Identification of Novel Tumor-Associated Antigens for Chronic Lymphocytic Leukemia (CLL) Based On HLA Ligandome Analysis – New Targets for Peptide Based Immunotherapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4119-4119
Author(s):  
Daniel Johannes Kowalewski ◽  
Heiko Schuster ◽  
Claudia Berlin ◽  
Lothar Kanz ◽  
Helmut R Salih ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
B Cells ◽  
Affinity Chromatography ◽  
Hla Class I ◽  
T Test ◽  
Class I ◽  
Isolation And Identification ◽  
Tumor Associated Antigens ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract Abstract 4119 Recurrent disease, selection for chemo resistant clones and inhibition of immune effector functions are limitations of chemotherapeutic treatment of cancer including CLL. This is even more since graft-versus-leukemia effects and remissions after donor lymphocyte infusions have been correlated to long-term CLL free survival. Clonotype analysis of such cases suggested clonally expanded CD8+ T cells that recognize tumor associated antigens (TAAs) presented on HLA (human leukocyte antigen) as mediators of the observed effects, thus making CLL an attractive target for peptide vaccine based immunotherapy. For this goal we established the approach of direct isolation and identification of naturally processed and presented HLA ligands from tissues of interest by affinity chromatography and mass spectrometry. Comparative, semi-quantitative analysis of the HLA ligandomes of malignant and benign samples provided the rationale for selection of potential targets. 42 CLL patients (ages 48–85; median 70 years) of different HLA types and disease stages (Binet A, 24 patients; Binet B, 11 patients; Binet C, 7 patients) were enrolled in this study. Furthermore we collected blood samples from 50 healthy volunteers. HLA class I ligands were isolated from CLL cells as well as benign B cells from healthy donors and unsorted healthy PBMC using a standard affinity chromatography protocol implementing the pan-HLA class I specific antibody W6/32. Liquid chromatography coupled mass spectrometry (LC-MS/MS) based peptide analysis was performed on an LTQ Orbitrap hybrid mass spectrometer followed by annotation of fragment spectra using the MASCOT search algorithm against the human proteome comprised in the SwissProt database. Spectral counting based analysis provided semi-quantitative information regarding the abundance of HLA ligands and their source proteins in the respective ligandomes. In addition, HLA quantification experiments on the cell surface of CLL cells and autologous healthy B cells were performed using a flow cytometric indirect immunofluorescence assay. For this interim analysis we completely analyzed the HLA ligandomes of 7 CLL patients and 10 healthy controls. In total, we identified more than 15.000 different HLA ligands representing more than 7300 different proteins. This comprised more than 6,500 different ligands from CLL cells representing a total of 4,149 source proteins. Comparative analysis of representation in the HLA ligandomes of CLL cells, healthy B cells and healthy PBMC identified 1741 different source proteins as being exclusively expressed in CLL. Semi-quantitative evaluation revealed 138 of these proteins as being highly expressed on CLL (e.g. SET proto-oncogene, Pim-1 Oncogene, Mucin 1). Flow cytomerty based quantification of surface HLA expression revealed similar amounts of HLA class I (p=0.23, unpaired t-test) and II (p=0.33, unpaired t-test) molecules on CLL cells and autologous benign B lymphocytes, with a trend to higher HLA expression on CLL cells. Taken together, the presented strategy enabled the identification of a vast array of both known and novel TAAs and their corresponding naturally processed and presented HLA ligands in CLL. It pinpointed highly overrepresented TAAs for further analysis and immunogenicity testing. Expansion of the dataset after analysis of all enrolled patients will provide an unprecedented in-depth characterization of the HLA ligandome of CLL for future immunotherapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4123-4123 ◽  
Author(s):  
Daniel J. Kowalewski ◽  
Heiko Schuster ◽  
Claudia Berlin ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
B Cells ◽  
Cd8 T Cells ◽  
Target Identification ◽  
Peptide Vaccine ◽  
Hla Class I ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Class I ◽  
Hla Ligands

Abstract 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.

scholarly journals Identification of Naturally Presented HLA Ligands of Enriched Leukemic Progenitor Cells for Peptide-Based Immunotherapy in Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4046-4046 ◽  
Author(s):  
Annika Nelde ◽  
Heiko Schuster ◽  
Daniel J. Kowalewski ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Progenitor Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Isolation And Identification ◽  
Hla Ligands ◽  
Hla Ligandome

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.

