Antibody-mediated delivery of a viral MHC-I epitope into the cytosol of target tumor cells repurposes virus-specific CD8+ T cells for cancer immunotherapy

Abstract

Background Redirecting pre-existing virus-specific cytotoxic CD8+ T lymphocytes (CTLs) to tumors by simulating a viral infection of the tumor cells has great potential for cancer immunotherapy. However, this strategy is limited by lack of amenable method for viral antigen delivery into the cytosol of target tumors. Here, we addressed the limit by developing a CD8+T cell epitope-delivering antibody, termed a TEDbody, which was engineered to deliver a viral MHC-I epitope peptide into the cytosol of target tumor cells by fusion with a tumor-specific cytosol-penetrating antibody. Methods To direct human cytomegalovirus (CMV)-specific CTLs against tumors, we designed a series of TEDbodies carrying various CMV pp65 antigen-derived peptides. CMV-specific CTLs from blood of CMV-seropositive healthy donors were expanded for use in in vitro and in vivo experiments. Comprehensive cellular assays were performed to determine the presentation mechanism of TEDbody-mediated CMV peptide-MHC-I complex (CMV-pMHCI) on the surface of target tumor cells and the recognition and lysis by CMV-specific CTLs. In vivo CMV-pMHCI presentation and antitumor efficacy of TEDbody were evaluated in immunodeficient mice bearing human tumors. Results TEDbody delivered the fused epitope peptides into target tumor cells to be intracellularly processed and surface displayed in the form of CMV-pMHCI, leading to disguise target tumor cells as virally infected cells for recognition and lysis by CMV-specific CTLs. When systemically injected into tumor-bearing immunodeficient mice, TEDbody efficiently marked tumor cells with CMV-pMHCI to augment the proliferation and cytotoxic property of tumor-infiltrated CMV-specific CTLs, resulting in significant inhibition of the in vivo tumor growth by redirecting adoptively transferred CMV-specific CTLs. Further, combination of TEDbody with anti-OX40 agonistic antibody substantially enhanced the in vivo antitumor activity. Conclusion Our study offers an effective technology for MHC-I antigen cytosolic delivery. TEDbody may thus have utility as a therapeutic cancer vaccine to redirect pre-existing anti-viral CTLs arising from previously exposed viral infections to attack tumors.

Improved intratumoral penetration of IL12 immunocytokine enhances the antitumor efficacy

“…To produce HCT-mono-mIL12, three plasmids encoding the two different HC variants [VH-EW HC(γ1/4) CH3A -p40 and VH-RVT HC(γ1/4) CH3B -p35] and one of three LC variants for HCT/0.5 (HCT/46, HCT/130, or HCT/217) were transiently co-transfected at an equivalent molar ratio into cultured HEK293F cells in FreeStyle 293F medium (Invitrogen) following the standard protocol ( 19 , 25 ). HCT-mono-mIL12 was purified from the culture supernatants after 6 to 7 d on a protein A–agarose chromatographic column (GE Healthcare) and extensively dialyzed to switch the solution to Dulbecco’s phosphate-buffered saline (PBS) buffer (2.67 mM KCl, 1.47 mM (KH 2 PO 4 ), 137 mM NaCl, 8.1 mM Na 2 HPO 4 , pH 7.4).…”

“…Before cell treatment, the purified proteins were sterilized using a cellulose acetate membrane filter (0.22 μm; Corning) and Mustang Q membrane filter (0.8 μm; Pall, MSTG25Q6). Protein concentration was determined with the bicinchoninic acid kit (Thermo Fisher Scientific) and by measuring absorbance at 280 nm using the molar extinction coefficient calculated from the primary sequence ( 25 ). To determine the size and assembly pattern, size-exclusion chromatography (SEC) analysis of purified proteins was performed on the Agilent 1100 high-performance liquid chromatography system with a Superdex 200 10/300 GC column (10 mm × 300 mm, GE Healthcare) ( 19 , 27 ).…”

“…Peripheral blood mononuclear cells (PBMCs) from healthy donors were acquired using protocols approved by the Institutional Review Board of Ajou University (approval ID: 201602-HM-001-01) ( 25 ). All donors provided written informed consent before blood collection into a BD Vacutainer (BD Biosciences, 367874).…”

“…All donors provided written informed consent before blood collection into a BD Vacutainer (BD Biosciences, 367874). PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare, 17-5442-03) density-gradient centrifugation ( 25 ). For long-term storage, PBMCs were resuspended with 10% dimethyl sulfoxide in FBS and stored in liquid nitrogen at 1–5 × 10 6 cells/mL ( 28 ).…”

The Stroma and Tumor Microenvironment Are Not the Most Instrumental Players in Tumor Cells Immune Evasion, Growth, and Resistance to Therapy

“…Future directions should mine such associations, which have now been documented, 111,112 and exploited with advantage. 113 Of importance too, is to examine in depth the metabolic changes that render the cell, and especially the cell surface membrane deaf to contact inhibition signals, and unable to properly display MHC class I molecules, Fas ligand and other activators and targets of cytotoxic immune cells. 4,102 Very recently, abundant cholesterol sulfate-producing cancer cells were found to exhibit remarkable resistance to cancer-specific T-cell transfer and immunotherapeutic interventions.…”