Generation of low-toxicity interleukin-2 fusion proteins devoid of vasopermeability activity

Abstract

Because of its key role in immunity, interleukin-2 (IL-2) has been studied extensively for the adoptive immunotherapy of cancer. Although systemic administration of IL-2 has been shown to stimulate antitumor responses in vivo, its efficacy in the clinic has been limited by the development of serious side effects, including the induction of vascular leak syndrome. Previously, we have identified a small peptide fragment of IL-2 that was found to contain the entire vasopermeability activity of the cytokine. The identification of the location of this potentially undesirable property of IL-2 enabled us to focus on the generation of mutant derivatives that might be lacking vasopermeability activity but that retain cytokine functionality. In addition to this discovery, our laboratory has constructed monoclonal antibody/IL-2 fusion proteins that can target this potent cytokine directly to tumor for the immunotherapy of both solid and lymphoid malignancies. Using this fusion protein technology, we have constructed a series of point mutations in the newly identified vasopermeability region of IL-2 for the purpose of deleting this activity.

Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells

“…for IL-2-mediated VLS (12,24). For anti-mouse IL-2 mAb S4B6, it is known that this mAb binds to amino acids 26-45 of mouse IL-2 (25), which overlaps with amino acids 22-31 of hIL-2 (26).…”

“…We measured NO 2 -in culture supernatants by using the Griess assay, as reported (24). Briefly, 1-2 × 10 5 Lin -CD31 + cells per well were left untreated or stimulated with 10 6 IU/mL IL-2.…”

Interleukin-2: Biology, Design and Application

“…More recent data showed that NK cells are implicated in VLS; however, the more proximal events of endothelial cell damage and VLS include the direct binding of IL-2 to CD25 + pulmonary endothelial cells [31]. Thus, use of IL-2/ S4B6 complexes or of the IL-2 muteins R38E, R38G, R38W, and F42A (all predicted or shown to impair binding to CD25) disfavors contact with endothelial cells and significantly reduces VLS [10,31,77].…”

IL-2/anti-IL-2 antibody complexes show strong biological activity by avoiding interaction with IL-2 receptor α subunit CD25

“…Thus, for exogenously-administered IL-2, renal elimination results in a very short in vivo half-life of IL-2 in the range of minutes. This has been a major limitation for IL-2-based strategies in the treatment of metastatic cancer and chronic viral infections, thus requiring the use of high doses of IL-2, which, however, can cause severe toxic side effects, termed vascular leak syndrome (11,12). In an attempt to extend in vivo halflife and increase biological activity, IL-2 has been coupled to larger proteins such as albumin and unrelated antibodies to create IL-2-IgG fusion proteins (IL-2-FP) (12,13), although with limited success.…”