Checkpoint inhibitors in cancer immunotherapy: Cross reactivity of a CTLA-4 antibody and IDO-inhibitor L-1MT in pigs

Blockade of checkpoint inhibitors has recently shown very convincing results in the treatment of cancer. One key target is CTLA-4, which has been demonstrated to be a potent negative regulator of lymphocyte activation. The treatment with the FDA-approved fully human CTLA-4 monoclonal antibody Ipilimumab increases anticancer T-cell reactivity and overall survival of metastatic cancer patients. Indole-amine 2,3-dioxygenase (IDO) is another checkpoint inhibitor which suppresses T-cell immunity by the depletion of tryptophan in the T-cell microenvironment, and also inhibition of IDO by L-1-Methyltryptophan (L-1MT) has shown promising results in clinical phase I/II studies of human cancer such as epithelial ovarian cancer.Pre-clinical immune therapeutic studies are usually performed with mice, but Ipilimumab is not reactive with mouse cells. Recent studies indicate that the pig may be a more suitable animal model for studies of immune reactivity due to higher similarity of the immunome between pig and man. This study is part of the CANVACPIG project “Accelerating development of vaccines against cancer with pigs as a large animal model” and investigates the reactivity of a fully human monoclonal anti CTLA-4 antibody and L-1MT on porcine immune cells.At the genome level, the homology between human and pig CTLA-4 and IDO is 86% and 73%, respectively, while the homology to the mouse is 75% and 63%. Our preliminary in vitro studies indicate that the monoclonal anti CTLA-4 antibody induces a non-specific activation of porcine T cells. This will be further investigated to provide the basis for in vivo studies investigating checkpoint inhibitor blockade in combination with other cancer immunotherapies. Eventually our goal is to establish pigs as an alternative large animal model for development and formulation of new human cancer vaccines.<br/

Broadening the repertoire of melanoma-associated T-cell epitopes

Immune therapy has provided a significant breakthrough in the treatment of metastatic melanoma. Despite the remarkable clinical efficacy and established involvement of effector CD8 T cells, the knowledge of the exact peptide-MHC complexes recognized by T cells on the tumor cell surface is limited. Many melanoma-associated T-cell epitopes have been described, but this knowledge remains largely restricted to HLA-A2, and we lack understanding of the T-cell recognition in the context of other HLA molecules. We selected six melanoma-associated antigens (MAGE-A3, NY-ESO-1, gp100, Mart1, tyrosinase and TRP-2) that are frequently recognized in patients with the aim of identifying novel T-cell epitopes restricted to HLA-A1, -A3, -A11 and -B7. Using in silico prediction and in vitro confirmation, we identified 127 MHC ligands and analyzed the T-cell responses against these ligands via the MHC multimer-based enrichment of peripheral blood from 39 melanoma patients and 10 healthy donors. To dissect the T-cell reactivity against this large peptide library, we used combinatorial-encoded MHC multimers and observed the T-cell responses against 17 different peptide-MHC complexes in the patient group and four in the healthy donor group. We confirmed the processing and presentation of HLA-A3-restricted T-cell epitopes from tyrosinase (TQYESGSMDK) and gp100 (LIYRRRLMK) and an HLA-A11-restricted T-cell epitope from gp100 (AVGATKVPR) via the cytolytic T-cell recognition of melanoma cell lines and/or K562 cells expressing the appropriate antigen and HLA molecule. We further found T-cell reactivity against two of the identified sequences among tumor-infiltrating lymphocytes from melanoma patients, suggesting a potential clinical relevance of these sequences. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00262-015-1664-x) contains supplementary material, which is available to authorized users

Characterization of T-cell responses against IκBα in cancer patients

The nuclear factor κ light chain enhancer of activated B cells (NFκB) is constitutively active in most cancers, controlling multiple cellular processes including proliferation, invasion and resistance to therapy. NFκB is primarily regulated through the association with inhibitory proteins that are known as inhibitors of NFκB (IκBs). Increased NFκB activity in tumor cells has been correlated with decrease stability of IκB proteins, in particular IκBα. In responso to a large number of stimuli, IκB proteins are degraded by the proteasome. Cytotoxic T lymphocytes (CTLs) recognize HLA-restricted antigenic peptides that are generated by proteasomal degradation in target cells. In the present study, we demonstrate the presence of naturally occurring IκBα -specific T cells in the peripheral blood of patients suffering from several unrelated tumor types, i.e., breast cancer, malignant melanoma and renal cell carcinoma, but not of healthy controls. Furthermore, we show that such IBα-specific T cells are granzyme B-releasing, cytotoxic cells. Hence, the increased proteasomal degradation of IκBα in cancer induces IκBα-specific CTLs