Fig 2: 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.

Fig 3: 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.