Abstract B69: Novel MHC class I antibody fusions for cancer treatment.
Authors: Schmittnaegel Martina , Hoffmann Eike , Mundigl Olaf , Niederfellner Gerhard , Bosslet Klaus , Umana Pablo , Levitsky Victor , Klein Christian , Knoetgen Hendrik 
Published: August 2014

MHC (major histocompatibility complex) class I-restricted CD8 cytotoxic T cells recognize tumor cells or virus infected cells as foreign and consequently initiate a cascade of events resulting in their destruction. An immunotherapeutic strategy based on antibody-mediated targeting of virus-derived peptide-MHC class I complexes to tumor cells aiming at the subsequent elimination of these tumor cells by peptide-specific cytotoxic T cells has been proposed a few years ago. A crucial limitation hampering advances of this therapeutic concept was the recombinant expression of MHC class I fused full IgG immunoglobulins.

We have identified novel recombinant fusion formats that allow the expression of full-length peptide-MHC class I IgG fusion molecules. These formats contain a single recombinant human pMHC class I complex consisting of a viral peptide (CMV or EBV derived), beta-2-microglobulin and HLA heavy chain (A*0201, lacking the transmembrane domain) fused to one of the two immunoglobulin heavy chains of a complete antibody molecule. These fusions can be expressed at high levels in standard mammalian expression systems overcoming several former technical hurdles (e.g. prokaryotic expression, low refolding yield, chemical coupling of components). By flow cytometry we show that the new fusion proteins successfully deliver virus-peptide complexed MHC class I molecules to tumor cells. The fusion proteins are potent CD8 T cell recruiters for human donor derived specific T cells or human PBMCs from chronically infected donors. A low frequency of virus peptide specific CD8 T cells in PBMCs (0.25 to 3.8 % of all CD8 positive T cells) effectively triggers in a peptide specific manner the killing of tumor cells at sub-nanomolar concentrations (cytotoxic T cell assay using real time analysis with xCelligence confirmed by classical LDH release). No unspecific activation of T cells was observed even at high concentrations, indicating a favorable safety profile. Confocal time-lapse microscopy showed T cell synapse formation on the tumor cells and serial killing by the T cells. We experimentally compare the killing of tumor cells of the MHC class I fused antibodies with bispecific anti-CD3 mediated T cell recruiters and characterized the virus specific T cells from human donors in detail.

Our results show for the first time that recombinant pMHC class I antibody fusions in our novel full human IgG format can be expressed with a significant yield in common mammalian production cell lines. The redirection of MHC class I mediated recruitment of pre-existing virus specific CD8 T cells from human PBMCs is potent mechanism to attack tumor cells with a low risk of unspecific T cell activation making the concept now suitable as a therapeutic option.

Citation Format: Martina Schmittnaegel, Eike Hoffmann, Olaf Mundigl, Gerhard Niederfellner, Klaus Bosslet, Pablo Umana, Victor Levitsky, Christian Klein, Hendrik Knoetgen. Novel MHC class I antibody fusions for cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B69.