Cancer testis antigen Cyclin A1 harbors several HLA-A*02:01-restricted T cell epitopes, which are presented and recognized in vivo

Abstract

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8⁺ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410-420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application.

Keywords: Acute myeloid leukemia; CD8+ T cell; Cyclin A1; Epitope; HLA immunopeptidome; Ovarian carcinoma.

Fig. 1

Selection of peptide candidates: a In silico prediction and epitope selection, *13 candidates include pre-described epitope p227–235, b HLA-A*02:01 stabilization assay, gating on HLA-A*02:01 high cells, positive (p227–235) and negative (no peptide) control, c HLA-A*02:01 stabilization assay: percentage of HLA-A*02:01 high cells

Fig. 2

Generation of specific T cell lines in vitro: a Highest reached frequencies of specific T cells quantified by IFNγ ICS after three (*) or four stimulations with DC or DC followed by PBMC. The successful clone source is marked by an arrow. b Vital FR toxicity assay normalization panel without effector cells (left) and samples with effector CTL (middle, right) against Cyclin A1 p289–297 and target THP-1 cells with and control THP-1 cells without IFNγ exposure. c Specific lysis calculated from three different Vital FR settings for target pairs: T2 peptide-pulsed with Cyclin A1 p289–297 as target versus T2 with irrelevant peptide as control (gray), THP1 with and without IFNγ exposure (black) and THP1 versus T2 both with IFNγ exposure (white)

Fig. 3

Spontaneous T cell responses against Cyclin A1 epitopes in treatment-naïve OC patients: a Example of intracellular TNFα staining of CD8-positive cells of patient OC69. b Specific cytokine production of all analyzed patients. Values are given as specific cells per unspecific cells