HLA Ligandome Analysis of Acute Myeloid Leukemia (AML) Revealed Novel Tumor-Associated Antigens for Immunotherapy.
Authors: Stickel Juliane Sarah , Berlin Claudia , Kowalewski Daniel Johannes , Schuster Heiko , Kanz Lothar , Salih Helmut R , Rammensee Hans-Georg , Stevanovic Stefan 
Published: October 2019

Abstract
Abstract 3012

Introduction:
Eradication of minimal residual disease (MRD) by antigen-specific immunotherapy after conventional chemotherapy and also after allogeneic stem cell transplantation may improve overall survival in acute myeloid leukemia (AML). To achieve this goal there is a need for novel leukemia-associated HLA-presented peptides, which are able to induce a tumor-specific T cell response. Accordingly we here aimed to identify novel leukemia associated antigens employing, for the first time, the approach of direct elution and analysis of naturally processed and presented HLA ligands from the surface of primary AML cells. In particular we focused on peptides derived from AML specific mutated proteins, which are produced only by leukemia cells, only.
Methods: HLA class I ligands were isolated from MACS-purified PBMCs of AML patients using immunoprecipitation. Liquid chromatography tandem mass spectrometry (LC-MS/MS) based peptide sequencing was performed to identify HLA presented peptides. The obtained data were mined for leukemia associated peptides by comparison of HLA ligandomes of AML cells with that of PMBCs and granulocytes from healthy donors, investigation of gene expression databases and literature research. All acquired peptide sequences were blasted against the COSMIC database (http://www.sanger.ac.uk/genetics/CGP/cosmic/), to identify sequences from AML specific mutated proteins. Identified mutations were confirmed by PCR. In addition, HLA quantification experiments on the cell surface of AML cells and autologous healthy monocytes and granulocytes were performed using a flow cytometric indirect immunofluorescence assay.

Results:
Quantitative preliminary results showed similar amounts of HLA class I molecules on AML cells as compared to autologous healthy monocytes and granulocytes lymphocytes (p=0.56, unpaired t-test). For HLA class II we could show a clear upregulation on AML cells compared to autologous healthy leukocytes, not reaching level of significance due to the small cohort examined till now (p= 0.12, unpaired t-test).
We were able to identify a total of more than 10.000 peptides from 6 AML patients, including 2 NPM1 mutated AMLs, 1 FLT3-ITD mutated AML, 1 JAK2 mutated AML from osteomyelofibrosis and two AMLs from myelodysplastic syndrome without any cytogenetical aberrations. Using a new approach of spectral counting the obtained sequences could be compared semi-quantitatively to the same amount of peptides obtained from HLA matched healthy volunteers. Thus several new naturally processed and presented leukemia-associated peptides (LEUMAPs) were identified. This comprised, amongst others, ligands of p53, c-myc oncogene, PRAME, RHAMM and NPM1.
The sequences of identified LEUMAPs were all verified by LC-MS/MS-based peptide sequencing of their synthetic counterparts. Testing for immunogenicity by ELISPOTs-assay and an in vitro T cell priming approach is presently ongoing.

Conclusions:
Our analysis revealed several new leukemia associated antigens, which were, for the first time, directly obtained from the HLA ligandomes of AML patients. Quantitative HLA analysis showed no loss or downregulation of HLA molecules on the surface of AML cells, thereby excluding a potential major obstacle for successful immunotherapy. Our results may pave the way for future peptide based immunotherapy approaches to eradicate MRD after obtaining a remission by presently available conventional therapies in AML patients.

Disclosures:
No relevant conflicts of interest to declare.