Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21.

Cesare Di Nitto,Tobias Weiss,Michael Weller,Dario Neri,Roberto De Luca

doi:10.3390/antib11010019

Cesare Di Nitto, Tobias Weiss + Show 3 more

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https://doi.org/10.3390/antib11010019

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Abstract

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory activity of NK and CD8+ T-cells. Here we describe four formats of novel IL21-based antibody–cytokine fusion proteins, targeting the extra domain A (EDA) of fibronectin and explore their potential for cancer treatment. The fusion proteins were designed, expressed, and characterized. F8 in single-chain diabody (scDb) format fused to IL21 at its C-terminus exhibited a promising profile in size exclusion chromatography (SEC) and SDS-PAGE. The lead candidate was further characterized in vitro. A cell-based activity assay on murine cytotoxic T-cells showed that human IL21, compared to murine IL21 partially cross-reacted with the murine receptor. The prototype was able to recognize EDA as demonstrated by immunofluorescence analysis on tumor sections. In an in vivo quantitative biodistribution experiment, F8(scDb)-murine IL21 did not preferentially accumulate at the site of disease after intravenous injection, suggesting that additional protein engineering would be required to improve the tumor-homing properties of IL21-based product.

Highlights

A first protein consisting of the F8 antibody in single chain diabody format [24] fused at the C-terminus of human IL21 was cloned and produced in CHO-S cells (Figure 1A) with a yield of 3.9 mg/L
A second fusion protein, in which human IL21 was linked at its C- and N-terminuses by a peptide linker to the F8 antibody in single chain variable fragment exhibited a yield of 2.9 mg/L (Figure 1B)
A third fusion protein featuring of the F8 antibody in single-chain diabody (scDb) format fused at the N-terminus by a peptide linker to human IL21 was cloned and produced (Figure 1C) with a yield of 1.2 mg/L

Summary

Introduction

The systemic administration of recombinant cytokines can induce potent anti-cancer activity, leading to their marketing authorization for certain indications [4,5] These immunomodulatory products may cause side effects already at low concentrations, precluding dose escalation to treatments, which would be required to meliorate the therapeutic benefit [6–8]. The fusion of cytokines to tumor-specific antibodies has been suggested as a strategy to increase the therapeutic efficacy of the cytokine payload, as a consequence of a specific accumulation at malignant lesions, while sparing healthy tissues [9,10]. These products ( called “immunocytokines”) are designed to reduce toxicity in patients, compared to untargeted recombinant cytokines. The validation of tumor associated antigens simplify the development of immunocytokines that localize in the tumor mass as a result of a specific binding to the target antigen

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