tigit cd155  (Galectin Therapeutics)

Journal: Frontiers in Immunology

Article Title: Recent advances in preclinical studies combining hyperthermia therapy with novel immune checkpoint targeting agents

doi: 10.3389/fimmu.2026.1722115

Figure Lengend Snippet: The interaction between CD4+/CD8+ T cells and antigen-presenting cells (APCs) or tumor cells is regulated by various immune checkpoints, such as the activating checkpoint CD226-CD155, and inhibitory checkpoints including TIGIT-CD155, Tim-3-Galectin-9, and PD1-PDL1. Activation of theseinhibitory checkpoints results in T cell exhaustion and/or functional impairment. Hyperthermia treatment has been shown to upregulate the expressionof TIGIT, Tim-3, and PD1 molecules on T cells, thereby enhancing their responsiveness to immune checkpoint blockade with monoclonal antibodies.

Article Snippet: The inhibitory checkpoints, including PD-1/PD-L1, CTLA-4/CD80/CD86, TIGIT/CD155, Tim-3/Galectin-9, CD47/SIRPα, LAG3/MHC, negatively regulate the activity of immune cells to avoid excessive response such as “cytokine storm”.

Techniques: Activation Assay, Functional Assay, Bioprocessing

Journal: Frontiers in Immunology

Article Title: Enhancing anti-tumor immunity through co-blocking PD-L1 and TIGIT by facilitating tumor-directed responses and additional VEGF inhibition

doi: 10.3389/fimmu.2025.1746155

Figure Lengend Snippet: PD-L1 and TIGIT targeting bispecific antibody HB0036 is more potent in reversing immune inhibition than combination of two parental mAbs: (A) Activation induced expression of co-receptors: Human PBMCs were stimulated in vitro with mitogenic antibodies against CD3 and CD28. Harvested cells were stained for surface expression of the indicated markers. FACS plots show expression of CD226, TIGIT, CD96 and PD-1. (B–F) Enhancement of T cell responses in vitro . (B) CTV-labelled PBMCs were cultured with mitomycin-treated HT-1080 cells in 96-well plates with indicated Abs at equal molar concentrations. Cells were then stimulated with mitogenic antibodies against CD3 and CD28 for 5 days. Cell proliferation, expression of surface markers and cytokine production were determined. (C) Histograms display cell division; line graphs show cell numbers at given cell divisions and bar graphs show total recovered proliferating T cells. (D) Bar graphs show cytokine levels from culture supernatants. (E) Bar graphs show the expression (mean MFI ± SEM) of CD226, CD96 and CD226 MFI/CD96 MFI ratio by CD4 + and CD8 + T cells. (F) Bar graphs show recovered NK cell numbers, expression of CD107a and CD226 by NK cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by One-way ANOVA multiple parameter comparison, n.s., not significant.

Article Snippet: Slides were then blocked, incubated with primary antibodies: anti- PanCK (Sigma, Cat#: C2562), anti-PD-L1 (CST, Cat#: 13684), anti-CD155 (CST, Cat#: 81254S), anti- TIGIT (CST, Cat#: 99567S), anti-CD226 (CST, Cat#: 66631), anti-PD-1 (CST, Cat#: 86163), anti-CD4 (ZSGB-Bio, Cat#: ZM0418), anti-CD8 (CST, Cat#: 70306), and HRP-secondary antibodies (Panovue), and subjected to signal amplification using Diaminobenzidine (Biolynx).

Techniques: Inhibition, Activation Assay, Expressing, In Vitro, Staining, Cell Culture, Comparison

Journal: Frontiers in Immunology

Article Title: Enhancing anti-tumor immunity through co-blocking PD-L1 and TIGIT by facilitating tumor-directed responses and additional VEGF inhibition

