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

Abstract

Cancer immunotherapy has already shown significant improvements by combining different antibodies specific for distinct immune checkpoints, such as Ipilimumab and Nivolumab. Here, we tested combinatorial treatments of immunomodulatory antibodies, previously generated in our laboratory, for their effects on hPBMC activation, either upon stimulation with SEB or in co-cultures with tumor cells by cytokine secretion assays. We found that some of them showed additive or synergistic effects, and on the basis of these observations, we constructed, for the first time, four novel bispecific tribodies (TR), made up of a Fab derived from one anti-IC mAb and two scFvs derived from another mAb targeting a different IC. All four TRs cotargeting either programmed cell death protein 1 (PD-1) and Lymphocyte Activating 3 (LAG-3) or programmed death-ligand 1 (PD-L1) and LAG-3 retained binding affinity for their targets and the antagonistic effects of their parental mAbs, but some of them also showed an increased ability to induce lymphocyte activation and increased in vitro cytotoxicity against tumor cells compared to parental antibodies used either alone or in combinatorial treatments. Furthermore, none of the tribodies showed significant increased cytotoxicity on human cardiomyocytes. Considering that the tribody format reduces production costs (as only one construct provides the inhibitory effects of two antibodies), has an intermediate molecular size (100 kDa) which is well suited for both tumor penetration and an acceptable half-life, we think that these novel immunomodulatory TRBs have the potential to become precious tools for therapeutic applications, particularly in monotherapy-resistant cancer patients.

Combinatorial immune checkpoint blockade increases myocardial expression of NLRP-3 and secretion of H-FABP, NT-Pro-BNP, interleukin-1β and interleukin-6: biochemical implications in cardio-immuno-oncology

“…To mimic the lymphocyte infiltration that is clinically found in cancer patients treated with ICIs who develop myocarditis, we have developed a cellular model of lymphocyte-cardiomyocyte interaction, already described in previous works ( 19 , 22 , 40 ) ( Figure 1 ). To this aim, human cardiomyocyte/hPBMCs co-culture was exposed or unexposed to 100 nM Relatlimab, Nivolumab, Atezolizumab, Ipilimumab or their combinations as used in clinical trials or approved by FDA.…”

“…In light of the new combinatorial immune checkpoint blockade therapies, basic pathophysiological studies are needed to predict possible adverse events in clinic scenario. This study focalized, for the first time, on the potential cardiotoxic side effects of novel combinatorial ICIs treatments on models of lymphocyte-cardiomyocyte interaction, such as cardiomyocyte/hPBMCs co-cultures ( 22 ), through selective analysis of cardiac cell lysis and quantification of several pro-inflammatory and conventional cardiotoxic biomarkers.…”

Novel tri-specific tribodies induce strong T cell activation and anti-tumor effects in vitro and in vivo

“…After 5 days post-transfection, each tribody was purified from cell culture supernatants by His-tag affinity chromatography followed by gel filtration chromatography. This process recovers the Tribody L/Fd chain heterodimer while limiting impurities such as product variants [ 21 , 22 , 30 ]. The purity of the tribodies was analyzed by SDS-PAGE, and size-exclusion chromatography (SEC) analysis was performed to evaluate the tribody heterodimer purity and freeze–thaw stability.…”

“…Human Peripheral Blood Mononuclear Cells (hPBMCs) were isolated from blood of healthy donors by using Greiner Leucosep® tube (Sigma-Aldrich, St Louis, Missouri USA) and cryopreserved by following the manufacturer’s instructions. Cryopreserved cell vials were thawed out, collected for resting and counted before use, as previously reported [ 30 ].…”

Strategies for clinical dose optimization of T cell-engaging therapies in oncology

“… 114 Other TCEs have been designed to target both PD-1 and CD3, in addition to a specific TAA, thereby aiming to mimic the anti-PD-1 combination therapy approach to reduce T cell dysfunction or to overcome the immunosuppressive tumor microenvironment. 115 , 116 When developing molecules with multiple target antigens on immune cells, particularly in the case of inhibitory versus agonistic mechanisms such as PD-1 and CD3, it will be important to combine data generated using a range of appropriate in vitro assays with modeling and simulation, in order to achieve the optimal receptor occupancy balance.…”