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.

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.

Synergistic Reactivation Of Cancer/Testis Antigens By Combination Treatment With Decitabine and Histone Deacetylase Inhibitors (Valproate, Panobinostat) In Myeloid Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3763-3763
Author(s):  
Nadja Blagitko-Dorfs ◽  
Tobias Bauer ◽  
Maren Prinz ◽  
Wolfram Brugger ◽  
Gesine Bug ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Myeloid Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Leukemia Cells ◽  
Cancer Testis Antigens ◽  
Dose Dependent ◽  
Hdaci Treatment ◽  
Cancer Testis

Abstract Introduction Epigenetic therapies with azanucleoside DNA hypomethylating agents, alone or in combination with histone deacetylase inhibitors (HDACi), show clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), particularly when given at non-cytotoxic doses. They are able to reactivate epigenetically silenced genes including, among others, a number of highly immunogenic proteins dubbed Cancer/testis antigens (CTAs), predominantly the CTAs located on the X chromosome. We have previously shown that decitabine can induce expression of several CTAs, including MAGEB2 and NY-ESO-1, in myeloid cells in vitro and thereby trigger an immune response (Almstedt et al., Leuk. Res. 2010). Induction of a CTA-specific cytotoxic T cell response in vivo was reported also in AML patients treated with azacitidine and sodium valproate (VPA) and correlated with clinical response (Goodyear et al., Blood 2010). To the best of our knowledge, no data have yet been reported on the effect of combination treatment with decitabine and panobinostat or sodium valproate (VPA) on CTA reactivation in myeloid leukemia. Aim We hypothesized that by combining decitabine with HDACi we could further enhance expression of CTAs in myeloid leukemia cells and thereby boost recognition of the malignant cells by the cytotoxic T lymphocytes. Methods The myeloid cell lines U937 and Kasumi-1 were treated with decitabine alone or in combination with the HDACi VPA or panobinostat applied at non-toxic concentrations (>80% cell viability). Expression of CTAs was analyzed by RT-qPCR and Western blot after 48 hours of HDACi treatment. DNA methylation of NY-ESO-1 and MAGEB2 promoter regions was quantified by pyrosequencing. Bone marrow mononuclear cells from 19 AML patients (treated with or without VPA as add-on to decitabine in the ongoing randomized phase II DECIDER clinical trial, NCT00867672) were collected before and on day 15 of treatment, in some patients also after 2 treatment cycles. CTA mRNA expression and promoter DNA methylation were quantified as described above. Results VPA or panobinostat alone did not induce MAGEB2 or NY-ESO-1 expression in vitro. However the pretreatment of cells with decitabine prior to addition of either HDACi resulted in a synergistic dose-dependent reactivation of MAGEB2 and NY-ESO-1 on the mRNA level (confirmed for the latter on the protein level). Pyrosequencing analysis of the heavily methylated NY-ESO-1 and MAGEB2 promoters revealed, as expected, no methylation changes upon HDACi treatment, but a dose-dependent hypomethylation upon decitabine. In recently initiated in vivo studies (DECIDER trial), until now cells from 19 AML patients receiving epigenetic treatment were sequentially analyzed. Induction of MAGEB2 mRNA was observed in 9 patients (from absent to a median of 0.002 relative to GAPDH, range 0.0004-0.043), with concomitant DNA hypomethylation of the MAGEB2 promoter from median 83% pretreatment methylation (range 63%-90%) to 63% posttreatment (range 44%-74%). In 5 patients modest hypomethylation without changes in MAGEB2 expression was observed (from median pretreatment values of 89% [72%-92%] to 82% [58%-87%] posttreatment). Another 5 patients disclosed neither hypomethylation nor reexpression of MAGEB2 (results as yet blinded to treatment arm and clinical response). Conclusions Combined epigenetic treatment with the hypomethylating agent decitabine and the HDACi VPA or panobinostat synergistically induced a dose-dependent reactivation of the CTAs MAGEB2 and NY-ESO-1 in vitro, accompanied by promoter hypomethylation. First translational results of the DECIDER AML trial also indicate in vivo effects of the epigenetic treatment on CTA induction. The unmasking of CTAs to the immune system by epigenetically active drugs can increase anti-tumor immune responses, and thus has clear implications for future clinical trials combining epigenetic therapy and specific immunotherapy in myeloid neoplasia. 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.

Thrombocyte HLA molecules retain nonrenewable endogenous peptides of megakaryocyte lineage and do not stimulate direct allocytotoxicity in vitro

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3168-3175
Author(s):  
Cécile Gouttefangeas ◽  
Marianne Diehl ◽  
Wieland Keilholz ◽  
Rainer Frank Hörnlein ◽  
Stefan Stevanović ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Hla Class I ◽  
T Cell Response ◽  
Third Party ◽  
Class I ◽  
Cytotoxic T Cell ◽  
Endogenous Peptides ◽  
Hla Class I Molecules ◽  
Hla Molecules

The origin and the function of HLA class I molecules present on the surface of human platelets are still unclear. In particular, it is controversial which fraction of these class I molecules represents integral membrane components derived from the megakaryocyte-platelet lineage versus soluble plasma HLA molecules acquired by adsorption. Results of the present study show that HLA-A2 ligands isolated from platelets possess the same peptide motif as described for HLA-A2-associated peptides obtained from nucleated cells. Sequencing of these platelet-derived peptides reveals that they originate mainly from ubiquitously expressed proteins also present in the megakaryocyte-platelet lineage. Moreover, one of these peptides derives from the GPIX protein, which is specifically expressed by platelets and their precursors. Platelet HLA molecules are unstable in vitro at 37°C, but can be partially stabilized by addition of exogenous β2-microglobulin and HLA class I binding peptide, suggesting that platelets cannot load HLA molecules with endogenous peptides. In in vitro experiments platelets were used to stimulate peripheral blood mononuclear cells. No allospecific cytotoxicity was observed after primary stimulation, or secondary restimulation, with allogenic resting or activated platelets, even in the presence of additional third-party helper activity. These data indicate that HLA class I molecules from platelets cannot directly induce allogenic CD8+ cytotoxic T-cell response in vitro.