doi: 10.3389/fimmu.2025.1746155

Figure Lengend Snippet: HB0036 enhances intratumoral accumulation of Anti-TIGIT antibody via PD-L1 engagement. (A) Experimental Design for Assessing Intratumoral Antibody Distribution>: hTIGIT/hPD-L1/hPD-1 humanized C57BL/6 mice were inoculated with hPD-L1 + and hPD-L1 - WT MC38 cells on contralateral flanks. After 13 days, biotinylated antibodies were administered, and tumors were harvested 24 hours later for analysis of antibody accumulation. (B) In Vivo Antibody Binding to Tumor Cells. Tumor digests were incubated with TIGIT-His protein, then stained with PE-Streptavidin (to detect biotinylated antibodies), APC anti-His (to identify anti-TIGIT antibodies), and mCD45. Flow cytometry plots show antibody binding to tumor cells (gated on live mCD45 - cells). (C) Quantification of TIGIT Antibody Accumulation in Tumors. Total TIGIT antibody levels in tumor digests were measured by ELISA. (D) In Vitro Antibody Binding Assay. CHOK1-hPD-L1 cells were incubated with equimolar concentrations of biotinylated HB0036, HB0030, 900458, control IgG, or biotin-free HB0036 for 30 min on ice. Cells were then treated with His-tagged human TIGIT protein, followed by staining with PE-Streptavidin and APC anti-His antibodies. *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Slides were then blocked, incubated with primary antibodies: anti- PanCK (Sigma, Cat#: C2562), anti-PD-L1 (CST, Cat#: 13684), anti-CD155 (CST, Cat#: 81254S), anti- TIGIT (CST, Cat#: 99567S), anti-CD226 (CST, Cat#: 66631), anti-PD-1 (CST, Cat#: 86163), anti-CD4 (ZSGB-Bio, Cat#: ZM0418), anti-CD8 (CST, Cat#: 70306), and HRP-secondary antibodies (Panovue), and subjected to signal amplification using Diaminobenzidine (Biolynx).

Techniques: In Vivo, Binding Assay, Incubation, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, In Vitro, Control

Journal: Frontiers in Immunology

Article Title: Enhancing anti-tumor immunity through co-blocking PD-L1 and TIGIT by facilitating tumor-directed responses and additional VEGF inhibition

doi: 10.3389/fimmu.2025.1746155

Figure Lengend Snippet: HB0036 with ADCC capability reduces Tregs in vitro and in vivo . (A) NK cell-mediated target cell killing. Human NK cells (1×10 5 /well) were co-cultured with Jurkat-TIGIT-22G8 target cells (1×10 5 /well) in 96-well U-bottom plates for 5 hours in the presence of serially diluted antibodies. Line graph depicts Jurkat cell lysis (%). (B) NK cell-mediated Treg killing. Tregs (~40% TIGIT + ) were seeded at 5×10 4 cells/well in 96-well round-bottom plates and treated with indicated antibodies: isotype control (wild-type Fc), HB0030, HB0036, HB0030 KO (ADCC-silent, #900541), or HB0036 KO (ADCC-silent, #700001). Bar graph shows Treg lysis (%). *** p < 0.001, **** p < 0.0001 (one-way ANOVA). (C) In vivo Treg reduction. Experimental setup as in Figure 2 . Humanized mice bearing contralateral hPD-L1 + /hPD-L1 - MC38 tumors were treated with biotinylated antibodies for 1 day after 13 days of tumor growth. Tumor-infiltrating mCD45 + cells were analyzed for CD4 + FoxP3 + Tregs. FACS plots show Tregs within CD4 + cells; bar graphs depict Treg frequency (% of CD4 + cells, mean ± SEM). ** p < 0.01, ns (not significant; one-way ANOVA).

Article Snippet: Slides were then blocked, incubated with primary antibodies: anti- PanCK (Sigma, Cat#: C2562), anti-PD-L1 (CST, Cat#: 13684), anti-CD155 (CST, Cat#: 81254S), anti- TIGIT (CST, Cat#: 99567S), anti-CD226 (CST, Cat#: 66631), anti-PD-1 (CST, Cat#: 86163), anti-CD4 (ZSGB-Bio, Cat#: ZM0418), anti-CD8 (CST, Cat#: 70306), and HRP-secondary antibodies (Panovue), and subjected to signal amplification using Diaminobenzidine (Biolynx).

