A novel fusion protein of IL-4 and IL-10 (IL4-10 FP) was effective in resolving chronic pain in multiple preclinical models and showed synergistic effects as compared to the activity of the two individual cytokines IL-4 and IL-10. The fusion protein was found to be effective against inflammatory, neuropathic and osteoarthritic pain, indicating that it targets common pain pathways in different types of chronic pain. This thesis by Cristine Steen-Louws describes the development of IL4-10 FP and the evaluation of its therapeutic potential in inflammatory diseases, as studied at the UMC Utrecht.

Inflammatory diseases, such as rheumatoid arthritis (RA), osteoarthritis (OA), and chronic pain are difficult to treat because of the complexity of the immune system. Regulatory cytokines are attractive candidates for therapeutic drugs to treat chronic inflammatory diseases as they potentially inhibit the formation and release of multiple inflammatory mediators. However, as stand-alone preparations, study outcomes with regulatory cytokines have been disappointing, largely due to poor pharmacokinetics (e.g. short half-life). Cytokines have complementary and synergistic effects and preferably act in networks to achieve optimal effects. It is, therefore, attractive to combine cytokines to increase their therapeutic effect.

Fusion protein

In this PhD thesis, Cristine Steen-Louws (Center for Translational Immunology, UMC Utrecht) investigated in pre-clinical studies the IL4-10 fusion protein. The IL4-10 fusion protein consists of the regulatory cytokines IL-4 and IL-10. In the fusion protein, these cytokines are covalently linked to each other using a linker sequence rich in serine and glycine residues to allow maximal flexibility between both cytokine moieties. Her PhD work describes the production and characterization of IL4-10 FP, as well as its therapeutic potential in inflammatory diseases.

Key findings from pre-clinical studies

The PhD research by Cristine Steen-Louws and colleagues resulted in the following findings:

• The IL4-10 fusion protein demonstrates to potent inhibition of pro-inflammatory cytokines, shifting a pro-inflammatory phenotype towards a more immuno-regulatory phenotype.
• IL-4 and IL-10 in the IL4-10 fusion protein neutralize each other’s unfavorable effects.
• Administration of IL4-10 fusion protein in experimental OA and RA animal models mitigated the disease.
• IL4-10 fusion protein better reduced pain in inflammatory pain models than the combination therapy of both individual cytokines. Upon repeated administration, the fusion protein was even able to permanently and completely resolve pain.
• Combining IL-4 and IL-10 in a fusion protein, also prolongs the half-life and improves its effects upon administration. In a pharmacokinetics study a 4-fold improved half-life of IL4-10 fusion protein as compared to the individual cytokines was shown.
• Modification of the biochemical properties of IL4-10 fusion protein by so-called glycol-engineering, modifying glycosylation sites and the degree of sialylation, further improved its characteristics.

In conclusion, this thesis describes several unique effects of IL4-10 fusion protein, which suggest that it is a promising potential therapeutic drug for the treatment of inflammatory diseases. The drug candidate is currently in pre-clinical development for osteoarthritis and inflammatory pain.

PhD defense

Cristine Steen-Louws (1985, Vlissingen) defended her PhD thesis on November 4, 2020 at Utrecht University. The title of her thesis was “SYNERKINE | IL4-10 fusion protein for the treatment of inflammatory diseases”. Supervisors were prof. dr. Erik Hack (Center for Translational Immunology, UMC Utrecht) and prof. dr. Floris Lafeber (department of Rheumatology and Clinical Immunology, UMC Utrecht). Co-supervisors were dr. Niels Eijkelkamp (Center for Translational Immunology, UMC Utrecht) and dr. Jelena Popov-Celeketic (Department of Rheumatology and Clinical Immunology, UMC Utrecht). Cristine works as a scientist at Genmab where she coordinates and supervises the bioanalysis of pre-clinical studies.