Abstract 2842: A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adjustable cytokine activity.
Authors: Gillies Stephen D. 
Published: August 2014

The clinical use of interleukin 2 (IL2) for cancer immune therapy has been limited by a lack of tumor targeting that requires the administration of dose levels sufficient to activate anti-tumor effector cells in the tumor microenvironment. These doses, in turn, are limited by several side effects including vascular toxicities of hypotension and vascular leak syndrome. One way to increase the accumulation of IL2 at the tumor site is to genetically fuse it to the heavy chains of a tumor specific antibody, and several such molecules (immunocytokines or ICs) have been shown to have improved anti-tumor activity compared to free IL2, the targeting antibody, or the combination. This is especially true of ICs with longer circulating half lives that increase the amount of drug delivered to the tumor. Unfortunately, this increases the vascular exposure and toxicity of the IL2 component that, again, limits the amount that can be dosed and the rate of infusion. One way to reduce the toxicity of long-acting ICs is to dose them subcutaneously (greatly reducing Cmax levels in the blood) rather than intravenously. Another way is to make the IC more selective for the high affinity IL2 receptor (IL2Rhi) than the intermediate form (IL2Rint) by mutagenesis. Both approaches are based on the hypothesis that activating cells expressing IL2Rint should be limited to reduce toxicity, while activating cells expressing IL2Rhi should be maintained for anti-tumor activity. I have recently developed a novel method for constructing ICs that utilizes fusion of cytokines to the C terminus of the Ig light (L) chain, rather than the traditional method of fusing to the heavy (H) chain. The fusion junction of IL2, in this case, is located adjacent to the antibody hinge region, where the L chain forms a disulfide bond through its C-terminal cysteine residue to the H chain. This constrains both the N-terminal region of IL2, as well as the hinge-CH2 region of the H chain where both Fc receptors and the C1q subunit of complement bind when they mediate antibody-dependent cytotoxic activities (ADCC and CDC). Surprisingly, such ICs are expressed well in transfected cells, are very stable in normal buffers and have biological properties that are superior to ICs made by fusion to the H chain. These properties include longer circulating half life, increased uptake following subcutaneous dosing, and similar or improved antibody effector activities of ADCC and CDC, respectively. Furthermore, the sequestering effect of this fusion junction allows one to adjust IL2Rint binding and activation by shortening the N-terminus of IL2 at the fusion point. This appears to limit access of the critical contact residue Asp20 of IL2 to the β chain of IL2Rint, while maintaining binding and activation of IL2Rhi expressing cells as well as antibody effector functions.

Citation Format: Stephen D. Gillies. A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adjustable cytokine activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2842. doi:10.1158/1538-7445.AM2013-2842