Techniques: In Vitro, In Vivo, Cell Culture, Lysis, Control

Journal: Frontiers in Immunology

Article Title: Enhancing anti-tumor immunity through co-blocking PD-L1 and TIGIT by facilitating tumor-directed responses and additional VEGF inhibition

doi: 10.3389/fimmu.2025.1746155

Figure Lengend Snippet: HB0036 Exhibits a stronger anti-tumor effect than combination therapy in two preclinical models. hPD1/hPDL1/hTIGIT BALB/c mice were inoculated with 1×10 6 CT26-hPDL1 cells on the flank. Treatment ( n=8-9 ) was started when tumour sizes were an average TV of 160 mm 3 . Equimolar dose of Abs (13.7 mg/kg body weight HB0036 or 10 mg/kg 900458 and 10 mg/kg body weight HB0030, twice a week, total 4 doses) were given intraperitoneally. At the end of the experiment, tumour weights were recorded. Tumours were digested for TIL analysis. (A) Tumor volumes and tumour weights. P values for tumour volumes were calculated with Prism two-way ANOVA Tukey’s multiple-comparisons test. P values for tumour weights were generated with one-way ANOVA multiple multiple-comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) tumoral cytokine content. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by one-way ANOVA multiple multiple-comparisons test. (C–H) BxPC-3 (1×10 7 cells) were inoculated subcutaneously into NPG mice on the right flank. When tumours had grown to an average size of 80 mm 3 , tumour-bearing mice were divided into 4 groups ( n=8 each) and injected i.v. with 1×10 7 PBMCs. Treatments started on the same day and antibodies were dosed twice weekly for 4 weeks intraperitoneally. (C) Tumour volume. ** p < 0.01 by two-way ANOVA Tukey’s multiple-comparisons test. (D, E) . mouse myeloid TILs: Analysis of FACS plots show the gating of mouse myeloid cells within mouse CD45 + TILs. (F–H) human lymphocytic TILs: (F) The percentages and the numbers of human TILs, (G) FACS plots show the expression of TIGIT and PD-1 by human CD8 + and CD4 + T cells. (H) Bar graphs show the percentages of human T-cell subsets with expression of TIGIT, CD226 or CD96. * p < 0.05, ** p < 0.01, **** p < 0.0001 by one-way ANOVA multiple-comparisons test.

Article Snippet: Slides were then blocked, incubated with primary antibodies: anti- PanCK (Sigma, Cat#: C2562), anti-PD-L1 (CST, Cat#: 13684), anti-CD155 (CST, Cat#: 81254S), anti- TIGIT (CST, Cat#: 99567S), anti-CD226 (CST, Cat#: 66631), anti-PD-1 (CST, Cat#: 86163), anti-CD4 (ZSGB-Bio, Cat#: ZM0418), anti-CD8 (CST, Cat#: 70306), and HRP-secondary antibodies (Panovue), and subjected to signal amplification using Diaminobenzidine (Biolynx).

Techniques: Generated, Injection, Expressing

Journal: Frontiers in Immunology

Article Title: Enhancing anti-tumor immunity through co-blocking PD-L1 and TIGIT by facilitating tumor-directed responses and additional VEGF inhibition

doi: 10.3389/fimmu.2025.1746155

Figure Lengend Snippet: Triple blockade of PD-L1, TIGIT, and VEGF enhances anti-tumor efficacy in preclinical models. (A–D) H22-hPD-L1 tumor model in hPD-1/hPD-L1/hTIGIT BALB/c mice: Mice (n=6–8/group) were inoculated with 1×10 6 H22-hPD-L1 cells and treated at an average tumor volume (TV) of 100 mm³. Treatment groups: isotype control (10 mg/kg), HB0036 (4.1 mg/kg), 2.1T (VEGF TRAP, 1 mg/kg), HB0036 + 2.1T, HB0025 (anti-PD-L1/VEGF, 2 mg/kg), HB0030 (anti-TIGIT mAb, 3 mg/kg), or HB0025 + HB0030. Antibodies were administered intraperitoneally ( i.p. ) twice weekly (4 total doses). (A, B) Tumor volumes and partial responses (defined as tumor volume reductions exceeding two-thirds of the mean tumor volume in the isotype control group at the final measurement) for isotype, HB0025, HB0030, and HB0025 + HB0030. (C, D) Tumor volumes and partial responses for isotype, HB0036, 2.1T, and HB0036 + 2.1T. (E) hPD-L1 MC-38 tumor model in hTIGIT/hPD-L1/hPD-1 humanized C57BL/6 mice: Mice ( n=6 /group) were inoculated with 1×10 6 hPD-L1-expressing MC-38 cells and treated at TV ≈100 mm³. Groups: PBS, HB0025 (3.6 mg/kg), HB0030 (3 mg/kg), or combination. Antibodies were given IP twice weekly (4 doses). Tumor volumes shown. (F) EMT6-hPD-L1 model in hPD-1/hPD-L1/hTIGIT BALB/c mice: Mice (n=6/group) were inoculated with 2×10 6 hPD-L1-expressing EMT6 and treated at TV ≈100 mm³. Treatment groups: isotype (3 mg/kg), HB0025 (1 mg/kg), HB0030 (3 mg/kg), or combination. Tumor volumes shown. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA, Tukey’s multiple comparisons test).

