Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

Abstract

Proinflammatory cytokines have been used for several years in patients with advanced cancer but their administration is typically associated with severe toxicity hampering their application to therapeutically active regimens. This problem can be overcome by using immunocytokines (cytokines fused to antibody or antibody fragments) which selectively deliver the active cytokine to the tumor environment. Preclinical and recent clinical results confirmed that this approach is a very promising avenue to go. We designed an immunocytokine consisting of the scFv(F8) specific to extra-domain A of fibronectin and the very potent human cytokine interleukin-12 (IL12). The heterodimeric nature of IL12 allows the engineering of various immunocytokine formats, based on different combinations of the two subunits (p35 and p40) together with the scFv. In comparison to monomeric or homodimeric cytokines, the construction of a heterodimeric immunocytokine poses many challenges, e.g. gene dosing, stable high-yield expression as well as good manufacture practice (GMP) purification and characterization. In this paper, we describe the successful construction, characterization and production of the heterodimeric immunocytokine F8-IL12. The positive outcome of this feasibility study leads now to GMP production of F8-IL12, which will soon enter clinical trials.

The Dose-Dependent Tumor Targeting of Antibody–IFNγ Fusion Proteins Reveals an Unexpected Receptor-Trapping Mechanism In Vivo

“…This discrepancy may reflect different activities of the cytokines among species, making clinical development more difficult. Indeed, while we have demonstrated a strong single-agent tumor growth inhibition with F8-IFNg in the three syngeneic immunocompetent mouse models of cancer tested, it remains to be considered whether the fully human F8-IFNg immunocytokine or the F8-IL12 immunocytokine would be the better candidate for clinical development (20,38). Indeed, F8-IL12 also targets tumors efficiently at low doses ($0.5 mg/kg) and is able to potently induce IFNg overexpression at the tumor site (18,20).…”

New insights into IL-12-mediated tumor suppression

“…105 The administered IL-12 was found to activate myeloid cells by increasing the expression of Fas and cross-presentation, leading to the stimulation of tumor antigen-specific CD8 T cells and regression of established tumors 102,106 (Figure 1). More recently, the development of novel approaches that direct IL-12 activity to the tumor site focus on immunocytokines, for example, the fusion of the cytokine to an antibody that binds specifically to the tumor vasculature, 107,108 or to exposed deoxyribonucleic acid (DNA) in the necrotic core of a tumor. 109 The targeting of necrotic areas within the tumor is of special interest due to the lack of perfusion of solid tumors and subsequent cell death.…”

Immunocytokines: a novel class of products for the treatment of chronic inflammation and autoimmune conditions

“…members of the IL12 superfamily) can offer additional design possibilities, depending on the assembly of the two cytokine subunits (Fig. 1c) [40][41][42][43]. When considering IgG-based immunocytokines, C-terminal fusions to the heavy or to the light chains have been proposed [44].…”