HLA Peptidomes Research Articles

Deciphering HLA-I motifs across HLA peptidomes improves neo-antigen predictions and identifies allostery regulating HLA specificity.

The precise identification of Human Leukocyte Antigen class I (HLA-I) binding motifs plays a central role in our ability to understand and predict (neo-)antigen presentation in infectious diseases and cancer. Here, by exploiting co-occurrence of HLA-I alleles across ten newly generated as well as forty public HLA peptidomics datasets comprising more than 115,000 unique peptides, we show that we can rapidly and accurately identify many HLA-I binding motifs and map them to their corresponding alleles without any a priori knowledge of HLA-I binding specificity. Our approach recapitulates and refines known motifs for 43 of the most frequent alleles, uncovers new motifs for 9 alleles that up to now had less than five known ligands and provides a scalable framework to incorporate additional HLA peptidomics studies in the future. The refined motifs improve neo-antigen and cancer testis antigen predictions, indicating that unbiased HLA peptidomics data are ideal for in silico predictions of neo-antigens from tumor exome sequencing data. The new motifs further reveal distant modulation of the binding specificity at P2 for some HLA-I alleles by residues in the HLA-I binding site but outside of the B-pocket and we unravel the underlying mechanisms by protein structure analysis, mutagenesis and in vitro binding assays.

A meta-analysis of HLA peptidome composition in different hematological entities: entity-specific dividing lines and "pan-leukemia" antigens.

Hematological malignancies (HM) are highly amenable targets for immunotherapeutic intervention and may be effectively treated by antigen-specific T-cell based treatment. Recent studies demonstrate that physiologically occurring anti-cancer T-cell responses in certain HM entities target broadly presented non-mutated epitopes. HLA ligands are thus implied as prime targets for broadly applicable and antigen-specific off-the-shelf compounds. With the aim of assessing the presence of common targets shared among different HM which may enable addressing a larger patient collective we conducted a meta-analysis of 83 mass spectrometry-based HLA peptidome datasets (comprising 40,361 unique peptide identifications) across four major HM (19 AML, 16 CML, 35 CLL, and 13 MM/MCL samples) and investigated similarities and differences within the HLA presented antigenic landscape. We found the cancer HLA peptidome datasets to cluster specifically along entity and lineage lines, suggesting that the immunopeptidome directly reflects the differences in the underlying (tumor-)biology. In line with these findings, we only detected a small set of entity-spanning antigens, which were predominantly characterized by low presentation frequencies within the different patient cohorts. These findings suggest that design of T-cell immunotherapies for the treatment of HM should ideally be conducted in an entity-specific fashion.

Purification of soluble HLA class I complexes from human serum or plasma deliver high quality immuno peptidomes required for biomarker discovery.

Soluble human leukocyte antigen class I (sHLA)-peptide complexes have been suggested to play a role in the modulation of immune responses and in immune evasion of cancer cells. The set of peptides eluted from sHLA molecules could serve as biomarker for the monitoring of patients with cancer or other conditions. Here, we describe an improved sHLA peptidomics methodology resulting in the identification of 1816 to 2761 unique peptide sequences from triplicate analyses of serum or plasma taken from three healthy donors. More than 90% of the identified peptides were 8-11mers and 74% of these sequences were predicted to bind to cognate HLA alleles, confirming the quality of the resulting immunopeptidomes. In comparison to the HLA peptidome of cultured cells, the plasma-derived peptides were predicted to have a higher stability in complex with the cognate HLA molecules and mainly derived from proteins of the plasma membrane or from the extracellular space. The sHLA peptidomes can efficiently be characterized by using the new methodology, thus serving as potential source of biomarkers in various pathological conditions.

