Abstract 2580: Construction of immunoglobulin light chain IL2 immunocytokines and mouse-specific surrogates for studying the effects of IL2 receptor selectivity in mouse tumor models
Authors: Gillies Stephen D. 
Published: May 2015

Abstract
Armed antibodies are an increasingly important approach to cancer therapy and include the use of anti-tumor antibodies for the delivery of radioactivity (RIT), cytotoxic drugs (ADCs), additional antibody domains (bi-specifics) and immune-stimulatory cytokines (immunocytokines, or ICs). While these approaches have been in development for more than two decades, recent successes have come only after refinements that have balanced tumor targeting with minimal systemic toxicity. For ADCs, this has come from new linker technology that minimizes cytotoxic drug activity until the conjugates have been taken up by target cells. Immunocytokines are also intended to act locally, within the tumor microenvironment, but their systemic delivery by intravenous injection is complicated by their ability to activate circulating immune cells expressing the cytokine receptor(s). For IL2 containing ICs, pre-clinical mouse tumor models have shown excellent anti-tumor activity while in cancer patients, the therapeutic index is lower, due to a greater (10-20 fold) ability to activate the human intermediate affinity IL2 receptor (βγIL2R). This suggests it would be advantageous to construct IL2 based ICs with normal activity on the high affinity IL2R (αβγL2R), but reduced (but not absent) ability to trigger βγIL2R. A new IC platform has been developed in which cytokines (or other proteins) are fused to the C terminus of the IgG light (L) chain. This greatly restricts the freedom of movement of the N-terminal region of the fused protein. For IL2, this restricts access to aspartic acid 20 (D20) of IL2, a key site of interaction with the β chain of the IL2R, and reduces the ability of the IC to trigger βγIL2R activation. For human cells expressing only βγIL2R, activity is reduced 10-20 fold, but normal for cells expressing αβγIL2R. This receptor selectivity can be increased by shortening the N-terminal region of IL2. Interestingly, an IC with a 100 fold reduced activity for βγIL2R appears to have full activity once the IC binds to antigen coated beads, at least for human immune cells. Unfortunately, the same molecules have very low activity for mouse immune cells expressing only βγIL2R, making mouse models non-predictive. To address this, a set of ICs (mouse model surrogates) have been made using a mutant IL2 with increased affinity to βγIL2R that is independent of the sterically-restricted D20 contact site. The L-chain fusion protein with a full length IL2 N-terminus is 50-100 times more potent than the same IC with native IL2 on human βγIL2 expressing cells and is being tested for activity with mouse splenocytes. Further modifications of the length of the IL2 N-terminus will be used to create molecules with the receptor specificity matching the ICs intended for human clinical trials. Once identified, these molecules will be tested for antigen-induced activation of βγIL2R.
Citation Format: Stephen D. Gillies. Construction of immunoglobulin light chain IL2 immunocytokines and mouse-specific surrogates for studying the effects of IL2 receptor selectivity in mouse tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2580. doi:10.1158/1538-7445.AM2014-2580