HLA Class I Ligandome Analysis In Acute Myeloid Leukemia – Novel T-Cell Epitopes For Peptide-Based Immunotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5431-5431
Author(s):  
Stickel S. Juliane ◽  
Claudia Berlin ◽  
Daniel J. Kowalewski ◽  
Heiko Schuster ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
T Cell ◽  
Conflicts Of Interest ◽  
Peptide Vaccine ◽  
T Cell Responses ◽  
Hla Class I ◽  
Class I ◽  
Isolation And Identification ◽  
Cell Responses ◽  
Healthy Donors ◽  
Hla Ligands

Abstract Data regarding the graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion strongly suggest that T lymphocytes play a major role in the rejection of leukemic cells. Immunotherapy directed against leukemia- associated antigens might elicit specific immune responses that may serve to eliminate minimal residual disease after chemotherapy, or enhance the GVL effect after SCT. To achieve this goal there is need to identify appropriate leukemia associated HLA ligands, which are able to induce specific T cell responses. We here aimed to characterize the HLA class I ligandome in AML patients to provide novel tumor associated antigens (TAA) for peptide-based immunotherapy employing our recently implemented approach of direct isolation and identification of naturally presented HLA ligands by affinity chromatography and mass spectrometry (LC-MS/MS) in AML (Stickel et.al., abstract in Blood 2012). Absolute HLA surface expression on AML cells and autologous monocytes and granulocytes was quantified by flow cytometry. HLA class I ligands were isolated from AML cells as well as bone marrow and peripheral blood mononuclear cell (BMNCs/PBMCs) of healthy donors. LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived TAA (LiTAA) and to select peptide vaccine candidates. The most abundantly detected peptide candidates were checked for immunogenicity by ELISpot and confirmed by intracellular interferon-g staining of CD8+ T-cells. Meanwhile 15 AML patients (8 FLT3-ITD mutant) and 35 healthy donors were analyzed. We observed overexpression of HLA class I and II on AML cells as compared to autologous monocytes and granulocytes, with the level of significance reached for HLA class II (p=0,04). A total of more than 12,000 AML derived HLA ligands representing >6,000 different source proteins were identified; of which 2,220 were exclusively represented in AML, but not in healthy PBMC/BMNC. Data mining for broadly represented LiTAA pinpointed 98 TAA as most promising targets. HLA ligands derived from these TAA were presented exclusively on more than 33% of all analyzed AML samples, amongst them already described TAA (e.g. JUP, FAF1) as well as several new leukemia-associated proteins (e.g. MTCH2, METTL7A). Subset analysis of the FLT3-ITD positive AML cohort revealed 21 LiTAA presented exclusively on more than 50% of FLT3-ITD positive AML cases. Additional screening for HLA ligands derived from described leukemia associated antigens revealed overrepresentation for e.g. FLT3, NUSAP, RHAMM and RGS5. Specific CD8+ T cell responses were detected against two A*03 epitope pools (pool 1: APLP2, DKGZ, FAF1, MTCH2; pool 2: KLF2, METTL7A, VCIP1, WIPI1) in AML patients. Notably, the chosen A*03 epitope pools did not elicit specific responses of CTL from healthy donors. Taken together, our HLA class I ligandome analysis in AML for the first time identified naturally presented HLA ligands from patients including a vast array of new leukemia associated antigens representing promising targets for a multipeptide-based immunotherapy approach in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mass Spectrometry-Based Immunopeptidome Analysis of Acute Myeloid Leukemia Cells Under Decitabine Treatment Delineates Induced Presentation of Cancer/Testis Antigens on HLA Class I Molecules

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5223-5223
Author(s):  
Jens Bauer ◽  
Nora Zieger ◽  
Annika Nelde ◽  
Leon Bichmann ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Myeloid Leukemia ◽  
Hla Class I ◽  
Class I ◽  
U937 Cells ◽  
Cancer Testis Antigens ◽  
Hla Class I Molecules ◽  
Hla Ligands ◽  
Cancer Testis

