Abstract
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). 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. 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). 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.
Highlights
Many proinflammatory cytokines have exhibited promising anticancer properties in preclinical experiments, but their administration to patients is often associated with substantial toxicities that hinder an escalation to the dose needed for therapeutic activity
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
Whereas clinical development programs in oncology have so far focused on the proinflammatory cytokines IL2, IL12, and TNF as active payloads, several other cytokines have been considered for immunocytokine construction, and their therapeutic activity has been tested in rodent models of cancer [1]
Summary
Many proinflammatory cytokines have exhibited promising anticancer properties in preclinical experiments, but their administration to patients is often associated with substantial toxicities that hinder an escalation to the dose needed for therapeutic activity. A promising avenue to improve the therapeutic index of anticancer cytokines consists in the fusion of cytokines with a suitable antibody serving as a vehicle for targeted delivery to the tumor environment A number of antibody–cytokine fusion proteins ("immunocytokines") have been moved to clinical trials using, in most cases, antibodies specific to splice isoforms of fibronectin or of tenascin-C [1, 5]. These components of the modified subendothelial tumor extracellular matrix are strongly expressed in the cancer neovasculature and stroma but are virtually undetectable in normal adult tissues [6]. Whereas clinical development programs in oncology have so far focused on the proinflammatory cytokines IL2 (interleukin 2), IL12, and TNF as active payloads, several other cytokines have been considered for immunocytokine construction, and their therapeutic activity has been tested in rodent models of cancer [1]
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