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

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by a higher mortality rate among breast cancer subtypes. Poly(ADP-ribose) polymerase (PARP) inhibitors are used in clinics to treat a subgroup of TNBC patients, but other targeted therapies are urgently needed. Programmed death-ligand 1 (PD-L1), involved in tumor immune escape, was recently identified as a target for TNBC; accordingly, the anti-PD-L1 monoclonal antibody (mAb), atezolizumab, has been approved by FDA in combination with Paclitaxel for the therapy of metastatic TNBC. Here, we tested novel combinations of fully human immunomodulatory mAbs, including anti-PD-L1 mAbs generated in our laboratory and atezolizumab, on TNBC and other tumor cell lines. We evaluated their anti-tumor efficacy when used as single agents or in combinatorial treatments with anti-CTLA-4 mAbs in in vitro co-cultures of hPBMCs with tumor cells, by measuring tumor cell lysis and IL-2 and IFNγ cytokines secretion by lymphocytes. In parallel, by using co-cultures of hPBMCs and cardiomyocytes, we analyzed the potential cardiotoxic adverse side effects of the same antibody treatments by measuring the cardiac cell lysis and the secretion of pro-inflammatory cytokines. We identified novel combinations of immunomodulatory mAbs endowed with more potent anti-cancer activity on TNBC and lower cardiotoxic side effects than the combination of atezolizumab and ipilimumab.

Novel Bi-Specific Immuno-Modulatory Tribodies Potentiate T Cell Activation and Increase Anti-Tumor Efficacy

“…The supernatants were then tested by ELISA assays to measure the secretion of IL-2 and IFNγ as markers of T cell activation. As shown in Figure 1 , both the combinations of anti-PD-1 or anti-PD-L1 with LAG-3_1 mAb induced the secretion of cytokines more efficiently than the single agent treatments, as also reported in previous studies [ 32 , 35 , 36 ].…”

“…Since PD-1, PD-L1 and LAG-3 were also found to be expressed on different types of tumor cells, such as breast cancer cells [ 33 , 35 , 36 ], and the parental mAbs PD-L1_1 and PD-1_1 have previously shown the ability to bind to IC-positive tumor cells by inhibiting their growth, even in the absence of immune cells, we investigated the effects of the novel tribodies on tumor cell viability in the absence of lymphocytes. To this end, MDA-MB-231 and BT-549 triple negative breast tumor cells, expressing satisfactory levels of PD-L1 and PD-1 and moderate levels of LAG-3 [ 35 , 36 ], were plated in 96-well plates and treated with the tribodies, or their corresponding parental mAbs, at a concentration of 200 nM for 72 h. As shown in Figure 7 A, the novel bispecific tribodies slightly inhibited tumor growth, i.e., by about 20–35%, even in the absence of immune cells, in a similar fashion to the parental mAbs, thus confirming that they had retained their antitumor activity.…”

“…To verify whether the tribodies affected the survival pathways previously identified for their parental mAbs [ 33 , 35 , 36 ], we performed Western Blotting analyses of protein extracts from MDA-MB-231 tumor cells treated for 72 h at 37 °C in the absence or presence of the tribodies, or the parental PD-L1_1 mAb, used at a concentration of 200 nM. As shown in Figure 7 B, the levels of phosphorylated Erk and Jnk proteins decreased when the cells were treated with TR0304 and TR0506, whereas the levels of Cleaved Caspase-3 increased after treatments, confirming the involvement of the same intracellular pathways downstream of PD-L1 and PD-1, affected by their parental mAbs [ 33 ].…”

“…Since the novel tribodies targeting PD-L1, PD-1 and LAG-3 efficiently induced the activation of lymphocytes, we further investigated their antitumor effects on co-cultures of tumor cells and lymphocytes. To this end, MDA-MB-231 and BT-549 breast cancer cells expressing high levels of PD-L1 [ 36 ] were cocultured with hPBMCs in the absence or presence of the TR0304 and TR0506 tribodies at a concentration of 66 nM for 48 h at 37 °C. In parallel assays, we tested the parental mAbs, used in combination at the same concentrations.…”

“…Herein, we investigated the efficacy of novel combinations of human anti-PD-L1, anti-PD-1 and anti-LAG-3 mAbs, previously generated in our laboratory [ 32 , 33 , 34 , 35 , 36 ], in order to choose the most effective combinations. On the basis of these findings, we constructed four novel different bispecific immuno-modulatory tribodies by fusing the Fab portion of one anti-IC antibody with two scFv domains of a different mAb, targeting a distinct IC.…”