Elsevier

Journal of Biotechnology

Volume 271, 10 April 2018, Pages 29-36
Journal of Biotechnology

Novel antibody-cytokine fusion proteins featuring granulocyte-colony stimulating factor, interleukin-3 and interleukin-4 as payloads

https://doi.org/10.1016/j.jbiotec.2018.02.004Get rights and content

Highlights

  • 3 novel fusion proteins GCSF-F8, F8-IL3 and F8-IL4-F8 were produced and characterized.

  • F8-IL4-F8 showed potent tumor growth retardation in a murine model of cancer.

  • GCSF-F8 and F8-IL3 may be applied therapeutically for neutrophil recruitment.

Abstract

Neutrophils can strongly influence disease activity in cancer and in chronic inflammation. Here, we report for the first time the construction and characterization of antibody-fusion proteins featuring granulocyte-colony stimulating factor and interleukin-3 as payloads capable of enhancing neutrophil activity and a novel antibody-interleukin-4 fusion protein with neutrophil inhibitory potential. We used the F8 antibody specific to the alternatively-spliced extra domain A (EDA) of fibronectin as a targeting agent, since the cognate antigen is strongly upregulated in diseases characterized by angiogenesis.

The fusion proteins GCSF-F8, F8-IL3 and F8-IL4-F8, were cloned, expressed, and their targeting ability assessed, exhibiting preferential tumor uptake with tumor:blood ratios at 24 h after injection of 3.3, 18.2 and 27.3, respectively.

In F9 tumor bearing-mice GCSF-F8 and F8-IL3 did not provide a therapeutic benefit, while F8-IL4-F8 showed a potent tumor growth retardation. In the collagen-induced model of arthritis, GCSF-F8 and F8-IL3 induced a worsening of the disease, while F8-IL4-F8 slowed arthritis progression but, surprisingly, exhibited substantial toxicity when used in combination with dexamethasone.

Collectively, the results indicate that the novel fusion proteins could be expressed and efficiently delivered to the site of disease. However, they were not superior to other antibody-cytokine fusions previously described by our laboratory.

Introduction

Cytokines are a class of immunomodulatory proteins which play an important role in health and disease by regulating the number, activity and behavior of cells (Murphy et al., 2012). Besides interleukins, produced by lymphocytes, some hematopoietic growth factors (e.g., Granulocyte-colony stimulating factor (G-CSF) and Granulocyte-macrophage colony-stimulating factor (GM-CSF)) are nowadays also classified as members of the cytokine family (Murphy et al., 2012). Various recombinant cytokines have gained marketing authorization for the treatment of different types of conditions (Bootz and Neri, 2016; Kontermann, 2012). In most cases, these agents show activity at sub-milligram doses in patients and the escalation to therapeutically-active regimens is sometimes prevented by the onset of toxicity. For this reason, considerable research efforts have been devoted to the generation of disease-targeting antibody-cytokine fusions (“immunocytokines”), with the aim to increase the therapeutic index of the immunomodulatory payload (Kontermann, 2012; Neri and Sondel, 2016; Pasche and Neri, 2012). A number of immunocytokine products have been investigated in clinical trials for the therapy of cancer and of other conditions and some agents are currently being investigated in Phase III clinical studies [clinical trial identifier: NCT02938299 (www.clinicaltrial.gov)] (Neri and Sondel, 2016; Pasche and Neri, 2012).

Various types of antibodies can be considered for the generation of cytokine fusions. Our laboratory has previously described the use of the F8 antibody, specific to the alternatively-spliced extra domain A (EDA) of fibronectin, as a vehicle for pharmacodelivery applications (Schwager et al., 2009; Villa et al., 2008). EDA(+)-fibronectin is virtually undetectable in normal tissues (exception made for placenta, the endometrium and some vessels of the ovaries), while the antigen is strongly expressed at sites of tissue remodeling, with a distinctive perivascular pattern of staining in various types of cancer and in rheumatoid arthritis (Rybak et al., 2007; Schwager et al., 2009).

For some biotechnological applications, it is convenient to use antibodies in full IgG format (Becker et al., 1996; Fallon et al., 2014; Mossner et al., 2010; Xuan et al., 2010). Alternatively, antibody fragments (e.g., scFv fragments or diabodies) may be preferable in order to achieve a long residence time at the site of disease and a fast clearance from circulation (Holliger et al., 1993; Huston et al., 1988). Our laboratory has previously described the fusion of disease targeting antibody fragments with various cytokine payloads, including several interleukins (e.g., IL1β, IL2, IL4, IL6, IL7, IL9, IL10, IL12, IL13, IL15, IL22), members of the TNF superfamily, interferons and growth factors (e.g., VEGF-A, GM-CSF) (Kiefer and Neri, 2016; Pasche and Neri, 2012).

