As IFN- triggers the transcription of PD-L1 [25], the tumor cells will produce an abundant amount of PD-L1 mRNA
As IFN- triggers the transcription of PD-L1 [25], the tumor cells will produce an abundant amount of PD-L1 mRNA. labeled Nbs. Nb C3 and E2 showed specific antigen binding and beneficial biodistribution. Through the use of CRISPR/Cas9 PD-L1 knock-out TC-1 lung epithelial cell lines, we demonstrate that SPECT/CT imaging using Nb C3 and E2 identifies PD-L1 expressing tumors, but not PD-L1 non-expressing tumors, thereby confirming the diagnostic potential of the selected Nbs. In conclusion, these data show that Nbs C3 and E2 can be used to non-invasively image PD-L1 levels in the tumor, with the strength of the signal correlating with PD-L1 levels. These findings warrant further research into the use of Nbs as a tool to image inhibitory signals in the tumor environment. = 1). (C) Representative flow cytometry results, showing staining of unmodified HEK293T cells (grey line) or HEK293T cells lentivirally modified to express mouse PD-L1 (293T moPD-L1, red line) with mAbs specific for mouse PD-L1 or Nbs C3, C7, E4 and E2 (= 3). The selected anti-PD-L1 and control Nbs Catechin were then produced and purified as C-terminally His6-tagged proteins from E. coli fermentation cultures. Quality control confirmed good purity and low LPS content (Table ?(Table1).1). The affinity (KD) of the purified Nbs for the immobilized mouse Catechin PD-L1 antigen was determined using SPR (Figure ?(Figure1B).1B). The KD values are in the low nanomolar range as shown in Figure ?Figure11 and Table ?Table1.1. The ability of the different Nbs to bind to the mouse PD-L1 antigen when expressed on lentivirally modified HEK293T cells, was confirmed in flow cytometry (Figure ?(Figure1C).1C). We also used SPR to determine the affinity of the different Nbs for the human PD-L1 antigen. The affinity of the Nbs for the human PD-L1 antigen was substantially lower than for the mouse PD-L1 antigen (Supplementary Figure 2A). The ability of the different Nbs to bind to the human PD-L1 antigen when expressed on lentivirally modified HEK293T cells is shown in Supplementary Figure 2B. Table 1 Summary of the endotoxin content, affinity for mouse PD-L1 and radiochemical purity of Catechin 99mTc-Nb complexes of purified anti-PD-L1 Nbs C3, C7, E4 and E2 = 3). (B) Gamma counting of isolated organs from WT or KO mice injected with 99mTc-Nbs C3, C7, E4 or E2. The graph summarizes the organ uptake of the Nb per gram organ as the mean SEM (= 3). (C) Percentage PD-L1 positive cells in spleen, lymph node (LN) and brown adipocyte tissue (BAT) of WT and PD-L1 KO mice using flow cytometry. The graph summarizes the percentage PD-L1 positive cells as mean SEM (= 3). = 5). The graph summarizes the percentage PD-L1 positive cells as mean SEM. (B) TC-1 KD or KI cells were injected subcutaneously at the tail Mouse monoclonal to FOXD3 base of C57BL/6 mice. Tumor growth was followed every other day. The evolution of tumor size is shown as mean SEM (= 5). (C) Mice were sacrificed on day 12 and tumors were isolated, after which expression of PD-L1 on tumor cells (CD45?, white bar) and tumor-infiltrating immune cells (CD45+, black bar) was evaluated Catechin in flow cytometry. The graph summarizes the percentage of PD-L1 as mean SEM (= 5). (D) Images of SPECT/CT scans to determine the accumulation of 99mTc-Nbs C3 and E2 in C57BL/6 mice bearing KI (left panel) or KD (right panel) tumors (= 6). The red arrow indicates the tumor on the images. (E) Graphs showing the quantified analysis to determine the accumulation of 99mTc-Nbs C3 and E2 in PD-L1 KI (black bar) or KD (white bar) tumors. The graph shows the percentage radioactivity per gram tumor as Catechin mean SEM (= 6). The PD-L1 knock-down and knock-in TC-1 cells were transplanted subcutaneously in wild type mice, and tumor growth was evaluated every other day. We observed a delayed outgrowth of TC-1 tumors in the PD-L1 knock-down model, highlighting the critical role of PD-L1 in tumor development (Figure ?(Figure3B).3B). We performed SPECT/CT imaging 1 hour after injection of the 99mTc-labeled Nbs C3 and E2, followed by evaluation of accumulation of these Nbs in individual organs.