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Image: Dendritic cells from Broz ML & Krummel MF. Cancer Immunol Res 2015; 3:313-19Cross-presentation of tumor antigens is key to the activation of T cells by dendritic cells. Siglec-G was found to interfere with peptide–MHC class I complex formation through inhibition of NOX2-mediated phagosome alkalinization, a condition conducive to optimal peptide loading.Ding Y,…, Cao X. Nature Immunol 2016 Oct;17:1167–75.Image: https://commons.wikimedia.org/wiki/File:Non-small_cell_lung_carcinoma_--_high_mag.jpgThe Keynote-024 trial was designed to compare the mAb pembrolizumab with chemotherapies as first-line treatments for NSCLC. Patients, preselected to have >50% PD-L1+ tumors, survived longer and with fewer serious adverse effects with pembrolizumab than chemotherapy.Reck M,…, Brahmer JR, M.D., for the KEYNOTE-024 Investigators. NEJM 2016 Oct 9; doi:10.1056/NEJMoa1606774.Image: Photo by David Dixon http://www.geograph.org.uk/photo/3492475Melanomas responsive to anti–CTLA-4 blockade are sensitive to IFNγ. IFNGR1-knockdown tumors in mice, and tumors from nonresponder patients, jump this hurdle by deleting or accumulating mutations in their IFNγ response pathways, which promotes tumor survival despite CTLA-4 blockade. This may represent a common mechanism for resistance to checkpoint blockade therapy.Gao J, …, Sharma P. Cell 2016 Oct 6;167:397–404.Image: https://ghr.nlm.nih.gov/art/large/squamous-cell-carcinoma.jpegPatients with HNSCC with tumors resistant to standard platinum-based treatment were randomized to receive anti-PD-1 (nivolumab) or another standard therapy. Nivolumab significantly prolonged survival, PD-L1 status notwithstanding, compared with standard therapies, and patients experienced fewer serious adverse effects.Ferris RL,…, Gillison ML. NEJM 2016 Oct 9; doi: 10.1056/NEJMoa1602252.Image: Türeci, Ö,…, Sahin U. Clin Can Res 2016; 22: 1885–96The proteasome cleaves proteins into peptides presented by class I on the cell surface. About a third of the HLA peptidome was found to consist of spliced-together peptides, creating new epitopes and a mechanism for presenting a broader spectrum of epitopes relevant to immunobiology and autoimmunity that are not accounted for in current prediction algorithms.Liepe J,…, Mishto M. Science 2016 Oct 21;354:354–8.Image: https://commons.wikimedia.org/wiki/File:Myeloblast_with_Auer_rod_smear_2010-01-27.JPGNK memory cells were tested as antitumor therapeutics in vitro and in vivo in a xenografted mouse model. These cells were effective cancer cell killers and also proved safe and promisingly effective in a small phase I clinical trial.Romee R,…, Fehniger TA. Science Transla Med 2016 Sept 21; 8:357ra123.

Unsupervised HLA Peptidome Deconvolution Improves Ligand Prediction Accuracy and Predicts Cooperative Effects in Peptide-HLA Interactions.

Ag presentation on HLA molecules plays a central role in infectious diseases and tumor immunology. To date, large-scale identification of (neo-)Ags from DNA sequencing data has mainly relied on predictions. In parallel, mass spectrometry analysis of HLA peptidome is increasingly performed to directly detect peptides presented on HLA molecules. In this study, we use a novel unsupervised approach to assign mass spectrometry-based HLA peptidomics data to their cognate HLA molecules. We show that incorporation of deconvoluted HLA peptidomics data in ligand prediction algorithms can improve their accuracy for HLA alleles with few ligands in existing databases. The results of our computational analysis of large datasets of naturally processed HLA peptides, together with experimental validation and protein structure analysis, further reveal how HLA-binding motifs change with peptide length and predict new cooperative effects between distant residues in HLA-B07:02 ligands.

Abstract 2654: GAPVAC-101 phase I trial: First data of an innovative actively personalized peptide vaccination trial in patients with newly diagnosed glioblastoma

