target gene and incubated with the candidate or reference antibodies. The target expression was monitored with a PE-coupled anti-Flag antibody and the binding of the antibodies was followed with an APC-coupled anti-IgG. Percentage of APC+ PE+ cells among the total PE+ cell population was indicated through a color gradient. (D–F) Binding of endogenous TIM3 by 6E9 in immune-relevant cells. NK lines NKL and NK-92 (D), activated PBMC (E) and TRM cultured with TGF-b1 and IL-15 (F) were incubated with 6E9 or 7KQL as a control. The T and NK lymphocytes subsets of the PBMC and TRM were optically isolated after staining of CD3 and CD56 (CD3+ T lymphocytes and CD3-CD56+ NK lymphocytes).

Article Snippet: Phage display libraries screening was assessed on recombinant human His-tagged TIM3 or human His-Tagged TIGIT (ACROBiosystems) coated at 25 μg/ml for the first round, and 10 μg/ml for the second round on a Nunc MaxiSorp plates (Thermo Fisher).

Techniques: Binding Assay, Biomarker Discovery, Produced, Recombinant, Incubation, Software, Transfection, Expressing, Cell Culture, Control, Isolation, Staining

Journal: Frontiers in immunology

Article Title: AI-enhanced profiling of phage-display-identified anti-TIM3 and anti-TIGIT novel antibodies.

doi: 10.3389/fimmu.2025.1499810

Figure Lengend Snippet: FIGURE 3 Post-translation modifications analysis. (A) PTM motifs shown on VH-VL 3D representations. The main PTM-prone residues of the identified deamidation, isomerization, and methionine or tryptophan oxidation sites are shown in colored spheres. The antibodies surfaces are shown in grey (frameworks) and pale green (CDRs). (B, C) Comparison with INN antibodies. The number of PTM motifs with solvent accessibility higher than 30% were computed from 735 INN antibodies. The number of motifs identified for our anti-TIGIT (B) and anti-TIM3 (C) candidates and their references are shown as colored lines are compared with the distribution of the INN antibodies shown in blue.

Article Snippet: Phage display libraries screening was assessed on recombinant human His-tagged TIM3 or human His-Tagged TIGIT (ACROBiosystems) coated at 25 μg/ml for the first round, and 10 μg/ml for the second round on a Nunc MaxiSorp plates (Thermo Fisher).

Techniques: Comparison, Solvent

Journal: Frontiers in immunology

Article Title: AI-enhanced profiling of phage-display-identified anti-TIM3 and anti-TIGIT novel antibodies.

doi: 10.3389/fimmu.2025.1499810

Figure Lengend Snippet: FIGURE 4 Aggregation parameters analysis. (A) Surface physicochemical patches. Surface properties (i.e. hydrophobicity, electrostatic forces, charges and shape) of the VH-VL pairs were computed and moderate (orange) to intense (red) regions were shown on the VH-VL structures. (B, C) Aggregation scores comparison with publicly-disclosed antibodies. The surface properties were used to train a model allowing to predict an aggregation score. Scores were computed for our anti-TIGIT (B) and anti-TIM3 (C) candidates and their references, and compared with the scores distribution computed from 735 INN antibodies (blue) and 31,712 patented antibodies (green).

Article Snippet: Phage display libraries screening was assessed on recombinant human His-tagged TIM3 or human His-Tagged TIGIT (ACROBiosystems) coated at 25 μg/ml for the first round, and 10 μg/ml for the second round on a Nunc MaxiSorp plates (Thermo Fisher).

Techniques: Comparison

Journal: Frontiers in immunology

Article Title: AI-enhanced profiling of phage-display-identified anti-TIM3 and anti-TIGIT novel antibodies.

doi: 10.3389/fimmu.2025.1499810

Figure Lengend Snippet: FIGURE 5 Humanness evaluation. A humanness score was computed from publicly available antibody sequences which allowed discrimination between human, humanized and non-human sequences (from chimeric antibodies). Separate scores were computed for the VH (A, C) and the VL (B, D). The scores obtained for our anti-TIGIT (upper panels) and anti-TIM3 (lower panels) candidates are compared with the benchmark distributions.

Article Snippet: Phage display libraries screening was assessed on recombinant human His-tagged TIM3 or human His-Tagged TIGIT (ACROBiosystems) coated at 25 μg/ml for the first round, and 10 μg/ml for the second round on a Nunc MaxiSorp plates (Thermo Fisher).

Techniques:

Journal: Frontiers in immunology

Article Title: AI-enhanced profiling of phage-display-identified anti-TIM3 and anti-TIGIT novel antibodies.

doi: 10.3389/fimmu.2025.1499810

Figure Lengend Snippet: FIGURE 6 Off-targets prediction. The numbers of predicted off-targets, either human (red) or non-human (blue), were computed for all FDA-approved antibodies and ranked as a probability of being recognized (high vs. medium). The number of off-targets predicted for our anti-TIGIT (left panel) and anti-TIM3 (right panel) candidates were compared with that scale.

Article Snippet: Phage display libraries screening was assessed on recombinant human His-tagged TIM3 or human His-Tagged TIGIT (ACROBiosystems) coated at 25 μg/ml for the first round, and 10 μg/ml for the second round on a Nunc MaxiSorp plates (Thermo Fisher).

Techniques:

Journal: Frontiers in immunology

Article Title: AI-enhanced profiling of phage-display-identified anti-TIM3 and anti-TIGIT novel antibodies.

doi: 10.3389/fimmu.2025.1499810

Figure Lengend Snippet: FIGURE 7 Affinity evaluation and structural model definition of the antibody-antigen complexes. (A, B) Experimental competition assays. Pairwise competition assays of TIGIT binders (A) or anti-TIM3 antibodies (B) were performed by flow cytometry. Briefly, the Fc fragments of our human IgG1 candidates were replaced by mouse IgG2a Fc fragments. HEK293 cells were transiently transfected with the Flag-tagged target gene and co-incubated the human IgG1-formatted and the mouse IgG2a-formatted candidates. In the case of TIGIT, the competition was also evaluated with its natural ligand CD155 fused to a mouse IgG2a Fc fragment. The target expression was monitored with a PE-coupled anti-Flag antibody and the binding of the human IgG1-formatted antibodies was followed with an APC-coupled antibody. The percentage of APC+PE+ cells (among the PE+ subset) was indicated as a color gradient. (C) Affinity predictions and structural model definition. The antibodies were docked on selected epitopes (indicated in the left boxes). The affinity of the obtained complexes was computed as the C-score (left column) which was a combination of PCC-score (middle column) and IR-score (right column). The upper panels show the affinity predicted for the anti-TIGIT candidates docked on 7VYT epitope. The middle and lower panels show the affinity predicted for the anti-TIM3 antibodies docked on 6TXZ and 7KQL epitope, respectively.

Article Snippet: Phage display libraries screening was assessed on recombinant human His-tagged TIM3 or human His-Tagged TIGIT (ACROBiosystems) coated at 25 μg/ml for the first round, and 10 μg/ml for the second round on a Nunc MaxiSorp plates (Thermo Fisher).

Techniques: Cytometry, Transfection, Incubation, Expressing, Binding Assay