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

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 (CDK2_167–175: WYRQPEILL) and CDK4 (CDK4_179–187: WYRQPEVLL), CDK2_167–175 and CDK4_179–187 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 CDK2_167–175- and CDK4_179–187-stimulated T cells from 3 of 5 individuals recognized each peptide specifically. ⁵¹ Cr-release assay showed that CDK2_167–175-stimulated T cells killed CDK2_167–175-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). CDK4_179–187-stimulated T cells also preferentially killed CDK4_179–187-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 CDK2_167–175-stimulated CTL line and 7.3% of the CDK4_179–187-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 CDK2_167–175 and CDK4_179–187 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 CDK2_167–175 and CDK4_179–187 peptides may be a promising approach to inducing leukemia-specific CTLs from donor-derived T cells in recipients of allogeneic stem cell transplantation.