In this study, we focused on the engineering and characterization of novel antibody-fusion proteins, featuring payloads capable of modulating neutrophil activity. G-CSF is responsible for the induction of neutrophil development, differentiation and mobilization (Murphy et al., 2012). It is produced by fibroblasts and monocytes and acts on neutrophils by binding to the homodimeric G-CSF-Receptor (G-CSFR) (Bendall and Bradstock, 2014; Murphy et al., 2012). In the clinic, recombinant G-CSF is used for the treatment of congenital and acquired (e.g. due to chemotherapy) neutropenia (Spiekermann et al., 1997). Similarly, interleukin-3 (IL3) acts on a multitude of cell types in early hematopoiesis and can induce a significant elevation of the neutrophil titer, although neutrophils eventually lose responsiveness at mature stages (Eder et al., 1997; Murphy et al., 2012; Oster et al., 1991). IL3 was so far both shown to induce a worsening as well as a beneficial effect in models of murine arthritis and osteoarthritis (Bruhl et al., 2009; Kour et al., 2016; Srivastava et al., 2011).

Interleukin-4 (IL4) is a crucial cytokine for the differentiation of CD4+ T cells into a TH2 lineage (Murphy et al., 2012). Furthermore, IL4 has recently been shown to inhibit neutrophil expansion and recruitment by interacting with neutrophil type 2 IL4 receptors to antagonize G-CSF and chemokine mediated signaling (Woytschak et al., 2016). An F8 diabody IL4 fusion protein was the first immunocytokine to induce complete cures in a collagen-induced arthritis (CIA) model in combination with dexamethasone (Hemmerle et al., 2014). In this study, a novel format of the fusion protein was generated.

We fused the F8 antibody with G-CSF, IL3 and IL4 (generating GSCF-F8, F8-IL3 and F8-IL4-F8), yielding proteins that could be purified to homogeneity and which retained full biological activity. In quantitative biodistribution studies, the fusion proteins efficiently localized to tumors after intravenous administration. However, in an immunocompetent mouse model of cancer, only the IL4 fusion displayed a potent anti-cancer activity. In the collagen-induced model of arthritis, GCSF-F8 and F8-IL3 worsened arthritis progression, while F8-IL4-F8 provided only a weak anti-arthritic activity.

Section snippets

Cell culture

For cell cultivation CHO-S cells (Invitrogen, Switzerland) were cultured in suspension in PowerCHO-2 CD (LZ-BE12-771Q; Lonza, Belgium) supplemented with 4 mM Ultraglutamine 1 (Lonza, Belgium), 1X HT Supplement (50X) (Gibco, UK) and 1X Anti-Anti (100X) (Gibco, UK) in shaking incubators. For protein production CHO-S cells were cultured in suspension in ProCHO 4 (LZ-BE12-029Q; Lonza, Belgium) supplemented with 4 mM Ultraglutamine 1 (Lonza, Belgium), 1X HT Supplement (50X) (Gibco, UK) and 1X

Production and characterization of novel fusion proteins

Previous work from our laboratory had shown that cytokine payloads could be fused either at the C-terminus or at the N-terminus of antibody fragments with retention of targeting ability (Bootz et al., 2016; Hemmerle and Neri, 2014a; Pasche et al., 2011; Schwager et al., 2009). We fused G-CSF at the N-terminus and IL3 at the C-terminus of the F8 antibody in homodimeric diabody format (Fig. 1A and B), since a similar topology had previously been described for fusions of these cytokines with other

Discussion

Our laboratory has a long-term goal to fuse and characterize most known immunomodulatory payloads (e.g., cytokines, chemokines) to antibodies of proven disease-homing activity (e.g., the anti-EDA F8 antibody). In this study, we reported the first antibody fusions with G-CSF and IL3, two cytokines which potently increase the number and activity of neutrophils. In addition, we described a novel format for IL4-based immunocytokines, as interleukin-4 is able to antagonize neutrophil activity (

Funding

We are grateful to ETH Zürich, the Swiss National Science Foundation [Project Nr. 310030B_163479/1], the Swiss Federal Commission for Technology and Innovation (KTI) [Project Nr. 17072.1] and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program [grant agreement No 607603] for the financial support.

Conflict of interest statement

DN is a cofounder and shareholder of Philogen SpA (Siena, Italy), the company that owns the F8 antibody and developed Dekavil (F8-huIL10). The experiments of this article have been co-financed by Philochem AG (Otelfingen, Switzerland), a fully-owned company of the Philogen group, in the frame of a collaborative Swiss Federal KTI MedTech Project with ETH (Kommission für Technologie und Innovation). All other authors declare that they have no conflicts of interest.

Acknowledgments

We thank Dr. Francesca Pretto for the support of the therapy experiments in the collagen-induced model of arthritis. We are also grateful to the laboratory staff of the Laboratory for Animal Model Pathology, (Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Switzerland) for skillful technical support.

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