Abstract The Glioma Actively Personalized Vaccine Consortium (GAPVAC; funded by the European Union Framework 7 Program) aims at treating newly diagnosed glioblastoma (GB) patients with two distinct actively personalized vaccines (APVACs). Resected tumor material is analyzed for multiple biomarkers to characterize the tumor in depth and to enable the design of APVACs tailored to each individual patient: Tumor-specific mutations, the HLA peptidome and gene expression profile are assessed by next-generation sequencing, mass spectrometry and RNA microarray analysis, respectively. Further, the patient-individual immune status is investigated by assessment of leukapheresis samples utilizing an in vitro immunogenicity platform. Data are integrated to define two distinct APVACs for each patient: APVAC1 is composed of up to ten peptides selected from a pre-manufactured “warehouse”. The warehouse contains 59 HLA class I-binding and three class II-binding tumor-associated peptides frequently over-presented in GB. APVAC2 is composed of one or two peptides that are de novo synthesized for a given patient and preferentially represent mutation-bearing neo-epitopes. After a preparation phase in which the warehouse was generated and setup of APVAC selection and manufacturing processes took place, the GAPVAC-101 phase I clinical trial was initiated. Primary endpoints of the study are assessment of safety, feasibility of APVAC manufacturing and biological activity. The trial is conducted at six European centers and recruits HLA-A*02:01 or A*24:02-positive patients with newly diagnosed GB after gross total resection. Patients receive APVAC1 and APVAC2 vaccinations plus immunomodulators (poly-ICLC and GM-CSF) three and six months post study enrolment, respectively, and concurrent to maintenance temozolomide (TMZ). As of November 2015, 11 patients have been enrolled, of whom six already received APVAC vaccines. Composition and manufacturing are ongoing for four patients. All APVACs were generated in time without ultimate failures. APVAC1 vaccines differ substantially with 31 out of 59 warehouse peptides have been selected at least once, indicating the need for personalization due to tumor heterogeneity even for non-mutated epitopes. In patients’ tumor samples an average of 40 non-synonymous mutations (including known driver mutations) were identified. Injection site reactions were the most frequent toxicities so far. One brain edema (Grade 3) and one allergic reaction (Grade 4)were observed, both potentially related to the vaccinations. First data on biological activity of APVACs and updated clinical data will be presented at the Annual Meeting. In conclusion, the GAPVAC concept has been successfully translated into the clinics and so far demonstrated to be safe and feasible with its level of personalization matching the observed tumor heterogeneity. Citation Format: Norbert Hilf, Katrin Frenzel, Sabrina Kuttruff-Coqui, Sandra Heesch, Sebastian Kreiter, Arie Admon, Valesca Bukur, Sjoerd van der Burg, Cecile Gouttefangeas, Judith R. Kroep, Marij Schoenmaekers-Welters, Jordi Piro, Berta Ponsati, Hans Skovgaard Poulsen, Ulrik Lassen, Francisco Martinez-Ricarte, Jordi Rodon, Juan Sahuquillo, Monika Stieglbauer, Stefan Stevanovic, Per thor Straten, Marco Skardelly, Ghazaleh Tabatabai, Michael Platten, David Capper, Andreas von Deimling, Valérie Dutoit, Hideho Okada, Christian Ottensmeier, Randi Kristina Feist, Jens Fritsche, Karoline Laske, Peter Lewandrowski, Martin Löwer, Regina Mendryzk, Miriam Meyer, Carsten Reinhardt, Bernhard Rössler, Anna Paruzynski, Nina Pawlowski, Colette Song, Stevermann Lea, Toni Weinschenk, Christoph Huber, Hans-Georg Rammensee, Pierre-Yves Dietrich, Wick Wolfgang, Ugur Sahin, Harpreet Singh-Jasuja. GAPVAC-101 phase I trial: First data of an innovative actively personalized peptide vaccination trial in patients with newly diagnosed glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2654.

A first dataset toward a standardized community-driven global mapping of the human immunopeptidome.

We present the first standardized HLA peptidomics dataset generated by the immunopeptidomics community. The dataset is composed of native HLA class I peptides as well as synthetic HLA class II peptides that were acquired in data-dependent acquisition mode using multiple types of mass spectrometers. All laboratories used the spiked-in landmark iRT peptides for retention time normalization and data analysis. The mass spectrometric data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001872. The generated data were used to build HLA allele-specific peptide spectral and assay libraries, which were stored in the SWATHAtlas database. Data presented here are described in more detail in the original eLife article entitled ‘An open-source computational and data resource to analyze digital maps of immunopeptidomes’.

An open-source computational and data resource to analyze digital maps of immunopeptidomes.

We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.

High-throughput characterization of the ovarian cancer HLA peptidome

High-throughput characterization of the ovarian cancer HLA peptidome

The nature and extent of contributions by defective ribosome products to the HLA peptidome

MHC class I peptides are products of endogenous cellular protein degradation. Their prompt presentation, after rapid degradation of their newly synthesized source proteins, is needed to alert the immune system during pathogen infection. A possible source for such rapidly degrading proteins can be defective ribosome products (DRiPs), which include polypeptides produced as part of the pioneer round of translation, premature translation termination, and proteins failing to fold properly or to assemble into their multisubunit protein complexes. However, the identities and relative contribution to the MHC peptidome of these mature or newly synthesized and rapidly degraded cellular proteins is not well understood. To clarify these issues, we used dynamic stable isotope labeling by amino acids in cell culture to define the relative rates of synthesis of the HLA class I peptidomes and the source proteomes of three cultured human hematopoietic cell lines. Large numbers of HLA class I peptides were observed to be derived from DRiPs, defined here as HLA peptides that shift from their light to heavy isotope forms faster than their source proteins. Specific groups of proteins, such as ribosomal and T-complex protein 1 (TCP-1), contributed a disproportionately large number of DRiPs to the HLA peptidomes. Furthermore, no significant preference was observed for HLA peptides derived from the amino terminal regions of the proteins, suggesting that the contribution of products of premature translation termination was minimal. Thus, the most likely sources of DRiPs-derived HLA peptides are full-sized, misassembled, and surplus subunits of large protein complexes.

