Novel Combinations of Human Immunomodulatory mAbs Lacking Cardiotoxic Effects for Therapy of TNBC

Figures

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  Figure 1. Effects of novel mAbs on the activation of stimulated lymphocytes. hPBMCs were stimulated for 66 h with SEB in the absence or in the presence of anti-CTLA-4 (black bars), anti-PD-L1 (dark grey bars) or anti-LAG-3 (striped bars) mAbs used alone (A) or in combination (B). In the panel B the levels of IL-2 and IFN-γ secreted were measured by ELISA (black bars for anti-CTLA-4 and anti-LAG-3 mAbs, dark grey bars for anti-PD-L1 and striped bars for combinations). Untreated hPBMCs or treated with an unrelated IgG were used as negative controls (white and grey bars, respectively). Error bars depict means ± SD. p-values for the indicated compounds are: ** p < 0.01; * p < 0.05.

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  Figure 2. Cytotoxic effects of novel immunomodulatory mAbs on tumor cells co-cultured with hPBMCs. BT-549 (A) or MDA-MB-231 (B) tumor cells were co-cultured with hPBMCs (Effector:Target cells ratio 5:1) and treated for 48 h with anti-CTLA-4 (black bars) and anti-PD-L1 (dark grey bars) mAbs or their combination (striped bars) at the indicated concentrations. Co-cultured cells untreated or treated with an unrelated IgG were used as negative controls (white and grey bars, respectively). Cell lysis was measured by detecting LDH release, as described in Materials and Methods. Error bars depict means ± SD. p-values for the indicated compounds are: *** p ≤ 0,001; ** p < 0.01; * p < 0.05.

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  Figure 3. Effects of novel immunomodulatory mAbs on the secretion of IL-2 and IFN-γ by co-cultures of BT-549 tumor cells with hPBMCs. IL-2 and IFN-γ secretion levels were measured by ELISA on supernatants of cells treated with anti-CTLA-4 (black bars), anti-PD-L1 (dark grey bars) mAbs or their combination (striped bars) for 48 h. Cells untreated or treated with an unrelated IgG were used as negative controls (white and grey bars, respectively). Error bars depict means ± SD. p-values for the indicated compounds are: ** p < 0.01; * p < 0.05.

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  Figure 4. Effects of novel immunomodulatory mAbs on the secretion of IFN-γ in co-cultures of MDA-MB-231 tumor cells with hPBMCs. Levels of IFN-γ secreted by hPBMCs co-cultured with tumor cells and treated with anti-CTLA-4 (black bars) or anti-PD-L1 (dark grey bars) mAbs and their combination (striped bars) at the indicated concentration for 48 h. Cells untreated or treated with an unrelated IgG were used as negative (white and grey bars, respectively). Error bars depict means ± SD. p-values for the indicated compounds are: ** p < 0.01; * p < 0.05.

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  Figure 5. Cardiotoxic and pro-inflammatory effects induced by immunomodulatory mAbs or their combination on human fetal cardiomyocytes. (A) Cardiotoxicity of novel immunomodulatory mAbs was analyzed by detecting the LDH released by HFC cells co-cultured with hPBMCs in the absence or presence of single mAbs (black and grey bars) or their combinations (striped bars) for 24 h. The pro-inflammatory effects of the indicated immunomodulatory mAbs were analyzed by evaluating the secretion of IL-6 in the supernatant of the co-cultures treated with anti-CTLA-4 (black bars), anti-PD-L1 mAbs (dark grey), or their combination (striped bars). IL-6 is expressed as pg/mL. (B) The release of Granzyme B was evaluated by detecting its levels in the supernatant of co-cultures treated as indicated. As negative controls, cells untreated or treated with unrelated IgG were used (white and grey bars, respectively). The values were reported as the mean of at least three determinations obtained in three independent experiments. Error bars depict means ± SD. *** p ≤ 0,001; ** p < 0.01; * p < 0.05.

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  Figure 6. Binding assays to define the epitopes recognized by the novel mAbs. ELISA assays performed on PD-L1/Fc (A) or CTLA-4/Fc (B) before and after heat denaturation treatment of immobilized proteins. Competitive ELISA assays were performed by testing the binding of the biotinylated antibodies atezolizumab (C) or ipilimumab (D) to PD-L1 or CTLA-4 chimeric proteins in the absence (white bars) or presence of molar excess of unlabeled competitive PD-L1_1 or ID-1 antibodies (black bars). As positive controls biotinylated atezolizumab or ipilimumab (grey bars) were used in the presence of their unlabeled counterparts. Binding values were reported as the mean of at least three determinations obtained in three independent experiments. Error bars depicted means ± SD.

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  Figure 7. Expression of PD-L1, PD-1, CTLA-4, and LAG-3 ICs on a panel of cancer cells. (A) Cell ELISA assays were performed on MDA-MB-231, BT-549, A-549, or HuT-78 tumor cells to measure the cell surface expression of the indicated ICs with commercial anti-PD-L1, anti-CTLA-4, or anti-LAG-3 mAbs. Binding values were reported as the mean of at least three determinations obtained in three independent experiments. (B) Western blotting analyses of extracts from MDA-MB-231, BT-549, A-549, and HuT-78 tumor cells, obtained by using the commercial anti-LAG-3 mAbs. The intensity of the bands corresponding to ICs was normalized to actin (see also Table S1).

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  Figure 8. Effects of novel immunomodulatory mAbs on viability of HuT-78 tumor cells in the absence of lymphocytes. (A,B) MTT assays were performed on the supernatant of HuT-78 tumor cells, treated for 72 h with anti-PD-L1 or anti-LAG-3 mAbs, used as single agents or in combinatorial treatments. Cell viability is expressed as percentage of viable cells with respect to control untreated ones. Error bars depict means ± SD ** p < 0.01; * p < 0.05. (C) LDH levels were detected in the supernatant of HuT-78 tumor cells co-cultured with hPBMCs (Effector:Target cells ratio 5:1), treated for 48 h with anti-PD-L1 or anti-LAG-3 mAbs, or their combination, at the indicated concentrations. (D) Levels of IFN-γ secreted by hPBMCs co-cultured with tumor cells and treated with anti-CTLA-4, anti-PD-L1, or anti-LAG-3 mAbs, or their combination, at the indicated concentration for 48 h. Cells untreated or treated with an unrelated IgG were used as negative controls. Error bars depict means ± SD. p-values for the indicated compounds are: ** p < 0.01; * p < 0.05.