Immunoprecipitation methods impact the peptide repertoire in immunopeptidomics

IntroductionMass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for characterizing novel tumor antigens for immunotherapeutic approaches. In recent years, improvements based on devices and methodology have been made to optimize sensitivity and throughput in immunopeptidomics. However, developments in ligand isolation, mass spectrometric analysis, and subsequent data processing can have a marked impact on the quality and quantity of immunopeptidomics data.MethodsIn this work, we compared the immunopeptidome composition in terms of peptide yields, spectra quality, hydrophobicity, retention time, and immunogenicity of two established immunoprecipitation methods (column-based and 96-well-based) using cell lines as well as primary solid and hematological tumor samples.ResultsAlthough, we identified comparable overall peptide yields, large proportions of method-exclusive peptides were detected with significantly higher hydrophobicity for the column-based method with potential implications for the identification of immunogenic tumor antigens. We showed that column preparation does not lose hydrophilic peptides in the hydrophilic washing step. In contrast, an additional 50% acetonitrile elution could partially regain lost hydrophobic peptides during 96-well preparation, suggesting a reduction of the bias towards the column-based method but not completely equalizing it.DiscussionTogether, this work showed how different immunoprecipitation methods and their adaptions can impact the peptide repertoire of immunopeptidomic analysis and therefore the identification of potential tumor-associated antigens

HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy

Genome sequencing has uncovered an array of recurring somatic mutations in different non-Hodgkin lymphoma (NHL) subtypes. If affecting protein-coding regions, such mutations may yield mutation-derived peptides that may be presented by HLA class I proteins and recognized by cytotoxic T cells. A recurring somatic and oncogenic driver mutation of the Toll-like receptor adaptor protein MYD88, Leu265Pro (L265P) was identified in up to 90% of different NHL subtype patients. We therefore screened the potential of MYD88(L265P)-derived peptides to elicit cytotoxic T cell responses as tumor-specific neoantigens. Based on in silico predictions, we identified potential MYD88(L265P)-containing HLA ligands for several HLA class I restrictions. A set of HLA class I MYD88(L265P)-derived ligands elicited specific cytotoxic T cell responses for HLA-B*07 and -B*15. These data highlight the potential of MYD88(L265P) mutation-specific peptide-based immunotherapy as a novel personalized treatment approach for patients with MYD88(L265P+) NHLs that may complement pharmacological approaches targeting oncogenic MyD88 L265P signaling

Immunopeptidomik - Entwicklung therapeutischer Vakzinen zur Behandlung von Leukämien

Dissertation ist gesperrt bis 07. Februar 2023 !With the development and clinical success of immune checkpoint inhibitors, T-cell-based immunotherapy has been revolutionized. Therefore, in recent years special emphasis was placed on the identification of the target structures of the anti-tumor T-cell responses. These target structures are represented by tumor-associated or tumor-specific antigens presented on the surface of tumor cells via human leukocyte antigen (HLA) molecules. Further, the tremendous evolving field of mass spectrometry enables nowadays the direct identification of naturally presented HLA ligands from primary human samples in undreamt depth. This thesis aims to provide an insight into the rapidly developing field of mass spectrometry-based immunopeptidomics and the development of T cell based immunotherapeutic approaches for leukemia patients. For chronic lymphocytic leukemia (CLL) we performed a longitudinal study on the influence and effect of lenalidomide on the immunopeptidome of primary CLL cells. Mass spectrometry-based profiling identified only minor effects on HLA-restricted peptide presentation and confirmed stable presentation of previously described CLL-associated antigens under lenalidomide treatment. Therefore, lenalidomide was validated as suitable combination partner for tailored T-cell-based immunotherapeutic approaches in CLL patients. For chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), we applied a mass spectrometry-based approach to identify naturally presented, HLA-restricted leukemia-associated peptides, respectively. Therefore, we utilized a comparative profiling approach using a comprehensive dataset of different benign tissues. Functional characterization revealed spontaneous T-cell responses against the novel identified CML- and AML-associated peptides in patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells. These antigens are thus prime candidates for T cell-based immunotherapeutic approaches for CML and AML patients