tim-3 protein  (Galectin Therapeutics)

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: 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-β1 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, 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: Binding constants.

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

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

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: 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-β1 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: Antibodies-containing HEK293 supernatants were diluted in PPI Terbium Detection buffer and the binding to TIM3 (6-His C-ter-tagged, Acro Biosystems) or TIGIT (TIT-H52H5 Human TIGIT Protein, His Tag, active dimer, AcroBiosystems) diluted at 0.6 ng/µl final concentration in small volume 384-wells plates (Greiner Bio-One).

Techniques: Binding Assay, 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: Binding constants.

Article Snippet: Antibodies-containing HEK293 supernatants were diluted in PPI Terbium Detection buffer and the binding to TIM3 (6-His C-ter-tagged, Acro Biosystems) or TIGIT (TIT-H52H5 Human TIGIT Protein, His Tag, active dimer, AcroBiosystems) diluted at 0.6 ng/µl final concentration in small volume 384-wells plates (Greiner Bio-One).

Techniques: Binding Assay

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: 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: Antibodies-containing HEK293 supernatants were diluted in PPI Terbium Detection buffer and the binding to TIM3 (6-His C-ter-tagged, Acro Biosystems) or TIGIT (TIT-H52H5 Human TIGIT Protein, His Tag, active dimer, AcroBiosystems) diluted at 0.6 ng/µl final concentration in small volume 384-wells plates (Greiner Bio-One).

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: 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: Antibodies-containing HEK293 supernatants were diluted in PPI Terbium Detection buffer and the binding to TIM3 (6-His C-ter-tagged, Acro Biosystems) or TIGIT (TIT-H52H5 Human TIGIT Protein, His Tag, active dimer, AcroBiosystems) diluted at 0.6 ng/µl final concentration in small volume 384-wells plates (Greiner Bio-One).

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: 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: Antibodies-containing HEK293 supernatants were diluted in PPI Terbium Detection buffer and the binding to TIM3 (6-His C-ter-tagged, Acro Biosystems) or TIGIT (TIT-H52H5 Human TIGIT Protein, His Tag, active dimer, AcroBiosystems) diluted at 0.6 ng/µl final concentration in small volume 384-wells plates (Greiner Bio-One).

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: 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: Antibodies-containing HEK293 supernatants were diluted in PPI Terbium Detection buffer and the binding to TIM3 (6-His C-ter-tagged, Acro Biosystems) or TIGIT (TIT-H52H5 Human TIGIT Protein, His Tag, active dimer, AcroBiosystems) diluted at 0.6 ng/µl final concentration in small volume 384-wells plates (Greiner Bio-One).

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: 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: Antibodies-containing HEK293 supernatants were diluted in PPI Terbium Detection buffer and the binding to TIM3 (6-His C-ter-tagged, Acro Biosystems) or TIGIT (TIT-H52H5 Human TIGIT Protein, His Tag, active dimer, AcroBiosystems) diluted at 0.6 ng/µl final concentration in small volume 384-wells plates (Greiner Bio-One).

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