Abstract In recent years, therapeutic approaches for acute myeloid leukemia (AML) have been improved, however the disease is still characterized by high relapse rates and a poor overall survival mainly in elderly patients aged 60 years and older. The standard therapy for these AML patients involves hypomethylating agents (HMAs) such as decitabine. With this, treatment remission can be achieved in some patients, however effective post-remission therapies are still overdue. Recent data suggests that HMAs induce gene expression of various cancer/testis antigens (CTAs), which could lead to the presentation of cancer/testis antigen-derived peptides on human leukocyte antigen (HLA) molecules. These CTA-derived peptides might serve as prime targets for tailored T cell-based immunotherapy approaches, which could represent an effective post-remission combination therapy. Here we present a mass spectrometry-based study, which longitudinally maps the HLA-presented immunopeptidome and in particular cancer/testis antigens of AML cells under in vitro decitabine treatment. To analyze the impact of decitabine on the presentation of HLA ligands we treated the AML cell lines U937 and MONO-MAC-6 as well as primary AML cells (n = 1) with decitabine for 48 h (t48) and 72 h (t72) in vitro. Upon flow cytometry-based quantification of HLA class I and II surface expression levels, no significant changes of HLA surface molecule levels under decitabine treatment compared to untreated controls were observed. Implementing label-free quantitation mass spectrometry, we then quantitatively assessed HLA class I ligand presentation under decitabine treatment. Only minor effects of decitabine on the whole HLA class I-restricted peptidome were observed: We detected a significant upregulation of 2.6 ± 0.9% of HLA class I ligands (fold change (FC) ≥ 4, p ≤ 0.01) after 48 h of decitabine treatment, whereas 9.6 ± 5.7% of the ligands were altered in their abundance over time without treatment. At t72 similar proportions of decitabine modulation were observed with 4.2 ± 2.7% of up-regulated HLA ligands. A total of 69 HLA class I ligands derived from 31 different CTAs were identified by mass spectrometric analysis, 9 of these ligands were presented exclusively under decitabine treatment. Furthermore, we showed that decitabine exposure caused a significantly increased presentation of 7/69 CTA-derived HLA ligands at least at one time point in the cell lines and the primary AML cells (FC ≥ 4, p ≤ 0.01). From the CTA cyclin A1, two HLA class I-presented peptides were significantly upregulated in U937 cells at t48 (FC 79.0 and 8.2) and t72 (FC 14.1 and 12.4). In primary AML cells, two peptides derived from JARID1B and KIAA0100 were significantly upregulated at either t48 (FC 21.8) or t72 (FC 6.6). In addition, we screened our dataset for HLA ligands derived from previously described decitabine-regulated genes and identified a HLA class I-presented peptide from DAZL, which was significantly upregulated in U937 cells at t72 under decitabine treatment (FC 57.2). Taken together, our results demonstrate a modulatory effect of the hypomethylating agent decitabine on the HLA ligandome of AML cells, enhancing the presentation of CTA-derived peptides on HLA class I molecules. The latter will be further evaluated for their eligibility as targets for tailored peptide-based immunotherapeutic approaches in AML patients undergoing HMA treatment. Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1.

scholarly journals Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry

2021 ◽  
Vol 22 (19) ◽  
pp. 10503
Author(s):  
Elena Lorente ◽  
Miguel Marcilla ◽  
Patricia G. de la Sota ◽  
Adriana Quijada-Freire ◽  
Carmen Mir ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Virus Infection ◽  
High Throughput ◽  
Antigen Processing ◽  
Hla Class I ◽  
T Cell Response ◽  
Class I ◽  
Hla Class I Molecules ◽  
Infected Cells ◽  
Hla Ligandome

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.

