Antibody-based targeted delivery of interleukin-4 synergizes with dexamethasone for the reduction of inflammation in arthritis.

Objectives: We have previously reported that F8-IL4, a fusion protein consisting of the F8 antibody specific to the alternatively-spliced extra domain A of fibronectin and of murine IL-4, cures mice with established arthritis, when used in combination with dexamethasone (DXM). The goal of this study was to assess whether other therapeutic agents, besides DXM, could induce cures in combination with F8-IL4 and to elucidate which leucocytes are most affected by the pharmacological treatment. Methods: We performed therapy experiments in mice with CIA, using intravenous administrations of F8-IL4 in combination with DXM, MTX, murine cytotoxic T-lymphocyte-associated protein 4 fused to the fragment crystallizable portion of murine IgG2a, as well as mAbs to murine IL17A or the p40 subunit of murine IL12/IL23. Histology and immunohistochemistry for the identification of the various leucocytes were performed on the paws of mice euthanized at different therapy time points. Results: Only the use of F8-IL4 in combination with DXM induced complete remissions, while all other combinations did not lead to cures. The light microscopical evaluation of paws with arthritis revealed a predominant infiltration of neutrophils, which substantially decreased 24 h after treatment with F8-IL4 and DXM. Conclusion: The combination of F8-IL4 with DXM promotes a rapid anti-arthritic action by potently inhibiting neutrophil activity. A fully human analogue of F8-IL4 may find clinical utility for the treatment of neutrophil-driven chronic inflammatory conditions.

Design and characterisation of a novel interleukin-15 receptor alpha fusion protein and analysis of interleukin-15 complexation.

Interleukin-15 (IL15) is one of the most important cytokines currently being considered for cancer therapy applications. It is thought that by administering IL15 in complex with its cognate receptor alpha chain (IL15Rα) its biological activity could be increased manifold. We produced a fusion protein of mouse IL15Rα and the F8 antibody, that targets the alternatively-spliced extra-domain A (EDA) of fibronectin, which is overexpressed in many types of cancer. The fusion protein F8IL15Rα was cloned, expressed and characterized in vitro and its ability to bind to mouse IL15 was assessed with both size exclusion chromatography (SEC) and surface plasmon resonance (SPR) experiments. Furthermore, mouse and human IL15 and their corresponding Fc fused IL15Rα subunits were purchased, characterized and used to compare the capacity of F8IL15Rα to generate complexes. Surprisingly, none of the IL15Rα fusion proteins showed IL15 complexation on SEC. However, on SPR, F8IL15Rα displayed the ability to bind IL15. In a cell-based activity assay none of the IL15Rα fusions were able to increase cellular proliferation in combination with IL15 compared to IL15 alone. A better understanding of the molecular requirements for effective IL15 signalling are likely to be important for the development of IL15-based biopharmaceuticals.

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

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.

Alternatively Spliced EDA Domain of Fibronectin Is a Target for Pharmacodelivery Applications in Inflammatory Bowel Disease.

The antibody-based pharmacodelivery of cytokines to sites of disease has been extensively studied for various indications but not for the treatment of inflammatory bowel diseases. Here, we report that the alternatively spliced EDA domain of fibronectin, a marker of angiogenesis and of tissue remodeling, is expressed in the dextran sodium sulfate mouse model of colitis and in patients with inflammatory bowel conditions, while being virtually undetectable in most normal adult tissues. Radiolabeled preparations of the F8 antibody, specific to the EDA domain of fibronectin, were shown to selectively localize to sites of inflammation in mice with colitis, as revealed by autoradiographic analysis. Fusion proteins of the F8 antibody with various murine payloads (interleukin-4, the p40 subunit of interleukin-12, interleukin-13) were administered to mice with colitis. IL12p40-F8 mediated an anti-inflammatory activity, which was comparable with the one of cyclosporine, whereas F8-IL4 did not inhibit colitis and F8-IL13 worsened the inflammatory conditions.