The antibody-based delivery of interleukin-12 to the tumor neovasculature eradicates murine models of cancer in combination with paclitaxel.

PURPOSE: Interleukin-12 (IL12) is a potent proinflammatory cytokine with antitumor activity. Its heterodimeric nature makes it compatible with a large variety of different immunocytokine formats. Here we report the design, production, and characterization of a novel immunocytokine, based on the fusion of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of tumor neovasculature) with IL12 (termed IL12-F8-F8). EXPERIMENTAL DESIGN: We developed a novel immunocytokine based on the sequential fusion of interleukin-12 as a single polypeptide with two F8 antibodies in single-chain Fv (scFv) format. The fusion protein was characterized in vitro, and its targeting performance was assessed in vivo. The immunocytokine antitumor activity was studied as monotherapy as well as in combination therapies in three different murine tumor models. Moreover, depletion experiments and tumor analysis revealed a dominant role of natural killer cells for the mechanism of action. RESULTS: IL12-F8-F8 can be produced in mammalian cells, yielding a product of good pharmaceutical quality, capable of selective localization on the tumor neovasculature in vivo, as judged by quantitative biodistribution analysis with radioiodinated protein preparations. The protein potently inhibited tumor growth in three different immunocompetent syngeneic models of cancer. The treatment was generally well tolerated. Moreover, the IL12-F8-F8 fusion protein could be produced both with murine IL12 (mIL12) and with human IL12 (hIL12). CONCLUSIONS: The potent antitumor activity of mIL12-F8-F8, studied alone or in combination with paclitaxel in different tumor models, paves the way to the clinical development of the fully human immunocytokine.

Immunocytokines: a novel class of potent armed antibodies.

Several cytokines have been investigated in clinical trials, based on their potent therapeutic activity observed in animal models of cancer and other diseases. However, substantial toxicities are often reported at low doses, thus preventing escalation to therapeutically active regimens. The use of recombinant antibodies or antibody fragments as delivery vehicles promises to enhance greatly the therapeutic index of pro-inflammatory and anti-inflammatory cytokines. This review surveys preclinical and clinical data published in the field of antibody-cytokine fusions (immunocytokines). Molecular determinants (such as molecular format, valence, target antigen), which crucially contribute to immunocytokine performance in vivo, are discussed in the article, as well as recent trends for the combined use of this novel class of biopharmaceuticals with other therapeutic agents.

The targeted delivery of IL17 to the mouse tumor neo-vasculature enhances angiogenesis but does not reduce tumor growth rate.

There has been a long controversy as to whether interleukin-17 (IL17) has an impact on tumor growth. In order to assess whether IL17 may affect tumor growth, it would be convenient to achieve high levels of this pro-inflammatory cytokine at the tumor neo-vasculature, since IL17 is known to promote angiogenesis. Here, we have generated and tested in vivo a fusion protein, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of angiogenesis) and of murine IL17 (mIL17). The resulting immunocytokine (termed F8-mIL17) was shown to selectively localize at the tumor neo-vasculature and to vigorously promote tumor angiogenesis, without however reducing or enhancing tumor growth rate both in immunocompetent and in immunodeficient mice.

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7.

We generated and characterized novel antibody-cytokine fusion proteins ("immunocytokines") based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed "F8-mIL7") of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed "F8-mIL7-F8"), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio=16:1, 24h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.

A novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin.

Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12.

Proinflammatory cytokines have been used for several years in patients with advanced cancer but their administration is typically associated with severe toxicity hampering their application to therapeutically active regimens. This problem can be overcome by using immunocytokines (cytokines fused to antibody or antibody fragments) which selectively deliver the active cytokine to the tumor environment. Preclinical and recent clinical results confirmed that this approach is a very promising avenue to go. We designed an immunocytokine consisting of the scFv(F8) specific to extra-domain A of fibronectin and the very potent human cytokine interleukin-12 (IL12). The heterodimeric nature of IL12 allows the engineering of various immunocytokine formats, based on different combinations of the two subunits (p35 and p40) together with the scFv. In comparison to monomeric or homodimeric cytokines, the construction of a heterodimeric immunocytokine poses many challenges, e.g. gene dosing, stable high-yield expression as well as good manufacture practice (GMP) purification and characterization. In this paper, we describe the successful construction, characterization and production of the heterodimeric immunocytokine F8-IL12. The positive outcome of this feasibility study leads now to GMP production of F8-IL12, which will soon enter clinical trials.