Abstract LB-264: Development of 3D microtissue models to study the activity of novel tumor-targeted immunotherapeutics.
Authors: Waldhauer Inja , Morra Laura , Lehmann Steffi , Agarkova Irina , Zumstein Philine , Kelm Jens M. , Umana Pablo , Klein Christian , Bacac Marina 
Published: August 2014

Abstract
IL-2 therapy can result in durable responses in a proportion of cancer patients, but the treatment is associated with significant toxicity. To improve the liabilities associated with IL-2 therapy, we recently developed a novel monomeric tumor-targeted immunocytokine (CEA-IL2v), which completely lacks binding to CD25 (IL-2Rα) but retains binding to IL-2Rβγ. When tested in classical 2D tumor/lymphocyte co-culture systems, CEA-IL2v induces concentration-dependent proliferation and activation of NK, CD4 and CD8 T cells, but differently from IL-2, does not lead to preferential activation of Tregs and induces lower levels Fas-induced apoptosis.
To further explore the properties of CEA-IL2v, including the assessment of antibody targeting and stimulation of immune-cell activation and infiltration, we developed a novel 3D heterotypic microtissues model consisting of tumor cells, fibroblasts and lymphocytes. To address the penetration and targeting, heterotypic 3D microtissues consisting of CEA-expressing human tumor cells and fibroblasts were incubated with tumor-targeted IL-2v (CEA-IL2v), stroma-targeted IL-2v (FAP-IL2v), and the corresponding untargeted control (DP47-IL2v). CEA-IL2v successfully penetrated into microtissues and specifically bound to CEA-positive tumor cells, FAP-IL2v was specifically retained by FAP-expressing fibroblasts whereas the DP47-IL2v was not detected in any of the components. The activity of the immunocytokines was further evaluated by addition of human lymphocytes to 3D microtissues. CEA-IL2v and DP47-IL2v activated different lymphocyte subsets, stimulated their infiltration and selectively promoted the elimination of tumor cells as shown by (a) reduction of microtissue size, (b) increase of LDH release, (c) elimination of CEA-positive tumor cells, (d) increase of lymphocytes infiltrating into the microtissues and (e) increase of cytokine and chemokine levels secreted in the supernatants, including MIP-1β, IL-6 and IFN-γ.
Taken together, the heterotypic tumor/fibroblast/lymphocyte 3D microtissue model presented here offers the possibility to monitor antibody penetration and targeting to tumor and stroma components, to study the interaction of tumor cells with immune cells in a system that more closely resembles the in vivo tumor microenvironment and thus provides an in vitro platform for optimal immunotherapy development. The study also points out that the tumor-targeted CEA-IL2v immunocytokine is an elegant tool for expansion and activation of immune effector cells, which may successfully be applied for cancer immunotherapy.
Citation Format: Inja Waldhauer, Laura Morra, Steffi Lehmann, Irina Agarkova, Philine Zumstein, Jens M. Kelm, Pablo Umana, Christian Klein, Marina Bacac. Development of 3D microtissue models to study the activity of novel tumor-targeted immunotherapeutics. [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 LB-264. doi:10.1158/1538-7445.AM2013-LB-264