Mapping The HLA Ligandome Of Chronic Lymphocytic Leukemia – Towards Peptide Based Immunotherapy

Accelerated clonal evolution and inhibition of immune effector functions are fundamental drawbacks of chemotherapeutic treatment of chronic lymphocytic leukemia (CLL) which contribute to increased clinical aggressiveness of relapsed disease. Anticancer immune responses such as graft versus leukemia effects and remissions after donor lymphocyte infusions, on the other hand, have been correlated to long-term CLL-free survival. Clonotype analysis in these cases suggested clonally expanded CD8+ T cells recognizing tumor associated antigens (TAAs) presented by HLA as mediators of the observed effects, thus making CLL an attractive target for peptide vaccine-based immunotherapy. We here report on our approach of direct isolation and identification of naturally processed and presented HLA ligands from tissues of interest by affinity chromatography and mass spectrometry. Comparative and semi-quantitative analysis of the HLA ligandomes of malignant and benign samples provided the rationale for the identification of ligandome derived TAAs (LiTAAs) and informed selection of peptide vaccine candidates. HLA class I ligands were isolated from MACS-sorted CLL cells as well as from normal B cells or PBMC of healthy volunteers using a standard immunoaffinity purification protocol. Liquid chromatography coupled mass spectrometry (LC-MS/MS) peptide analysis was performed on a LTQ Orbitrap hybrid mass spectrometer followed by database assisted processing of fragment spectra. Semi-quantitative data analysis provided information regarding the abundance of HLA ligands in the respective ligandomes. In addition, HLA surface expression on CLL cells and autologous normal B cells was quantitatively determined using a flow cytometric assay. Selected peptides were characterized functionally in IFN-γ ELISPOT assays using PBMC of healthy volunteers and CLL patients. No significant difference in HLA class I surface expression between CLL cells and autologous normal B cells was observed. So far, we were able to map the HLA class I peptidomes of 25 CLL patients and 35 healthy controls. In total, we were able to identify more than 25,000 different HLA ligands representing >8,500 different source proteins. More than 15,000 different ligands were derived from CLL cells representing a total of 6,500 source proteins. A twofold data mining approach was used to identify both, broadly presented LiTAAs suited for off-the-shelf vaccine development, and LiTAAs showing patterns of patient-specific overrepresentation allowing for actively personalized target identification. The former strategy enabled us to pinpoint the most frequently and abundantly represented targets from the bulk of over 2,000 source proteins, which were exclusively represented in the ligandomes of CLL cells. Several published CLL-associated antigens/epitopes were found to be presented (e.g. Pim-1 Oncogene, SET nuclear oncogene, Mucin-1), which served to validate our methodological approach as proof of principle. Beyond that we identified a vast array of novel proteins that are broadly and exclusively represented in the HLA peptidome of CLL cells. Based on these findings we selected HLA-A*02, A*03 and B*07 restricted ligands derived from top ranking LiTAAs (e.g.TP53I11, PARP3, CDCA7L) for immunological characterization. Using patient PBMC, we observed frequent, reproducible and specific immune recognition of the selected peptides by CD8+ T cells in recall ELISPOT assays. The observed reactivity to CLL-associated self-peptides indicates their potential as therapeutic vaccines while underlining the validity of our target identification and selection strategy. Currently we are expanding our analyses to cover a comprehensive spectrum of HLA types with the goal to develop a clinically applicable CLL-specific multi-peptide vaccine. Disclosures:No relevant conflicts of interest to declare.

Quantitating the role of the proteasome in generating the HLA peptidome

Quantitating the role of the proteasome in generating the HLA peptidome

Peptides Presented by HLA-DR Molecules in Synovia of Patients with Rheumatoid Arthritis or Antibiotic-Refractory Lyme Arthritis

