Novel Human Anti-PD-L1 mAbs Inhibit Immune-Independent Tumor Cell Growth and PD-L1 Associated Intracellular Signalling

Abstract

The novel antibody-based immunotherapy in oncology exploits the activation of immune system mediated by immunomodulatory antibodies specific for immune checkpoints. Among them, the programmed death ligand-1 (PD-L1) is of particular interest as it is expressed not only on T-cells, but also on other immune cells and on a large variety of cancer cells, such as breast cancer cells, considering its high expression in both ErbB2-positive and Triple Negative Breast Cancers. We demonstrate here that PD-L1_1, a novel anti-PD-L1 T -cell stimulating antibody, inhibits PD-L1-tumor cell growth also by affecting the intracellular MAPK pathway and by activating caspase 3. Similar in vitro results were obtained for the first time here also with the clinically validated anti-PD-L1 mAb Atezolizumab and in vivo with another validated anti-mouse anti-PD-L1 mAb. Moreover, we found that two high affinity variants of PD-L1_1 inhibited tumor cell viability more efficiently than the parental PD-L1_1 by affecting the same MAPK pathways with a more potent effect. Altogether, these results shed light on the role of PD-L1 in cancer cells and suggest that PD-L1_1 and its high affinity variants could become powerful antitumor weapons to be used alone or in combination with other drugs such as the anti-ErbB2 cAb already successfully tested in in vitro combinatorial treatments.

Aptamer targeted therapy potentiates immune checkpoint blockade in triple-negative breast cancer

“…As both PDGFRβ and PD-L1 are crucial targets for TNBC treatments [31,43,44], we tested whether their co-inhibition would enhance TNBC cells killing. To this aim, we used an aptamer and a mAb that we previously validated as high a nity binders and inhibitors of PDGFRβ [31] and PD-L1 [40,45], respectively, in different tumor types including TNBC. Speci cally, Gint4.T is a 2'F-Py RNA aptamer that binds to the extracellular domain of PDGFRβ and inhibits receptor activation and downstream phosphatidyl-3-kinase (PI3K)/Akt and ERK1/2 pathways [22,31,35], thus affecting growth, migration and invasion of mesenchymal TNBC cell lines [31].…”

“…Speci cally, Gint4.T is a 2'F-Py RNA aptamer that binds to the extracellular domain of PDGFRβ and inhibits receptor activation and downstream phosphatidyl-3-kinase (PI3K)/Akt and ERK1/2 pathways [22,31,35], thus affecting growth, migration and invasion of mesenchymal TNBC cell lines [31]. On the other side, the high a nity human anti-PD-L1 (10_12) mAb [38,46] not only interferes in the PD-L1/PD-1 interaction but also inhibits the immuneindependent PD-L1 positive TNBC cell growth by affecting the intracellular MAPK pathway [40].…”

“…First, we analyzed the effects of Gint4.T aptamer and 10_12 mAb used in combination, along with single agents, on the growth of human TNBC MDA-MB-231 cells, expressing high levels of both PDGFRβ [31,37] and PD-L1 [40], further enhanced by previous cell exposure to cisplatin and doxorubicin, two chemotherapeutics used in clinic for TNBC [41]. Based on our previous dose and time-response analyses in MDA-MB-231 cells for the aptamer [31] and antibody [40], we used them at a concentration of 200 nM and 100 nM, respectively, prolonging the incubation up to 96 h. Indeed, under these experimental conditions the inhibitory effects of both the aptamer and antibody is not so strong as it is at higher concentration and/or longer incubation time [31,40], thus allowing to appreciate a potential enhancement of their effects in combination. As shown in Fig.…”

“…As expected, no effects were observed in the presence of an unrelated IgG antibody and a scrambled aptamer (Scr), used as negative controls. Next, in order to assess whether the aptamer-mediated inhibition of PDGFRβ enhances the inhibitory effects on cell growth exerted by 10_12 mAb in the context of PD-1/PD-L1 interaction between TNBC cells and in ltrating lymphocytes [38,40], we tested the aptamer plus mAb on co-cultures of MDA-MB-231 cells with human lymphocytes by using hPBMCs (effector:target ratio 10:1) ( Fig. 1B-E).…”

Pemetrexed Enhances Membrane PD-L1 Expression and Potentiates T Cell-Mediated Cytotoxicity by Anti-PD-L1 Antibody Therapy in Non-Small-Cell Lung Cancer

“…For T cell-mediated cytotoxicity [39,40], after 3 days of incubation in the previous reported transwell system, PBMCs were transferred from the insert to the corresponding well in a contacting co-culture with A549 cells and maintained for 24 h in the absence or presence of 200 µg/mL atezolizumab. Finally, PBMCs and dead tumor cells were washed away and the crystal violet assay was performed.…”