Abstract B67: Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo
Authors: Sefrin Julian Peter , Hillringhaus Lars , Lifke Valeria , Lifke Alexander 
Published: March 2017

Recent success of T cell based cancer immunotherapy approaches like chimeric antigen receptor T cells (CAR-Ts) and especially bispecific T cell engagers (BiTEs) has suggested great potential for cancer therapy. However, these technologies have several drawbacks like high risks for side-effects, such as autoimmunity, as well as inappropriate T cell activation due to a non-natural synapse. Hence, we sought to use antibody-mediated delivery of viral peptides to tumor cells in order to mimic viral infection and induce tumor cell lysis by virus-specific cytotoxic CD8+ T cells. Here, we generated immunoconjugates called antibody-targeted pathogen-derived peptides (ATPPs), where mature virus-derived MHC class I peptides from Influenza A or Epstein-Barr virus were conjugated to a tumor-associated antigen-specific antibody via a disulfide bond. Using optical imaging in combination with fluorescence resonance energy transfer, we revealed that after ATPP binding to the target antigen and subsequent internalization, the peptides are released in endosomes and loaded onto recycling MHC class I complexes. These peptide-MHC complexes are subsequently presented on the cell surface and mediate activation of human peptide-specific CD8+ T cells as shown by Interferon-γ release using enzyme-linked immunosorbent assay. Moreover, ATPP-loaded cancer cells from different tumor types were efficiently killed in vitro by human CD8+ T cells in a dose-dependent manner, reaching up to 100% killing after 24h at 0,13nM with an effector-to-target ratio of 3:1. Tumor cell death was assessed by the xCELLigence system as well as lactate dehydrogenase quantification in the supernatant. Interestingly, ATPPs were about 1000-times more potent than free peptide, while antibody alone, a non-cleavable ATPP construct, as well as peptides conjugated to a non-binding antibody exhibited no effects. Importantly, all used antibodies carried the P329G/LALA mutation for abolished Fc-receptor binding. In addition, we performed an experimental subcutaneous xenograft in vivo study with PDL1-expressing MDA-MB231 breast cancer cells in NOG mice in combination with anti-PD1 treatment. We observed that ATPPs successfully recruited adoptively transferred human peptide-specific CD8+ T cells into the tumor and induced 60% tumor growth inhibition of an established tumor after 3 weeks in the anti-PD1 combination setting.

Altogether, we generated first in class immunoconjugates for antibody-targeted loading of cancer cells with foreign MHC-I restricted peptide antigens. Our results demonstrate potent ATPP-mediated antitumor efficacy in vitro and in vivo, by loading tumor cells with viral peptide antigens and redirecting virus-specific cytotoxic T cells against cancer.

Citation Format: Julian Peter Sefrin, Lars Hillringhaus, Valeria Lifke, Alexander Lifke. Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B67.