Two Cyclin-Dependent Kinase Derived Peptides Are Potential Leukemia-Associated-Antigens Able To Eradicate Acute Myeloid Leukemia Cells After Allogeneic Stem Cell Transplantation.

Abstract The success of PR1 vaccine trial for treatment of myeloid leukemia has demonstrated that aberrantly expressed self-antigens in leukemia cells serve as leukemia-associated-antigens (LAAs) of leukemia reactive T cells. We hypothesized that graft-versus-leukemia (GvL) could be separated from graft-versus-host disease (GvHD) if GvL targets such LAAs against which immune tolerance has broken down. Deregulation of the cell cycle machinery is a common finding in leukemia and proteins controlling the G1/S phase transition are often aberrantly expressed in leukemia cells. We previously identified heterologous Cyclin E1- and Cyclin E2-derived peptides as LAAs. We investigated whether the homologous proteins cyclin-dependent kinase 2 (CDK2) and cyclin-dependent kinase 4 (CDK4), which form a complex with Cyclin E and Cyclin D, respectively, are potential LAAs. We first examined expression of these two proteins in leukemia cell from 5 AML and 1 ALL patients using western blot. Three of 5 AML patient’s cells and the ALL patient’s cells aberrantly expressed both CDK2 and CDK4 proteins, 1 AML patient’s cells aberrantly expressed only CDK4 protein compared to peripheral blood mononuclear cells (PBMCs) from 2 healthy individuals. When stabilization of HLA-A24 was examined using HLA-A24-transfected T2 cells (A24-T2) and two deduced HLA-A24-restricted peptides from CDK2 (CDK2167–175: WYRQPEILL) and CDK4 (CDK4179–187: WYRQPEVLL), CDK2167–175 and CDK4179–187 increased the expression of HLA-A24 by 356.4% and 299.8% at 100μM peptide concentration, respectively. Naïve (>95% CD45RO− ) CD8+ T cells were enriched from PBMCs of HLA-A24+ healthy individuals using magnetic beads, and stimulated with each peptide-coated A24-T2 in the presence of low dose IL-2 for 3 weeks. Flow cytometry detecting cell-associated interferon-gamma (IFNg) and CD107a expression by T cells showed that CDK2167–175- and CDK4179–187-stimulated T cells from 3 of 5 individuals recognized each peptide specifically. 51 Cr-release assay showed that CDK2167–175-stimulated T cells killed CDK2167–175-coated A24-T2 more efficiently than non-peptide-coated A24-T2 (% specific lysis at an E/T ratio of 5; 26.6% vs. 10.5% specific lysis). CDK4179–187-stimulated T cells also preferentially killed CDK4179–187-coated A24-T2 compared to non-peptide-coated A24-T2 (13.7% vs. 4.1%). When peptide-specific T cells were generated in the same way from an HLA identical sibling donor of an AML patient, 2.6% of the CDK2167–175-stimulated CTL line and 7.3% of the CDK4179–187-stimulated CTL line secreted IFNg in response to stimulation by the patient’s AML cells which aberrantly express both CDK2 and CDK4 proteins. On the other hand, the frequency of IFNg+ CDK2-CTL and IFNg+ CDK4-CTL after stimulation by autologous EBV-LCL and the patient’s EBV-LCL were only 0.4%, 0.7%, 0.5% and 0.6% respectively. These findings suggests that CDK2167–175 and CDK4179–187 peptides are potential LAAs capable of inducing CTLs against leukemia cells from CD8+ T cells of an HLA-identical sibling donor whose naïve CD8+ cells retain high avidity to aberrantly expressed LAAs. Vaccination of CDK2167–175 and CDK4179–187 peptides may be a promising approach to inducing leukemia-specific CTLs from donor-derived T cells in recipients of allogeneic stem cell transplantation.