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Galectin Therapeutics protein 3 tim3
Protein 3 Tim3, supplied by Galectin Therapeutics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/protein+3+tim3/lgals3/pm41810858-249-22-8
Average 86 stars, based on 1 article reviews

protein 3 tim3 - by Bioz Stars, 2026-09

86/100 stars

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Article Title: Proteomic profiling reveals potential mechanisms regulating acquisition of luteolytic capacity in the bovine corpus luteum†.
Article Snippet: D ow naded rom http/academ ic.p.com /biolreprod/advance/10.1093/biolre/ioag059/8514081 by BD -FLC H -U SP user on 15 M arch 2026 UN CO RR EC TE D MA NU SC RI PT 3 The corpus luteum (CL) produces progesterone to support pregnancy but undergoes luteolysis in response to prostaglandin F2A (PGF2A) when pregnancy is absent.. Developing CL resist PGF2A-induced regression.. This acquisition of luteolytic capacity (ALC) occurs around day 5 of the estrous cycle in cattle.

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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).

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

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.

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.

Techniques: Comparison, Solvent

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).

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).

Techniques: Comparison

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.

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.

Techniques:

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.

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.

Techniques:

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.

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.

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

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).

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, 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: Binding constants.

Techniques: Binding Assay

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.

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.

Techniques: Comparison, Solvent

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).

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).

Techniques: Comparison

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.

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.

Techniques:

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.

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.

Techniques:

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.

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.

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

Alignment of the V H and V L amino acid sequences in TIM-3 antibodies. The framework regions (FRs) and complementarity-determining regions (CDRs) within the V H and V L regions were highlighted accordingly. The absence of no amino acid at a certain location is indicated by the symbol

Journal: Heliyon

Article Title: Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands

doi: 10.1016/j.heliyon.2024.e28126

Figure Lengend Snippet: Alignment of the V H and V L amino acid sequences in TIM-3 antibodies. The framework regions (FRs) and complementarity-determining regions (CDRs) within the V H and V L regions were highlighted accordingly. The absence of no amino acid at a certain location is indicated by the symbol "-".

Article Snippet: Microplate wells were coated with recombinant proteins overnight at 4 °C, including human TIM-3-His (hTIM-3; Sino Biological, Beijing, China; 10390-H08H), CD137-His (hCD137; Sino Biological, 10041-H08H), OX40-His (hOX40; Sino Biological, 10481-H08H), PD-L1-His (hPD-L1; Sino Biological, 10084-H08H), mouse TIM-3-His (mTIM-3; Sino Biological, 51152-M08H), or cynomolgus TIM-3-hFc (cynTIM-3; Sino Biological, 90312-C02H) at a concentration of 1 μg/mL.

Techniques:

Analysis of the specificities of TIM-3 antibodies. (A) The purified TIM-3-specific mouse IgG antibodies (Abs) were subjected to SDS-PAGE analysis under non-denaturing (left panel) and denaturing (right panel) conditions. The separate images were cropped from the same gel. The entire image of the gel is shown in The recognition characteristics of TIM-3 Abs for native and denatured TIM-3 protein in western blot analysis. The combined images were cropped from different images. The entire image of western blots is shown in The specificity of the anti-TIM-3 mAbs was assess by ELISAs. The columns represent mean ± standard deviation (SD) of triplicates. Statistical significance was indicated as ****p < 0.0001, ***p < 0.001, and *p < 0.05 compared with Blank. Consistent results were observed in three independent experiments. (D, E) Reactivities of anti-TIM-3 mAbs towards endogenous cell surface TIM-3 protein on the RPMI8226 multiple myeloma cell line were assessed using flow cytometry. The RPMI8226 cells without addition of TIM-3 mAb were used as control (Con). Representative dot plots (D) and histograms (E) depicting the TIM-3 expression by flow cytometry were generated. M: molecular weight marker; 1 = MsT001; 2 = MsT065; 3 = MsT229; 4 = MsT286; 5 = non-denatured TIM-3 protein; 6 = denatured TIM-3 protein; hTIM-3 = human TIM-3; cynTIM-3 = cynomolgus TIM-3; mTIM-3 = mouse TIM-3; hCD137 = human CD137; hPD-L1 = human PD-L1; hOX40 = human OX40.

