Fig 2: HB0036 Exhibits a stronger anti-tumor effect than combination therapy in two preclinical models. hPD1/hPDL1/hTIGIT BALB/c mice were inoculated with 1×106 CT26-hPDL1 cells on the flank. Treatment (n=8-9) was started when tumour sizes were an average TV of 160 mm3. 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 (1×107 cells) were inoculated subcutaneously into NPG mice on the right flank. When tumours had grown to an average size of 80 mm3, tumour-bearing mice were divided into 4 groups (n=8 each) and injected i.v. with 1×107 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.

Fig 3: 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/mm2) of TIGIT+ and CD226+ cells within the CD4+ (left) and CD8+ (right) T cell populations. R2 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.