Identification of Naturally Presented HLA Ligands of Enriched Leukemic Progenitor Cells for Peptide-Based Immunotherapy in Acute Myeloid Leukemia (AML)
Authors: Nelde Annika , Schuster Heiko , Kowalewski Daniel J. , Kanz Lothar , Salih Helmut R. , Rammensee Hans-Georg , Stevanovic Stefan , Stickel Juliane S. 
Published: October 2019

Abstract
Several studies demonstrated that peptide-based cancer immunotherapy can induce specific immune responses and affect clinical outcome in a variety of different cancer entities. We recently conducted a study, which directly characterized the antigenic landscape of acute myeloid leukemia (AML) by mass spectrometric analysis of naturally presented HLA ligands and identified a panel of AML-specific CD4+ as well as CD8+ T-cell epitopes as suitable targets for T-cell based immunotherapy (Berlin et al. Leukemia 2015). One main reason for the high relapse rates in AML patients after standard polychemotherapy and allogeneic stem cell transplantation is the presence of minimal residual disease (MRD), which is associated with the persistence of leukemic stem cells (LSCs) in the bone marrow of patients. For clinically effective immunotherapy it is therefore indispensable to target the highly chemotherapy resistant LSCs.
Here we present a mass spectrometry-based study, which for the first time analyzes the naturally presented HLA ligandome of stem cell enriched (LSCenr) fractions of primary AML samples to identify novel LSC-associated antigens using the approach of direct peptide isolation and identification. The enrichment of LSCs was performed using fluorescence-activated cell sorting of the originally described phenotype of lineage-negative CD34+CD38- cells of PBMCs from eight AML patients. The original stem cell containing population of 1-3% within the PBMCs of most patients was enriched to >90% purity with cell counts of 20-200x106 for the LSCenr fraction per sample. Consistent with our own previous results, all samples showed comparable expression levels of HLA class I molecules on primary leukemia blasts as well as for the LSCenr fractions, with HLA class I molecule counts ranging from 145,000 to 175,000 molecules/cell for the LSCenr fractions.
To specifically identify leukemia-associated antigens on LSCenr cells, the HLA ligandome results obtained from the sorted LSCenrfractions were combined with data acquired from AML blasts of 20 AML patients (HLA class I n=19, HLA class II n=20) in previous studies as well as our normal tissue database that comprises 153 HLA class I and 82 HLA class II ligandomes of various healthy tissues (e.g. blood, bone marrow, spleen, kidney, liver, brain, skin, ovary, bowl).
We identified more than 14,600 different naturally presented HLA class I ligands representing ̴6,500 source proteins on LSCenr fractions of primary AML samples (n=8) and their autologous blast cells by mass spectrometry. Overlap analysis of the HLA class I ligandomes of LSCenr fractions and autologous AML blasts with the benign peptidome revealed 45.4% (3,132/6,896) and 40.2% (4,922/12,244) of the LSCenr fraction and the autologous AML blast ligandomes to be represented in the benign-associated HLA ligandome, respectively. 79.1% (5,458/6,896) of the mapped LSCenr fraction ligandome was also presented on autologous AML blasts. 1,029 (14.9%) of these identified HLA class I ligands were presented exclusively on LSCenr fractions and not found on autologous AML blasts, previously analyzed AML blasts or any benign tissue.
Furthermore, we were able to identify more than 8,000 different naturally presented HLA class II ligands representing ̴1,700 source proteins. Overlap of the HLA class II ligandomes revealed 45.0% (2,800/4,624) and 39.9% (2,706/6,790) of the LSCenr fraction and autologous AML blast ligandomes to be represented in the benign-associated HLA ligands, respectively. The HLA ligandomes of the LSCenr fraction and the autologous AML blasts showed an overlap of 69.7% (3,224/4,624). 941 (11.5%) HLA class II ligands showed exclusive representation in the LSCenr fraction ligandomes and were never identified on AML blast or benign tissue.
These LSC-associated peptides represent highly interesting targets for immunotherapeutic approaches in AML patients and will be further evaluated for their potential to elicit a specific T-cell response.
Taken together these preliminary results prove the feasibility of our approach to enrich leukemic progenitor cells of primary AML samples for the successful isolation and identification of HLA presented peptides associated with enriched leukemic progenitor cells.

Disclosures
Schuster: Immatics Biotechnologies GmbH: Employment. Kowalewski:Immatics Biotechnologies GmbH: Employment.