Journal: Heliyon

Article Title: Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands

doi: 10.1016/j.heliyon.2024.e28126

Figure Lengend Snippet: Analysis of the specificities of TIM-3 antibodies. (A) The purified TIM-3-specific mouse IgG antibodies (Abs) were subjected to SDS-PAGE analysis under non-denaturing (left panel) and denaturing (right panel) conditions. The separate images were cropped from the same gel. The entire image of the gel is shown in The recognition characteristics of TIM-3 Abs for native and denatured TIM-3 protein in western blot analysis. The combined images were cropped from different images. The entire image of western blots is shown in The specificity of the anti-TIM-3 mAbs was assess by ELISAs. The columns represent mean ± standard deviation (SD) of triplicates. Statistical significance was indicated as ****p < 0.0001, ***p < 0.001, and *p < 0.05 compared with Blank. Consistent results were observed in three independent experiments. (D, E) Reactivities of anti-TIM-3 mAbs towards endogenous cell surface TIM-3 protein on the RPMI8226 multiple myeloma cell line were assessed using flow cytometry. The RPMI8226 cells without addition of TIM-3 mAb were used as control (Con). Representative dot plots (D) and histograms (E) depicting the TIM-3 expression by flow cytometry were generated. M: molecular weight marker; 1 = MsT001; 2 = MsT065; 3 = MsT229; 4 = MsT286; 5 = non-denatured TIM-3 protein; 6 = denatured TIM-3 protein; hTIM-3 = human TIM-3; cynTIM-3 = cynomolgus TIM-3; mTIM-3 = mouse TIM-3; hCD137 = human CD137; hPD-L1 = human PD-L1; hOX40 = human OX40.

Techniques: Purification, SDS Page, Western Blot, Standard Deviation, Flow Cytometry, Expressing, Generated, Molecular Weight, Marker

Binding characteristics of TIM-3-specific antibodies. (A) Identification of distinct epitopes of TIM-3 through sandwich ELISAs. (B) The verification of various epitopes on TIM-3 using the ForteBio Octet system. A sensorgram shows TIM-3-Fc on the anti-human IgG Fc capture sensors, which has been linked to an anti-TIM-3 antibody to achieve saturation, and then bind to all TIM-3 antibodies. Statistical significance was indicated as ****p < 0.0001 and *p < 0.05 compared with Blank.

Journal: Heliyon

Article Title: Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands

doi: 10.1016/j.heliyon.2024.e28126

Figure Lengend Snippet: Binding characteristics of TIM-3-specific antibodies. (A) Identification of distinct epitopes of TIM-3 through sandwich ELISAs. (B) The verification of various epitopes on TIM-3 using the ForteBio Octet system. A sensorgram shows TIM-3-Fc on the anti-human IgG Fc capture sensors, which has been linked to an anti-TIM-3 antibody to achieve saturation, and then bind to all TIM-3 antibodies. Statistical significance was indicated as ****p < 0.0001 and *p < 0.05 compared with Blank.

Techniques: Binding Assay

Sensitivities of TIM-3-specific antibodies. (A) Sensitivities of unlabeled (left) and biotin-labeled (right) TIM-3 monoclonal antibodies were determined by indirect ELISAs. (B) The sensitivities of anti-TIM-3 antibody pairs in sandwich ELISA assays.

Journal: Heliyon

Article Title: Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands

doi: 10.1016/j.heliyon.2024.e28126

Figure Lengend Snippet: Sensitivities of TIM-3-specific antibodies. (A) Sensitivities of unlabeled (left) and biotin-labeled (right) TIM-3 monoclonal antibodies were determined by indirect ELISAs. (B) The sensitivities of anti-TIM-3 antibody pairs in sandwich ELISA assays.

Techniques: Labeling, Sandwich ELISA

TIM-3 antibodies' affinities for human TIM-3 as assessed by the ForteBio Octet system. The original sensorgram shows TIM-3-Fc on the anti-human IgG Fc capture sensors, which bound to various concentrations of TIM-3 antibodies. The binding affinity parameter K D was determined by fitting the sensorgram. R 2 is the coefficient of determination to estimate the goodness of the curve fit as reported by ForteBio Data Analysis Software 9.0.