scholarly journals Mapping the HLA Ligandome Landscape of Chronic Myeloid Leukemia (CML)—Towards Peptide Based Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4518-4518 ◽  
Author(s):  
Daniel J. Kowalewski ◽  
Mirle Schemionek ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
Tim H. Brümmendorf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Myeloid Leukemia ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Peptide Vaccination ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract Despite the success of targeted therapy with tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable. Immunotherapy, and in particular multi-peptide vaccination, may be a promising approach to eliminate residual CML cells. As of now, a multitude of potential vaccine targets have been proposed based on reverse immunology and functional genomic approaches focusing either on BCR-ABL junction peptides, which represent CML-specific neo-antigens, or on aberrantly expressed self-proteins such as WT-1, PR and hTERT. However, the results of clinical studies employing such antigens have so far not been encouraging. This might in part be due to the inherent limitations of the above mentioned approaches: evidence of natural presentation of the predicted epitopes is lacking and the correlation of transcript abundance and HLA restricted presentation of the corresponding gene product has been shown to be skewed. Modern mass spectrometry, on the other hand, enables the comprehensive analysis of the entirety of naturally presented HLA ligands on tissues of interest, termed the HLA ligandome. Here we implemented this direct approach and comparatively mapped the HLA ligandome landscape of 16 primary CML samples and 40 healthy volunteer (HV) controls (30 blood and 10 bone marrow samples). We identified more than 30,000 different naturally presented HLA class I ligands representing ~10,000 source proteins. Regression analysis suggests source protein identifications on CML (4,337 different proteins) to attain >95% of maximum achievable coverage with the implemented analytical setup. Based on this extensive dataset, we investigated the HLA restricted presentation of established CML-associated/specific antigens and applied a novel approach defining tumor-associated antigens strictly based on exclusive and frequent representation in CML ligandomes. Strikingly, we found the vast majority of previously described antigens including wild-type BCR protein (6% CML, 5% HV), Myeloperoxidase (56% CML, 15% HV) and Proteinase 3 (38% CML, 11% HV) to be (also) represented on normal PBMC or BMNC. No evidence of naturally presented BCR-ABL junction peptides was found. However, we identified a panel of 7 LiTAAs (ligandome-derived tumor-associated antigens) represented by 16 different HLA ligands, showing CML-exclusive representation in ≥25% of CML patient ligandomes. As CD4+ T cells mediate important indirect and direct effects in anti-tumor immunity, we further applied our approach to HLA class II ligandomes of 15 CML patients and 18 HV (13 blood and 5 bone marrow samples), identifying more than 9,000 different naturally presented HLA class II ligands (1,900 source proteins). Applying the same antigen-ranking strategy as described for HLA class I, we identified 7 additional HLA class II LiTAAs represented by 50 corresponding LiTAPs (ligandome-derived tumor-associated peptides). Overlap analysis of CML-exclusive source proteins revealed 6 proteins to be represented both in HLA class I and II ligandomes. Notably, for Galectin-1, which shows CML-exclusive representation in 19% of HLA class I and 13% of HLA class II ligandomes, one of the HLA class II ligands was found to contain a complete, embedded HLA class I peptide. Such naturally presented embedded HLA ligands might present optimal vaccine candidates that are recognized by both, CD4+ and CD8+ T cells. Functional analysis of the here defined HLA class I and II LiTAPs with regard to induction of T cell responses is presently ongoing and serves to validate them as prime targets for the development of an off-the-shelf peptide vaccination in CML patients. Disclosures No relevant conflicts of interest to declare.

HLA Ligandome Analysis of Different Hematological Malignancies Identifies a Small Panel of "Pan-Leukemia"-Associated Antigens

