Influence of antigen density and immunosuppressive factors on tumor-targeted costimulation with antibody-fusion proteins and bispecific antibody-mediated T cell response.

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.

IL15-Based Trifunctional Antibody-Fusion Proteins with Costimulatory TNF-Superfamily Ligands in the Single-Chain Format for Cancer Immunotherapy.

IL15 and costimulatory receptors of the tumor necrosis superfamily (TNFRSF) have shown great potential to support and drive an antitumor immune response. However, their efficacy as monotherapy is limited. Here, we present the development of a novel format for a trifunctional antibody-fusion protein that combines and focuses the activity of IL15/TNFSF-ligand in a targeting-mediated manner to the tumor site.

Diabody-Ig: a novel platform for the generation of multivalent and multispecific antibody molecules.

Multivalent mono- or bispecific antibodies are of increasing interest for therapeutic applications, such as efficient receptor clustering and activation, or dual targeting approaches. Here, we present a novel platform for the generation of Ig-like molecules, designated diabody-Ig (Db-Ig). The antigen-binding site of Db-Ig is composed of a diabody in the V-V orientation stabilized by fusion to antibody-derived homo- or heterodimerization domains, e.

Production, Purification, and Characterization of Antibody-TNF Superfamily Ligand Fusion Proteins.

Antibody-fusion proteins with ligands, e.g., of the TNF superfamily (TNFSF) can be adequately produced in mammalian expression systems.

Tumor-targeted costimulation with antibody-fusion proteins improves bispecific antibody-mediated immune response in presence of immunosuppressive factors.

Therapeutic strategies aiming for the induction of an effective immune response at the tumor site can be severely hampered by the encounter of an immunosuppressive microenvironment. We investigated here the potential of concerted costimulation by tumor-directed antibody-fusion proteins with B7.1, 4-1BBL and OX40L to enforce bispecific antibody-induced T cell stimulation in presence of recognized immunosuppressive factors including IL-10, TGF-β, indoleamine 2,3-dioxygenase (IDO), PD-L1 and regulatory T cells.

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format.

Co-stimulation via receptors of the tumor necrosis factor superfamily (TNFSF) emerges as promising strategy to support antitumor immune responses. Targeted strategies with antibody-fusion proteins composed of a tumor-directed antibody part and the extracellular domain of a co-stimulatory ligand of the TNFSF constitute an attractive option to focus the co-stimulatory activity to the tumor site. Since TNFSF members intrinsically form functional units of non-covalently linked homotrimers, the protein engineering of suitable antibody-fusion proteins is challenging.