MLAA-34: A New Leukemia-Associated Antigen for Immunotherapy Against Acute Monocytic Leukemia

Introduction: Relapse after chemotherapy is inevitable in the majority of acute myeloid leukemia patients without allogeneic transplantation. Thus, it is necessary to develop novel therapies that have different antileukemic mechanisms. Recent advances in immunology and identification of promising leukemia-associated antigens open the possibilities for eradicating minimal residual diseases by antigen-specific immunotherapy after chemotherapy.

Identification of CTL epitopes that can induce CD8⁺ T cells to kill tumor cells is the fundamental step for development of cancer peptide vaccines. Our previous research showed that monocytic leukemia associated antigen-34(MLAA-34) was an anti-apoptotic molecule and overexpressed in patients with acute monocytic leukemia (M5), indicating MLAA-34 was a promising antigen for immunotherapy of M5. HLA-A*0201 is the main HLA class-I antigen in Chinese population. Therefore, we focused on identifying HLA-A*0201-restricted CTL epitopes of MLAA-34.

Methods: MLAA-34 HLA-A*0201 restricted CTL epitopes were screened by bioinformatics prediction. The actual affinity of predicted peptides to HLA-A*0201 molecules was evaluated by T2 cell surface HLA class I stabilization assay. Mature DCs were induced and cultured from HLA-A*0201⁺ PBMCs using recombinant GM-CSF, IL-4 and TNF-α. High-purity CD8⁺ T cells were selected from non-adherent cells of HLA-A*0201⁺ PBMCs by magnetic activated cell sorting. CTLs were induced by three stimulations with DCs pulsed with the MLAA-34-derived peptides. CTL activity was tested using IFN-γ ELISPOT assay and LDH release assay. Humanized immunity was reconstructed in SCID mice by HLA-A*0201 human peripheral blood lymphocytes (hu-PBL). Human acute monocytic leukemia cell THP-1-bearing hu-PBL-SCID mouse model was used for in vivo assay. MLAA-34 peptide vaccine was composed of MLAA-34 epitope CP701 (²³⁶ILDRHNFAI²⁴⁴), Th epitope (⁸³⁰QYIKANSKFIGITE⁸⁴³) and incomplete adjuvant. Different vaccines were applied to leukemia bearing hu-PBL-SCID mice to observe the tumor size, survival of mice and to determine the antileukemic mechanisms and effects of vaccines.

Results: 10 HLA-A*0201 restricted epitopes of MLAA-34 were predicted and synthesized. CP693 (²⁹³ILLKNQPKL³⁰¹), CP700 (³²⁴YLIKQIRDL³³²) and CP701 (²³⁶ILDRHNFAI²⁴⁴) showed the strongest HLA-A*0201 binding affinity. IFN-γ ELISPOT assay showed that peptides CP701, CP700 and positive peptide CML28 (173-181)-specific CTLs produced a higher amount of IFN-γ than CTLs stimulated with peptide CP693 and negative control. Comparing with peptide CP700 and CP 693-induced CTLs, peptide CP701-induced CTLs presented the stronger cytotoxic effect on THP-1 cells (HLA-A2⁺MLAA34⁺) and T2 cells pulsed with peptide CP701 at various effector to target ratios. The CP701-induced CTLs had significant higher specific lysis on THP-1 cells (HLA-A2⁺MLAA34⁺) than that of human monocyte leukemia cell line U937 (HLA-A2^-MLAA-34⁺), human breast cancer cells MCF-7 (HLA-A2⁺MLAA-34^-) and human lung adenocarcinoma cell A549 (HLA-A2^- MLAA-34^-). Leukemia bearing success rate of hu-PBL-SCID mice was 100%. MLAA-34 peptide vaccine could inhibit the tumor growth in leukemia-bearing mice. The mean survival time of tumor-bearing mice in MLAA-34 peptide vaccine group was significantly longer than that of WT1 peptide group, inactivated THP-1 cells group, Th peptide group and incomplete Freund's adjuvant group. At different effector to target ratios, killing efficacy of CTLs and NK cells derived from MLAA-34 peptide vaccinated group were significantly higher than other therapeutic groups. CTLs and NK cell derived from MLAA-34 peptide showed specific cytotoxicity to THP-1 cells (HLA-A2⁺MLAA34⁺), but not to U937 (HLA-A2^-MLAA-34⁺), MCF-7 (HLA-A2⁺ MLAA-34^-) or A549 (HLA-A2^-MLAA-34^-). Comparing with other therapeutic groups, there were significant higher percentage of CD3⁺CD8⁺ T cells and lower percentage of Treg cells in MLAA-34 peptide vaccine group. Furthermore, MLAA-34 peptide-stimulated T cells showed increased IFN-γ and IL-2 and decreased IL-10 and IL-4 expression compared to controls.

Conclusions: MLAA-34 peptide CP701 (²³⁶ILDRHNFAI²⁴⁴) is an effective HLA-A*0201-restricted CTL epitope and that it may serve as a promising strategy in designing antigen-specific immunotherapy against MLAA-34-positive leukemia.