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2169-2169
Author(s):  
Linus Backert ◽  
Daniel J. Kowalewski ◽  
Simon D. Walz ◽  
Heiko Schuster ◽  
Claudia Berlin ◽  
...  
Keyword(s):  
T Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Hematological Malignancies ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract Effective antigen-specific T-cell-based cancer immunotherapy requires exact knowledge of tumor-associated epitopes that can act as rejection antigens. While the current paradigm views mutation-derived neoantigens as the most promising targets, we have recently demonstrated that tumor-specific T-cell responses target panels of non-mutated tumor-associated self antigens in patients with hematological malignancies. Using the approach of direct HLA ligandome analysis by mass spectrometry, we were able to identify and characterize multiple immunogenic and naturally presented tumor-associated antigens for chronic lymphocytic leukemia (CLL, Kowalewski et. al., PNAS 2015), acute myeloid leukemia (AML, Berlin/Kowalewski et. al., Leukemia 2014), multiple myeloma (MM, Walz/Stickel et. al., Blood 2015) and chronic myeloid leukemia (CML, unpublished data). In this project we performed a comprehensive meta-analysis of our HLA ligandome data from different hematological malignancies (HM) to screen for the existence of "pan-leukemia" antigens for the broad application in T-cell based immunotherapy approaches in hematological malignancies. In a first step we performed unsupervised cluster analyses to identify similarities and differences in the HLA ligandome landscape of HM. To avoid skewed clustering due to HLA types of the samples, these analyses were performed specifically for the most common HLA allotypes in our datasets (A*02 (n=46 HM), A*03 (n=28 HM)). Distinct clustering was shown for the different entities (CLL, MM, CML, AML) as well as for the lymphoid versus myeloid malignancies on the HLA ligandome level. To identify leukemia-exclusive HLA ligands we compared the HLA ligandomes of CLL (HLA class I, n=35; HLA class II, n=30), AML (HLA class I, n=19; HLA class II, n=20), MM (HLA class I, n=15; HLA class II n=12) and CML (HLA class I, n=16; HLA class II n=15) with our normal tissue database including 153 HLA class I and 82 HLA class II ligandomes of various normal tissues (including normal blood, bone marrow and spleen). Cluster analysis of the leukemia-exclusive antigens showed identical clustering of the different entities and lymphoid/myeloid malignancies as shown before for the whole HLA ligandome and the respective source proteins. Overlap analysis revealed only 0.6% (16/2,716) and 0.3% (10/3,141) of the identified leukemia-exclusive HLA class I and class II antigens, respectively, to be represented across all analyzed hematological malignancies. These "pan-leukemia" antigens (n=26) include candidate antigens associated with T-cell activation (HSH2D), lymphoid development (IL2RF) and oncogenesis (LYN protooncogene, RAB5A). However, none of these "pan-leukemia" antigens shows frequent representation (>20%) across all 4 entities (CLL, AML, MM, CML). Furthermore, none of the "pan-leukemia" source proteins yielded corresponding peptides represented in all entities. To identify "pan-leukemia" HLA ligands, overlap analyses were performed in an allotype-specific fashion for the most frequent HLA allotypes (HLA-A*01, -A*02, -A*03, -A*24, -B*07, -B*08, -B*18) in our cohort. 0% (0/92) of HLA-A*01-, 1.6% (12/744) of HLA-A*02-, 1.4% (8/561) of HLA-A*03-, 0% (0/331) of HLA-A*24-, 0.1% (1/830) of HLA-B*07-, 0% (0/472) of HLA-B*08- and 0.8% (5/600) of the HLA-B*18-restricted peptides showed representation in all four entities. Out of these 26 "pan-leukemia" HLA ligands, only two (1 HLA-A*02-, 1 HLA-A*03-restricted peptide) showed frequent representation (>20%) in all entities. These peptides represent "pan-leukemia" targets that might be used for immunotherapeutic approaches in patients expressing the respective HLA allotype. Taken together, our approach of direct HLA ligandome analysis of hematological malignancies identified a small panel of "pan-leukemia"- proteins and peptides that show cancer-exclusive representation across all 4 included hematological malignancies. However, due to the low presentation frequencies of the candidate targets within the different entities, target discovery and compound development for the immunotherapy of HM may be more effectively achieved in an entity-specific or even patient-individualized manner. Disclosures Kowalewski: Immatics Biotechnologies GmbH: Employment. Schuster:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Niederwieser:Novartis Oncology Europe: Research Funding, Speakers Bureau; Amgen: Speakers Bureau. Weisel:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Onyx: Consultancy; BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Novartis: Honoraria.

HLA Ligandome Analysis Of Chronic Myeloid Leukemia (CML), Revealed Novel Tumor Associated Antigens For Peptide Based Immunotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3975-3975 ◽  
Author(s):  
Juliane S. Stickel ◽  
Daniel J. Kowalewski ◽  
Mirle Schemionek ◽  
Lothar Kanz ◽  
Tim H. Brümmendorf ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Myeloid Leukemia ◽  
Adhesion Molecule ◽  
Myeloid Leukemia ◽  
Hla Class I ◽  
Class I ◽  
Tumor Associated Antigens ◽  
Healthy Donors ◽  
Hla Ligands ◽  
First Time

Abstract Despite the success of targeted therapy using tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, most likely due to the treatment resistance of leukemic stem cells. Specific T-cell based immunotherapies for patients with CML might be able to eliminate these residual CML cells. For this goal the identification of tumor associated HLA-presented peptides, which are able to induce a tumor-specific T cell response, is indispensable. However, only few tumor associated antigens for CML are described till date. Thus the aim of this study was to identify for the first time naturally processed and presented HLA ligands from the cell surface of primary CML cells. HLA class I ligands from primary CML cells as well as from bone marrow mononuclear cell (BMNCs) and peripheral blood PBMCs of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. So far 10 CML patients (8 chronic phase, 2 accelerated phase) and 30 healthy donors were analyzed within this study. We were able to identify a total of more than 8200 CML derived HLA ligands representing >4500 different source proteins, of which 734 were exclusively represented in CML, but not in healthy PBMCs/BMNCs. 55 of these CML exclusive antigens are presented on 3 or more of all examined CML patients, representing, as broadly represented LiTAAs, promising targets for peptide vaccination. For the first time, previously predicted CML tumor associated antigens for example Myeloperoxidase (10 peptide sequences on 7 CML patients) and Proteinase 3 (5 peptide sequences on 4 CML patients) were here confirmed by direct elution from primary CML cells, which also served to validate our methodological approach. Notably, beyond that we also identified a vast array of novel proteins (e.g. Carcinoembryonic antigen-related cell adhesion molecule 8, CEACAM8; Matrix metallopeptidase 8, MMP8; intracellular adhesion molecule 3, ICAM3) that are broadly and exclusively represented in the ligandome of CML cells. By providing for the first time HLA class I tumor associated antigens, directly obtained from the HLA ligandomes of CML patients, this study may pave the way for the future development of a multipeptide-based immunotherapy approach to eradicate residual CML cells after conventional TKI therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HLA Ligandome Analysis in Primary Polycythemia Vera (PV) Samples Shows No Tumor-Association of Naturally Presented JAK2 Ligands but Reveals a Vast Array of Novel PV-Associated HLA Ligands