Article Snippet: Slides were then blocked, incubated with primary antibodies: anti- PanCK (Sigma, Cat#: C2562), anti-PD-L1 (CST, Cat#: 13684), anti-CD155 (CST, Cat#: 81254S), anti- TIGIT (CST, Cat#: 99567S), anti-CD226 (CST, Cat#: 66631), anti-PD-1 (CST, Cat#: 86163), anti-CD4 (ZSGB-Bio, Cat#: ZM0418), anti-CD8 (CST, Cat#: 70306), and HRP-secondary antibodies (Panovue), and subjected to signal amplification using Diaminobenzidine (Biolynx).

Techniques: Control, Expressing

Journal: Frontiers in Immunology

Article Title: Enhancing anti-tumor immunity through co-blocking PD-L1 and TIGIT by facilitating tumor-directed responses and additional VEGF inhibition

doi: 10.3389/fimmu.2025.1746155

Figure Lengend Snippet: Pattern of expression of PD-L1 and CD155 (PVR) and its correlation to blocking and immune landscape. (A) FACS plots show expression pattern of PD-L1 and CD155 by HT-1080 cells. (B) CTV-labelled PBMCs were cultured with mitomycin-treated HT-1080 cells in 96-well plates with indicated Abs at equal molar concentrations. Histograms show the numbers of proliferated cells under stimulated conditions. **** p < 0.0001 by one-way ANOVA multiple-comparisons test. (C–H). High-Content Analysis of human lung adenocarcinoma microenvironment. Multiplex immunofluorescence and quantitative analysis were used to profile the tumor (PanCK + ) and stromal (PanCK - ) compartments. (C) Representative High-Content analysis images illustrating the immune landscape in adenocarcinoma tissue. (D) Quantification of the percentage of cells expressing PD-L1 within the PanCK + tumor and PanCK - stromal compartments. Lines connect paired tumor and stroma samples from the same patient. The mean difference between compartments is plotted on the right axis for each graph. PD-L1 expression was then further expressed as Tumor Proportion Score (TPS), calculated as the ratio of PanCK + PD-L1 + cells to PanCK + tumor cells, and a Combined Positive Score (CPS), calculated as the ratio of all PD-L1 + cells to PanCK + tumor cells. (E) Quantification of the percentage of cells expressing CD155 within the PanCK + tumor and PanCK - stromal compartments. Lines connect paired tumor and stroma samples from the same patient. The mean difference between compartments is plotted on the right axis for each graph. (F) Co-expression analysis of PD-L1 and CD155 on PanCK + tumor cells. Each dot represents a patient sample. Dashed lines indicate the median density of the cohort for each marker, stratifying tumors into four subsets. (G) Graphs show Frequencies of CD4 + and CD8 + T cells, calculated as a percentage of total cells within the tissue; Frequencies of PD-1, TIGIT, and CD226 expression on CD4 + and CD8 + T cells, shown as a percentage of the parent T-cell population; and Linear regression analysis showing a significant positive correlation between the density (cells/mm 2 ) of TIGIT + and CD226 + cells within the CD4 + (left) and CD8 + (right) T cell populations. R 2 and P values are shown. (H) Correlation analysis between the density of total PD-L1 + cells (left) or total CD155 + cells (right) and the density of CD4 + (red line) or CD8 + (blue line) T cells.

Article Snippet: Slides were then blocked, incubated with primary antibodies: anti- PanCK (Sigma, Cat#: C2562), anti-PD-L1 (CST, Cat#: 13684), anti-CD155 (CST, Cat#: 81254S), anti- TIGIT (CST, Cat#: 99567S), anti-CD226 (CST, Cat#: 66631), anti-PD-1 (CST, Cat#: 86163), anti-CD4 (ZSGB-Bio, Cat#: ZM0418), anti-CD8 (CST, Cat#: 70306), and HRP-secondary antibodies (Panovue), and subjected to signal amplification using Diaminobenzidine (Biolynx).

Techniques: Expressing, Blocking Assay, Cell Culture, High Content Screening, Multiplex Assay, Immunofluorescence, Marker