Ab-IL2 fusion proteins mediate NK cell immune synapse formation by polarizing CD25 to the target cell-effector cell interface

Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI 53711, USA.
Cancer Immunology and Immunotherapy (Impact Factor: 3.94). 07/2011; 60(12):1789-800. DOI: 10.1007/s00262-011-1072-9
Source: PubMed


The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM(+) (ovarian) and GD2(+) (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells' IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50-200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.

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    • "Alternative approaches for IL-2 based immunotherapy e.g. toxin conjugates, antibodies, fusion proteins, gene therapy are therefore currently being explored in various cancers (Eigentler et al., 2011; Telang et al., 2011; Gubbels et al., 2011). However, since IL-2 is essential for the development, survival and function of regulatory T (Treg) cells, which function to inhibit immune responses and prevent autoimmune disease, IL-2 may have a role in promoting T cell tolerance (an important consideration is the dose of IL-2 used as a low dose appears to favour tolerance over autoimmunity). "
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    ABSTRACT: Two candidate preparations of human sequence recombinant Interleukin-2 (IL-2) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement international standard. The preparations were tested by eight laboratories using in vitro bioassays and immunoassays. The candidate preparation 86/500 was judged suitable to serve as a replacement international standard based on the data obtained for activity and stability. On the basis of the results reported here, the preparation coded 86/500 was established by the WHO Expert Committee on Biological Standardisation (ECBS) in 2012 as the WHO 2nd IS for human IL-2 with an assigned value for IL-2 activity of 210 IU/ampoule. Calibration of the 2nd IS is primarily based on the bioassay in use in various laboratories and relies exclusively on the estimates calculated relative to the WHO 1st IS for continuity of the IU.
    Full-text · Article · Nov 2013 · Journal of immunological methods
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    • "Some IL-2 centered strategies have already been approved by the FDA for the treatment of metastatic melanoma [16] and renal cell carcinoma [17]. Intense research (basic, translational, and clinical) is also underway on the use of IL-2 and IL-2-antibody conjugates (immunocytokines) to boost the anti-cancer activities of NK [18] [19] [20] [21]. Our initial studies on the characterization of the global proteome of naïve and IL-2-stimulated human NK cells reveal a large number of proteins exhibiting changes in expression levels upon IL-2 stimulation. "
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    ABSTRACT: Unlabelled: Natural killer (NK) cells efficiently cytolyse tumors and virally infected cells. Despite the important role that interleukin (IL)-2 plays in stimulating the proliferation of NK cells and increasing NK cell activity, little is known about the alterations in the global NK cell proteome following IL-2 activation. To compare the proteomes of naïve and IL-2-activated primary NK cells and identify key cellular pathways involved in IL-2 signaling, we isolated proteins from naïve and IL-2-activated NK cells from healthy donors, the proteins were trypsinized and the resulting peptides were analyzed by 2D LC ESI-MS/MS followed by label-free quantification. In total, more than 2000 proteins were identified from naïve and IL-2-activated NK cells where 383 proteins were found to be differentially expressed following IL-2 activation. Functional annotation of IL-2 regulated proteins revealed potential targets for future investigation of IL-2 signaling in human primary NK cells. A pathway analysis was performed and revealed several pathways that were not previously known to be involved in IL-2 response, including ubiquitin proteasome pathway, integrin signaling pathway, platelet derived growth factor (PDGF) signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway and Wnt signaling pathway. Biological significance: The development and functional activity of natural killer (NK) cells is regulated by interleukin (IL)-2 which stimulates the proliferation of NK cells and increases NK cell activity. With the development of IL-2-based immunotherapeutic strategies that rely on the IL-2-mediated activation of NK cells to target human cancers, it is important to understand the global molecular events triggered by IL-2 in human NK cells. The differentially expressed proteins in human primary NK cells following IL-2 activation identified in this study confirmed the activation of JAK-STAT signaling pathway and cell proliferation by IL-2 as expected, but also led to the discovery and identification of other factors that are potentially important in IL-2 signaling. These new factors warrant further investigation on their potential roles in modulating NK cell biology. The results from this study suggest that the activation of NK cells by IL-2 is a dynamic process through which proteins with various functions are regulated. Such findings will be important for the elucidation of molecular pathways involved in IL-2 signaling in NK cells and provide new targets for future studies in NK cell biology.
    Full-text · Article · Jun 2013 · Journal of proteomics
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    • "Recent findings describe the formation of activating immune synapses (AIS) between FcR-deficient NK cells and tumor cells mediated by IL2 immunocytokines. These functional AIS were formed by the interaction of the IL2 component of the immunocytokine and IL2 receptors on effector cells[26,27], demonstrating that IL2 immunocytokines can initiate direct target cell lysis even without Fc involvement. "

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