Improvement of biological and pharmacokinetic features of human interleukin-11 by site-directed mutagenesis Yuni Jung a,⇑ , Haesook Ahn a , Dong-Sik Kim a , Yu Ri Hwang a , Seong-Hyun Ho a , Jong-Mook Kim a , Sujeong Kim a,1 , Suyong Ma b , Sunyoung Kim c a ViroMed Co., Ltd., Seoul, Republic of Korea b Beijing Northland Biotech Co., Ltd., Beijing, China c Department of Biological Science, Seoul National University, Seoul, Republic of Korea article info Article history: Received 30 December 2010 Available online 14 January 2011 Keywords: Interleukin-11 Site-directed mutagenesis Stability Pharmacokinetics Thrombocytopenia abstract Recombinant human interleukin-11 (rhIL-11) has been shown to increase platelet counts in animals and humans and is the only drug approved for its use in chemotherapy-induced thrombocytopenia (CIT). However, due to its serious side effects, its clinical use has been limited. The current work presents sig- nificantly improved efficacy of rhIL-11 via knowledge based re-designing process. The interleukin-11 mutein (mIL-11) was found to endure chemical and proteolytic stresses, while retaining the biological activity of rhIL-11. The improved efficacy of mIL-11 was evident after subcutaneous administration of mIL-11 and rhIL-11 in the rodent and primate models. More than three-fold increase in maximum plasma concentration (C max ) and area-under-the curve (AUC) was observed. Furthermore, three-fold higher increase in the platelet counts was obtained after seven consecutive daily subcutaneous mIL-11 injec- tions than that with rhIL-11. The mIL-11 demonstrated not only improved stability but also enhanced efficacy over the currently used rhIL-11 regimen, thereby suggesting less toxicity. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Interleukin-11 (IL-11) is a multifunctional cytokine secreted from bone marrow stromal cells, which promotes megakaryocyto- poiesis and platelet formation in mammals [1–4]. At present, re- combinant human IL-11 (rhIL-11), which lacks the N-terminal proline of wild type IL-11, is the only approved drug for the treat- ment of chemotherapy-induced thrombocytopenia (CIT) [5]. Although the administration of rhIL-11 has been shown to have a demonstrable pharmacological effect by increasing the platelet counts in patients undergoing cancer chemotherapy, its clinical use has been restricted due to the reports of its serious adverse events including edema [6,7]. Therefore, less toxic and more effec- tive agents are required for the treatment of CIT. Protein stability is a critical concern in the development of a therapeutic protein drug, because degradation, aggregation, or unfolding can result in its loss of function or potential immunoge- nicity. It was reported that rhIL-11 undergoes complete hydrolysis at Asp 134 –Pro 135 in the acidic condition [8]. Interestingly, the same peptide bond at Asp 13 –Pro 14 stays relatively intact under the same condition. The acid-hydrolytic cleavage proceeds the formation of intramolecular imide-ring formation after rearrangement [9]. Additionally, it is generally accepted that IL-11 adapts 4-helical fold with non-structured amino-terminus [10,11], which can be deleted without any loss of activity [12]. Often the unstructured re- gion or flexible loop region is more susceptible to the proteolysis. Therefore, it may be possible to engineer IL-11 to make it more sta- ble against the chemical and proteolytic stresses by removing the potential degradation sites, using site-directed mutagenesis. In this study, we show that the mutations introduced to IL-11 could not only increase the in vitro stability, but also improve phar- macokinetics in rats and monkeys. This new mIL-11 produced effi- cacy similar to that of rhIL-11, at a considerably less amount, suggesting the possibility of lower toxicity than that of rhIL-11, the currently used regimen. Our data show the potential of using the mIL-11 for the reduction of hematological toxicities associated with anti-cancer chemotherapy as well as the prevention or treat- ment of thrombocytopenia. 2. Materials and methods 2.1. Preparation of recombinant human IL-11 mutein (mIL-11) N-terminus deletion and site-directed mutation were performed using site-specific mutagenesis kit and PCR methods [3,13]. The 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.01.041 ⇑ Corresponding author. Address: ViroMed Co., Ltd., Bldg. 203, 599 Gwanak-ro Gwanak-gu, Seoul 151-742, Republic of Korea. Fax: +82 2 880 0070. E-mail address: yunijung@viromed.co.kr (Y. Jung). 1 Present address: Kolon Life Science Inc., Seoul, Republic of Korea. Biochemical and Biophysical Research Communications 405 (2011) 399–404 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc