Abstract
Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-β, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells. Here, suboptimal activity of the bispecific antibody at diverse EpCAM expression levels could be effectively enhanced by targeting-mediated costimulation by B7.1, 4-1BBL and OX40L in a broad range of EGFR expression levels. Furthermore, the benefit of combined costimulation by B7.1/4-1BBL and 4-1BBL/OX40L was demonstrated. In addition, the expression of immunosuppressive factors was shown in all co-culture settings, where blocking of prominent factors led to synergistic effects with combined costimulation. Thus, targeting-mediated costimulation showed general promise for a broad application covering diverse target expression levels, with the option for further selective enhancement by the identification and blockade of main immunosuppressive factors of the particular tumor environment.
Highlights
Interfering with the regulatory network of the immune response has become an intensively investigated strategy for cancer immunotherapy
Antibody-fusion proteins composed of the antibody scFv and 4-1BBL or OX40L present as homotrimeric molecules, due to trimerization via the TNFSF ligand, while the antibody-fusion protein composed of the antibody Db and the B7.1 ligand presents as homodimeric molecule, due to the dimerization inherent of the diabody format (Fig. 1a)
Current developments of bispecific antibodies and chimeric antigen receptor (CAR)-T cells targeting Her2 and EGFR, respectively, have shown that tumor selectivity can be enhanced by lowering the affinity and enforcing the avidity of the antibody; T cell triggering becomes only effective at high antigen density [21, 22]
Summary
Interfering with the regulatory network of the immune response has become an intensively investigated strategy for cancer immunotherapy. Besides the development of checkpoint inhibitors that antagonize coinhibitory ligands/receptors (e.g., PD-L1, PD-1, CTLA-4), there is increasing interest in the development of agonists for costimulatory receptors (e.g., 4-1BB, OX40, GITR, CD40) [1, 2]. Enforcing directly the immune response by costimulation might be required in the presence of an immunosuppressive tumor microenvironment. Many immunosuppressive factors have been recognized so far (e.g., TGF-β, IL-10, IDO) [3], the composition and predominance in different tumor types/stages and the role of costimulation to counteract their negative influence on the antitumor response remain largely to be investigated. Costimulatory monoclonal antibodies are systemically active and prone for the development of immunerelated adverse events.
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