23rd Human Proteome Organization World Congress

Prof

The biology of antigen processing revealed through the lens of immunopeptidomics

Keynote Lecture

Human Leukocyte antigen (HLA) molecules play a key role in health and disease by presenting antigen to T-lymphocytes for immunosurveillance. Immunopeptidomics involves the study of the collection of peptides presented within the antigen binding groove of HLA molecules. Identifying their nature and diversity is crucial to understanding immunosurveillance especially during infection or for the recognition and potential eradication of tumours. I will discuss new insights into the fidelity of antigen processing and presentation and how novel informatics approaches and databases have shed light on the diversity of peptide antigens derived from unconventional sources including peptides derived from transcripts associated with frame shifts, long noncoding RNA, incorrectly annotated untranslated regions, post translational modifications and proteasomal splicing. Several challenges remain in successful analysis of immunopeptides, yet these developments point to unexplored biology waiting to be unravelled.

Keynote lecture

Immunopeptidomics-assisted determination of TCR-like antibody specificities

Keynote speaker

CAN-IMMUNE: a comprehensive platform for cancer-specific immunopeptidomics-based neoantigen mapping and evaluation

Immunopeptidomics

Neoantigens are formed due to the somatic mutations that arise during carcinogenesis; they give rise to unique neopeptides bearing cancer-specific mutations that are presented on the surface of the tumour in complex with human leukocyte antigen (HLA) molecules for scrutiny by T cell recognition. A subset of these neopeptides can be recognised by tumoricidal T cells. As such, the neoepitopes derived from these neoantigens can serve as targets for cancer vaccines and antigen-based immunotherapies. Liquid Chromatography-Mass Spectrometry (LC-MS) is the technology of choice for identifying HLA-presented peptides, generally through spectral matching to inferred peptide sequences generated from a reference proteome database. However, the reference proteome database only contains canonical wild-type protein/peptide sequences and lacks cancer-specific mutations, hindering the identification of cancer-specific neoantigens using conventional database searching approaches. To tackle this challenge, we developed a comprehensive platform, CAN-IMMUNE, for MS-based identification of cancer-specific neopeptides. To construct a cancer-specific mutant peptide library, we collected missense mutations identified by whole exome sequencing (WES) from various sources, including the Catalogue of Somatic Mutations in Cancer (COSMIC), the Cancer Cell Line Encyclopedia (CCLE), and published literature. To date, CAN-IMMUNE documents 854,813 mutant peptides across 33 cancer types from 1428 cancer cell lines and 52 tumour tissues. CAN-IMMUNE allows users to browse, search, and download bespoke mutant peptide libraries for their cancer of interest. In addition, CAN-IMMUNE provides customisable source codes for users to generate their mutant peptide libraries using WES data. To assess the utility of CAN-IMMUNE, we searched our raw spectra data of MDA-MB-231 (a triple-negative breast cancer cell line), HCT116 (a colorectal cancer cell line), and various melanoma cell lines using both PEAKS 11 Online and deep learning-rescoring-assisted MSFragger database search engines. The results demonstrate that our constructed databases successfully identified potential cancer-specific neoantigens that cannot be found using the canonical proteome library, providing promising neoantigen candidates for further immunogenicity assessment. We anticipate that CAN-IMMUNE can accelerate the identification of cancer neoantigens via its WES-based mutant peptide libraries, thereby better informing the development of targeted cancer immunotherapies.

Oral presentation

Prof. Dr.

PD, Dr.

