Figure 6.

BND-22 enhances anti-tumor functions of anti-PD-1 or tumor targeting antibodies. (A) Analysis of ILT2 and PD-1 expression in intratumoral CD8 T cells from patients with CRC documented in http://crctcell.cancer-pku.cn/. (B) A bioinformatic analysis of ILT2 expression in melanoma patients treated with Nivolumab from.47 The RNAseq data was used to calculate expression changes in patients with cancer post versus pre-Nivolumab treatment. Log2 ILT2 fold-change differences above 0.5 or below 0.5 were considered as increase and decrease in expression, respectively. (C) DCs were generated from monocytes and CD8 T cells were isolated from PBMCs of healthy human donors. The cells were combined in E:T of 5:1 ratio with the indicated treatments and IFN-γ was measured in culture supernatants at day 5. (D) Single-cell suspensions were prepared by enzymatic digestion of a fresh tumor isolated from a patient with colon cancer. PBMCs were isolated from the same patient and cocultured with the indicated treatments in presence of IL-2. TNFα secretion was detected by ELISA. Representative results are shown. The mean±SE of values from three repeats per treatment are displayed. Unpaired Student’s t-test compared with control IgG for BND-22 or anti-PD-1, compared with anti-PD-1+IgG for anti-PD-1+BND-22.*P<0.05; (E) 786-O cells, or (F) A253-HLA-G cells were stained with pHrodo red cell labeling dye, washed and added to macrophages generated from monocytes isolated from healthy donors, along with the various treatments in triplicates. A single representative time point of the experiment at 9.5 hours is presented. The percentage of increase in phagocytosis compared with control IgG is presented. Unpaired Student’s t-test compared with erbitux+medium. **P<0.01. CRC, colorectal cancer; DC, dendritic cell; IFN-γ, interferon gamma; ILT2, immunoglobulin-like transcript 2; PD-1, programmed cell death protein-1; T_CM, central memory T cells; T_EMRA, T cells re-expressing CD45RA; T_EX, exhausted T cells.