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4581-4581
Author(s):  
Juliane S. Stickel ◽  
Daniel J. Kowalewski ◽  
Steffen Koschmieder ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Polycythemia Vera ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Jak2v617f Mutation ◽  
Class I ◽  
Tumor Associated Antigens ◽  
Hla Ligands ◽  
Overlap Analysis

Abstract Polycythemia vera (PV) is a myeloproliferative disorder mainly driven by the JAK2V617F mutant kinase. Except allogeneic stem cell transplantation, the current standard therapies, including novel therapies aiming for specific inhibition of JAK2 that are presently under investigation, do not offer a chance for cure. Of note, clear clinical benefits of unspecific immunotherapy with pegylated IFN-α suggest that PV might also be targeted by specific peptide based vaccination. Here we aimed to identify tumor associated HLA ligands of PV with special focus on peptides derived from JAK2. Therefore we analyzed HLA class I and II ligands of primary PV samples using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry. We analyzed the HLA class I ligandomes of 7 PV patients with the JAK2V617F mutation as well as of 4 patients with secondary (JAK2V617F negative) polycythemia. As controls, we profiled the ligandomes of 40 healthy volunteers (30 blood and 10 bone marrow samples). We identified more than 7,000 unique HLA class I ligands representing 4,435 proteins on primary PV samples. Moreover, 3 different HLA ligands derived from JAK2 on PV (KYLINLETL, HLA-A*24; KIGDFGLTK, HLA-A*03; GQGTFTKIFK, HLA-A*11) were identified. None of these ligands was found to be PV-exclusive, with JAK2 being represented on 3/4 (75%) of PV samples (JAK2V617F positive) and 18/30 (60%) healthy controls. No JAK2-derived peptides were identified on JAK2V617F negative patients, and no peptides covering the JAK2V617F mutation were identified. Since our previous work strongly suggests that tumor exclusive representation of an antigen is a prerequisite for the immunogenicity of its corresponding T cell epitopes, JAK2-derived wild-type peptides are unlikely to constitute optimal vaccines in PV. On the other hand, comparative profiling of the HLA class I ligandomes identified 935 antigens to be exclusively presented in JAK2V617F positive PV with 12 ligandome-derived tumor-associated antigens (LiTAAs) representing 26 different HLA ligands with exclusive representation in ≥30% of PV patients. Overlap analysis of PV patients with and without JAK2V617F mutation revealed 92% of the exclusive antigens to be represented on both groups, while only 8% and 0% showed exclusive representation in JAK2V617F positive and negative PV patients, respectively. As CD4+ T cells play important indirect and direct roles in anti-tumor immunity, we further applied our approach to HLA class II ligandomes of 8 PV patients (6 with, 2 without JAK2V617F mutation) and 18 HV (13 blood and 5 bone marrow samples) Using this approach, we identified more than 7,700 different naturally presented HLA class II ligands (1,700 source proteins). Notably, no JAK2-derived HLA class II peptides were identified in PV patients and HV ligandomes. Applying the same antigen-ranking strategy as described for HLA class I, we identified 5 additional HLA class II LiTAAs (exclusive in >30% of PV ligandomes) represented by 19 corresponding LiTAPs (ligandome-derived tumor-associated peptides). Overlap analysis of PV-exclusive source proteins revealed 2 proteins (POLA1, polymerase (DNA directed), alpha 1 and RHOQ, ras homolog family member Q) to be exclusively represented both in HLA class I and II ligandomes of PV patients. The novel HLA class I and II ligandome-derived tumor associated antigens identified in this study may constitute prime candidates for peptide-based immunotherapy of PV. Disclosures No relevant conflicts of interest to declare.

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