The proteomic and immunopeptidomic landscape of non-small cell lung cancer

Introduction: Non-small cell lung carcinoma (NSCLC) is the most common type of lung cancer, with adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being the most prevailing histological subtypes with distinct biological signatures. Correct classification and accurate molecular profiling are essential for effective therapeutic guidance. In this research, we performed an in-depth mass spectrometry (MS)-based proteomic study of a large patient cohort representing the two aforementioned subtypes of NSCLC. This MS-based profiling of LUAD and LUSC revealed proteomic alterations that might determine their clinical behavior. High-coverage proteome analysis opens the possibility to unravel the mechanisms of immune evasion in NSCLC. Moreover, additional immunopeptidomics study should elucidate distinct tumor-associated antigens, that are pivotal for the development of epitope-specific cancer immunotherapies. Methods: In our work, the patient-derived samples were processed with an optimized, semi-automated single-pot, solid-phase enhanced sample preparation (SP3-beads) workflow utilizing a Bravo pipetting platform. Thereby, extracted proteins were digested and the resulting peptides were analyzed via liquid-chromatography tandem-mass spectrometry (LC-MS/MS).For the immunopeptidome study, we established and optimized a workflow to enrich HLA-I and HLA-II-bound antigens. This workflow involves automated affinity purification with crosslinked monoclonal antibodies, performed on a AssayMAP Bravo platform. This adaptable method enables processing of fresh frozen tissue from biopsies, among a variety of sample types.Results: The MS-analysis of the patient cohort (consisting of LUAD (n = 83), LUSC (n = 70), and tumor-adjacent (n = 138)) resulted in high proteome coverage of a total of over 10000 unique proteins. Initial statistical analysis shows significant differences between LUAD and LUSC and suggests a possible distinction based on patient's gender.In our pilot study of the immunopeptidome, we successfully identified &gt; 4000 peptides after HLA class I enrichment and &gt; 7500 peptides after HLA class II enrichment. As for the patient cohort, the initial analysis of HLA-I enriched samples revealed a total of 40k unique peptides acquired in data independet acquisition. This coverage enables a robust analysis of the immunopeptidome, providing valuable insights into antigen presentation. Furthermore, a de novo sequencing analysis of the HLA-I peptides was performed.Outlook: Deep proteomic coverage of a large patient cohort enables us to gain deeper insight into the fundamental molecular biology of LUAD and LUSC. The further immunopeptidomic investigation gives a promising outlook to reveal a subset of tumor-associated antigens unique to each NSCLC subtype, offering precise targets for epitope-specific cancer immunotherapies. This study should facilitate the development of personalized immunotherapies tailored to each patient's tumor profile.

Professor

Immunopeptidomics shows how deleterious knockouts in the HLA class I antigen processing and presentation machinery induce distinct changes in the antigen landscape

Antigen presentation is an indispensable trigger for T-cell-based immune responses where T-cell receptors recognize HLA-peptide complexes on the cell surface. In the case of HLA class I, cells constantly sample the cytoplasmic proteome for short peptides to present them as antigens. For antigen presentation, an entire network of proteins act in concert to uphold a molecular machinery that delivers a flow of HLA-peptide complexes to the cell surface to potentially bind T-cell receptors.Functionally impairing mutations of the antigen processing and presentation machinery (APPM) are a frequently observed immune evasion mechanism in cancer. Our aim is to understand how deleterious knock out of key players in the APPM affects the antigenic landscape, and, consequentially, potential to stimulate T-cells.HAP1 is a near-haploid leukemia-derived cell line model expressing highly frequent HLA class I alleles among the European population, and, as such, a suitable model to generate and study the immunopeptidome of APPM knock-outs. In total, a series of 11 knock-outs have been previously generated (IRF2, B2M, GANAB, CALR, CALX, TAPBP, PDIA3, ERAP1, TAP1, TAP2, SPPL3), including genes involved in modifying, chaperoning, translocating, and loading the HLA molecules with peptides.In order to study antigen presentation, we investigated each knock-out&quot;s immunopeptidome with our high-throughput 96-well plate-based immunopeptide enrichment workflow, in triplicates and we further measured the respective proteomes. Data was acquired in DDA and DIA on a HF-X Q Exactive. With help of the FragPipe search engine environment and using chimeric spectra to find PSMs with a database search, we accumulated a library of more than 22&quot;000 unique immunopeptides, mapping to more than 8&quot;000 unique proteins, for a subsequent library search of the DIA data (FDR 1%).Our data shows varying levels of impact on the overall ability to present antigens, ranging from complete elimination of antigen presentation capability, like in the case of B2M, to sampling depths resembling those of the wildtype controls as in CALR. We determined knock-out specific effects that are directly linked to the biological function, e.g., knocking-out ERAP shifted the peptide length distribution to longer peptides. In addition, due to the quantitative quality of DIA library searches, we also determined an HLA allele-dependent shift in antigen presentation levels in different knock-outs, directly linking the dependencies of HLA-alleles on APPM components regarding the regulation of their presentation. Proteomics data provided insights on the efficiency of sampling source proteins for presentation.We demonstrate that impairments in the APPM result in distinct features in the antigenic landscape. This research enhances our understanding of the roles played by various APPM actors in shaping the immunopeptidome and could lead to improved HLA binding predictions that account for defects in the machinery.

Conference room 5-6

Session

Immunobiology

Session
Immunobiology

Termin

Vorsitzende

Programm

SessionImmunobiology

Termin

Vorsitzende

Programm

Session
Immunobiology