Journal: Heliyon

Article Title: Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands

doi: 10.1016/j.heliyon.2024.e28126

Figure Lengend Snippet: TIM-3 antibodies' affinities for human TIM-3 as assessed by the ForteBio Octet system. The original sensorgram shows TIM-3-Fc on the anti-human IgG Fc capture sensors, which bound to various concentrations of TIM-3 antibodies. The binding affinity parameter K D was determined by fitting the sensorgram. R 2 is the coefficient of determination to estimate the goodness of the curve fit as reported by ForteBio Data Analysis Software 9.0.

Techniques: Binding Assay, Software

The sensitivities and affinities of TIM-3 antibodies to cynomolgus TIM-3 protein. (A) The sensitivities of the unlabeled and biotin-labeled TIM-3 antibodies were assessed by ELISAs. (B) The affinities of the TIM-3 antibodies for cynTIM-3 were assessed using the ForteBio Octet system.

Journal: Heliyon

Article Title: Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands

doi: 10.1016/j.heliyon.2024.e28126

Figure Lengend Snippet: The sensitivities and affinities of TIM-3 antibodies to cynomolgus TIM-3 protein. (A) The sensitivities of the unlabeled and biotin-labeled TIM-3 antibodies were assessed by ELISAs. (B) The affinities of the TIM-3 antibodies for cynTIM-3 were assessed using the ForteBio Octet system.

Techniques: Labeling

The binding characteristics of anti-TIM-3 antibodies to ligand/TIM-3 complex. The binding of mouse anti-human TIM-3 antibodies to human Gal-9/TIM-3 (B), CEACAM-1/TIM-3 (C) or HMGB-1/TIM-3 complex (D) was detected using anti-mouse antibodies in ELISAs. Prior to being treated with mouse anti-TIM-3 mAbs, TIM-3-His were firstly attached to immobilized Gal-9, CEACAM-1, or HMGB-1 in ELISA plates. Reaction complexes were identified using anti-Ms-(H + L)-HRP. NC = negative control without TIM-3 protein; Blank = blank control without an anti-TIM-3 antibody. Horizontal lines indicate the cut-off, which were determined based on 2.1 times the mean absorbance of the negative control or blank. Statistical significance was indicated as ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 compared with NC or Blank.

Journal: Heliyon

Article Title: Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands

doi: 10.1016/j.heliyon.2024.e28126

Figure Lengend Snippet: The binding characteristics of anti-TIM-3 antibodies to ligand/TIM-3 complex. The binding of mouse anti-human TIM-3 antibodies to human Gal-9/TIM-3 (B), CEACAM-1/TIM-3 (C) or HMGB-1/TIM-3 complex (D) was detected using anti-mouse antibodies in ELISAs. Prior to being treated with mouse anti-TIM-3 mAbs, TIM-3-His were firstly attached to immobilized Gal-9, CEACAM-1, or HMGB-1 in ELISA plates. Reaction complexes were identified using anti-Ms-(H + L)-HRP. NC = negative control without TIM-3 protein; Blank = blank control without an anti-TIM-3 antibody. Horizontal lines indicate the cut-off, which were determined based on 2.1 times the mean absorbance of the negative control or blank. Statistical significance was indicated as ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 compared with NC or Blank.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Anti-TIM-3 antibodies inhibited Gal-9 from binding to TIM-3. Binding of hTIM-3-His to plate-bound Gal-9 was evaluated via ELISA in the presence of anti-TIM-3 antibodies. The reaction complex was detected using anti-His-HRP or anti-Ms-(H + L)-HRP. Horizontal lines indicate the cut-off, which were determined based on 2.1 times the mean absorbance of the negative control without TIM-3 protein. Statistical significance was assessed using multiple unpaired t-tests (**p < 0.01 compared with negative control). NS indicates no statistically significance.

Journal: Heliyon

Article Title: Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands

doi: 10.1016/j.heliyon.2024.e28126

Figure Lengend Snippet: Anti-TIM-3 antibodies inhibited Gal-9 from binding to TIM-3. Binding of hTIM-3-His to plate-bound Gal-9 was evaluated via ELISA in the presence of anti-TIM-3 antibodies. The reaction complex was detected using anti-His-HRP or anti-Ms-(H + L)-HRP. Horizontal lines indicate the cut-off, which were determined based on 2.1 times the mean absorbance of the negative control without TIM-3 protein. Statistical significance was assessed using multiple unpaired t-tests (**p < 0.01 compared with negative control). NS indicates no statistically significance.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control

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