Disease-associated HLA-DR molecules, which may present autoantigens, constitute the greatest genetic risk factor for rheumatoid arthritis (RA) and antibiotic-refractory Lyme arthritis (LA). The peptides presented by HLA-DR molecules in synovia have not previously been defined. Using tandem mass spectrometry, rigorous database searches, and manual spectral interpretation, we identified 1,427 HLA-DR-presented peptides (220-464 per patient) from the synovia of four patients, two diagnosed with RA and two diagnosed with LA. The peptides were derived from 166 source proteins, including a wide range of intracellular and plasma proteins. A few epitopes were found only in RA or LA patients. However, two patients with different diseases who had the same HLA allele had the largest number of epitopes in common. In one RA patient, peptides were identified as originating from source proteins that have been reported to undergo citrullination under other circumstances, yet neither this post-translational modification nor anti-cyclic citrullinated peptide antibodies were detected. Instead, peptides with the post-translational modification of S-cysteinylation were identified. We conclude that a wide range of proteins enter the HLA-DR pathway of antigen-presenting cells in the patients' synovial tissue, and their HLA-DR genotype, not the disease type, appears to be the primary determinant of their HLA-DR-peptide repertoire. New insights into the naturally presented HLA-DR epitope repertoire in target tissues may allow the identification of pathogenic T cell epitopes, and this could lead to innovative therapeutic interventions.

Soluble plasma HLA peptidome as a potential source for cancer biomarkers

The HLA molecules are membrane-bound transporters that carry peptides from the cytoplasm to the cell surface for surveillance by circulating T lymphocytes. Although low levels of soluble HLA molecules (sHLA) are normally released into the blood, many types of tumor cells release larger amounts of these sHLA molecules, presumably to counter immune surveillance by T cells. Here we demonstrate that these sHLA molecules are still bound with their authentic peptide repertoires, similar to those of the membranal HLA molecules (mHLA). Therefore, a single immunoaffinity purification of the plasma sHLA molecules, starting with a few milliliters of patients' blood, allows for identification of very large sHLA peptidomes by mass spectrometry, forming a foundation for development of a simple and universal blood-based cancer diagnosis. The new methodology was validated using plasma and tumor cells of multiple-myeloma and leukemia patients, plasma of healthy controls, and with cultured cancer cells. The analyses identified thousands of sHLA peptides, including some cancer-related peptides, present among the sHLA peptidomes of the cancer patients. Furthermore, because the HLA peptides are the degradation products of the cellular proteins, this sHLA peptidomics approach opens the way for investigation of the patterns of protein synthesis and degradation within the tumor cells.

Correlation of T-cell response, clinical activity and regulatory T-cell levels in renal cell carcinoma patients treated with IMA901, a novel multi-peptide vaccine

3017 Background: Cancer vaccines have been able to induce T-cell responses in cancer patients but rarely demonstrated a correlation of immune responses with clinical benefit. Here, we present immunological results of a phase 1 study with IMA901, a therapeutic cancer vaccine based on multiple novel synthetic tumor-associated peptides (TUMAP) identified as being naturally presented in primary renal cell carcinoma (RCC) tissues. Methods: The HLA peptidome of 32 primary RCC samples was systematically investigated using a combination of mass spectrometry, gene expression profiling and in vitro human T-cell assays. 9 HLA-A*02- and 1 HLA-DR- restricted TUMAPs derived from 9 different tumor antigens were selected and designated IMA901. 28 HLA-A*02-positive stage III/IV RCC patients were enrolled in a single arm, multicenter study and received 8 vaccinations on days 1, 2, 3, 8, 15, 22, 36, and 64 each consisting of 4.5 mg IMA901 (including a HBV-derived viral marker peptide) and 75 μg GM-CSF as immune adjuvant. T-cell responses using IFN-γ ELISPOT and HLA multimer analysis and CD4+ Foxp3+ regulatory T cell (Treg) levels were measured in peripheral blood. Results: In vivo IMA901- induced specific T-cell responses were detected to the HBV marker peptide (52% of 27 evaluable patients), at least one TUMAP (74%) or multiple TUMAPs (30%). T-cell responses were detectable already at day 15, peaked subsequently and were sustainable until follow-up in the majority of patients. Most importantly, patients eliciting multiple responses to TUMAPs significantly showed a higher clinical benefit rate (SD+PR; p=0.018) and lower Treg levels at study onset (p=0.016). No correlation of HBV marker peptide responses with either clinical benefit or Treg levels was observed. Conclusions: IMA901 rapidly induced T-cell responses in a majority of advanced RCC patients. A clinical mode of action is strongly supported by the significant correlation of multiple T-cell responses with clinical benefit. CD4+ Foxp3+ Tregs seem to play an active role in limiting the broadness of T-cell responses. Furthermore, our data suggest that we can predict in vivo immunogenicity of cancer vaccine antigens by our in vitro drug discovery approach. No significant financial relationships to disclose.