Chimera of IL-2 Linked to Light Chain of anti-IL-2 mAb Mimics IL-2/anti-IL-2 mAb Complexes Both Structurally and Functionally

Abstract

IL-2/anti-IL-2 mAb immunocomplexes were described to have dramatically higher activity than free IL-2 in vivo. We designed protein chimera consisting of IL-2 linked to light chain of anti-IL-2 mAb S4B6 through flexible oligopeptide spacer (Gly(4)Ser)(3). This protein chimera mimics the structure of IL-2/S4B6 mAb immunocomplexes but eliminates general disadvantages of immunocomplexes like possible excess of either IL-2 or anti-IL-2 mAb and their dissociation to antibody and IL-2 at low concentrations. This novel kind of protein chimera is characterized by an intramolecular interaction between IL-2 and binding site of S4B6 mAb similarly as in IL-2/S4B6 mAb immunocomplexes. Our protein chimera has biological activity comparable to IL-2/S4B6 mAb immunocomplexes in vitro, as shown by stimulation of proliferation of purified and activated OT-I CD8(+) T cells. The protein chimera exerts higher stimulatory activity to drive expansion of purified CFSE-labeled OT-I CD8(+) T cells activated by an injection of a low dose of SIINFEKL peptide than IL-2/S4B6 mAb immunocomplexes in vivo.

IL-2/anti-IL-2 mAb immunocomplexes: A renascence of IL-2 in cancer immunotherapy?

“…51 This cluster of residues collocates with an interface where CD25 recognizes IL-2, thus giving a molecular gist to the previous functional studies. 77,80 All of these corresponding data support the assumption that the binding of S4B6 mAb (and JES6-5H4 mAb alike) blocks or seriously hinders the CD25 recognition of IL-2. Another study of Rojas et al 67 extended the dataset to JES6.1, identifying a completely different cluster of residues that are recognized by this antibody.…”

Interleukin-2: Biology, Design and Application

“…Notably, for clinical application, there is some interest in generating a fusion protein of IL-2 complex by using a flexible linker between IL-2 and the mAb, although others do not see a problem with using IL-2/mAb complexes that can dissociate in vivo (Box 1) [10]. Currently, there is only one report describing a fusion protein of the IL-2/S4B6 complex; however, it remains to be tested to what extent this fusion protein still allows association of mIL-2 with S4B6's antigen-binding groove [81]. Notably, it is worth considering the use of S4B6-like mAbs to modulate endogenous IL-2 for selective immune stimulation, as shown in mice [9]; whether similar effects can be achieved with JES6-1-like mAbs remains to be explored.…”

“…Even if all IL-2/mAb complexes were to dissociate following injection, the amount of IL-2 would reach only 2-5% of the IL-2 dose usually administered to patients; moreover, the anti-IL-2 mAb could then bind to endogenous IL-2 and direct it to the targeted immune cells. Potentially, IL-2/mAb complexes could be linked by a flexible linker [10,81]. With regard to IL-2 muteins, immunogenicity should be considered.…”

Fine epitope specificity of antibodies against interleukin-2 explains their paradoxical immunomodulatory effects

“…29 An extreme example of the bioactivity of the bound form is the recent report of a chimeric protein including IL-2 and the S4B6 mAb light chain in a single polypeptide able to bind the S4B6 heavy chain. 30 Such chimera stabilizes the immune complex between IL-2 and the antibody, because the physical linkage promotes reassociation. A related, and even more drastic, approach is the use of an IL-2-derived mutein in which the interface with the chain was severely disrupted by mutagenesis, resulting in permanent abrogation of this particular interaction.…”