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 Mapping the HLA Ligandome Landscape of Chronic Myeloid Leukemia Identifies Novel CD8+ and CD4+ T Cell-Epitopes for Immunotherapeutic Approaches

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4232-4232
Author(s):  
Tatjana Bilich ◽  
Annika Nelde ◽  
Daniel J. Kowalewski ◽  
Janet Peper ◽  
Mirle Schemionek ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Hla Ligands ◽  
Tki Treatment ◽  
Positive Controls

Abstract While the discovery of BCR-ABL and the respective tyrosine kinase inhibitors (TKI) resulted in a significant prolongation of patient survival rates, there still is no curative treatment for chronic myeloid leukemia (CML) except for allogeneic stem cell transplantation. The concept of T cell-based immunotherapy is a promising opportunity to eliminate residual leukemic cells, which might promote disease relapse after TKI discontinuation. As effective antigen-specific immunotherapy requires exact knowledge of tumor-associated epitopes that can act as rejection antigens, we have developed a mass spectrometry-based approach, which allows for the direct identification of naturally presented tumor-associated HLA ligands in hematological malignancies. In this study we used this approach to identify HLA class I and II CML-associated peptides as targets for T cell-based immunotherapy. Analysis of HLA class I ligandomes of primary CML cells (n=16) identified 8,291 HLA ligands representing 4,337 source proteins. Comparative ligandome profiling using a benign HLA class I database, which includes various healthy tissues (n=188, 65,949 HLA ligands, 14,030 source proteins) originating from peripheral blood, bone marrow, kidney, lung, liver, colon, spleen and others, revealed 38 CML-exclusive HLA class I ligands with frequencies ≥ 25% of CML patients. Because of the important indirect and direct roles of CD4+ T cells in anti-cancer immune responses, an optimal immunotherapy approach requires the inclusion of HLA class II epitopes. Hence we also analyzed the HLA class II ligandomes of primary CML cells (n=15, 2,822 HLA ligands, 794 source proteins). Comparative ligandome analysis (benign tissue, n=114, 54,149 HLA ligands, 8,584 source proteins) identified 44 CML-associated HLA class II ligands showing CML-exclusive representation in > 25% of the analyzed CML samples. To validate the immunogenicity of our HLA class I and II CML-associated peptides, we performed IFNγ- ELISPOT assays after 12-days of in vitro peptide stimulation. For HLA class II antigens, a panel of 4 peptides was implemented for stimulation of PBMCs obtained from CML patients and healthy volunteers (HV). The ELISPOT assay revealed peptide-specific immune recognition of 4/4 (100%) CML-exclusive peptides in CML patients. The frequencies of the detected immune responses ranged from 17% (4/23 patients) to 4% (1/23 patients) within the tested CML samples. These immune responses were mediated by functional CML patient-derived CD4+ T cells and strictly CML-directed, as no immune response against CML-associated peptides could be detected in HV (0/8). For HLA class I antigens, ELISPOT assays were performed using a panel of 8 peptides. Immune responses were only detected for 1/8 (13%) peptides with a low frequency of 6% (1/18 patients) of tested CML patient samples. A possible explanation for the observed weak immune response to our HLA class I CML-associated peptides compared to the immune responses shown for HLA class II peptides and for HLA class I peptides in other hematological malignancies (e.g. CLL (Kowalewski et. al. PNAS 2015)) might be an inhibition of CD8+ T cell-responses, that reportedly occurs upon TKI treatment of CML patients. To prove this hypothesis in our CML patient cohort (all patients included were under TKI treatment at the time of sample collection), we compared the ELISPOT positive controls (stimulated with a set of 5 Epstein-Barr viral peptides) of all analyzed CML samples with positive controls derived from HV and CLL samples. We could show a highly significant mean spot count reduction (per 100,000 cells) in CML samples (mean 74±16 spots, n=19) compared to HV (mean 241±24 spots, n=42, p<0.001, two-tailed t-test) or CLL (mean 218±16 spots, n=125, p=0.008) samples, confirming the general debilitated CD8+ T cell-response in CML patients under TKI treatment. To prove the immunogenicity of our HLA class I CML-associated peptides, we performed in vitro artificial antigen-presenting cell (aAPC)-based priming experiments of HV CD8+ cells. For one HLA-A*03-restricted peptide we observed tetramer-positive CD8+ populations with frequencies ranging from 0.12% to 1.41% of viable cells in 2/2 HVs so far. Taken together, these results are a first step towards a successful validation of these newly defined HLA class I and II CML-associated antigens as prime targets for further T cell-based immunotherapy approaches in CML patients. Disclosures Kowalewski: Immatics Biotechnologies GmbH: Employment. Brümmendorf:Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.

Inhibition of XIAP Enhances Specific Cytotoxic T Lymphocyte (CTL) Killing and CD20-Directed Antibody-Dependent Cellular Cytotoxicity of Chronic Lymphocytic Leukemia B Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3475-3475 ◽  
Author(s):  
Arnon P. Kater ◽  
Roman Rieger ◽  
Kate Welsh ◽  
Adel Nefzi ◽  
Richard Houghten ◽  
...  
Keyword(s):  
Nk Cells ◽  
Combinatorial Libraries ◽  
Hla Class I ◽  
Lymphocytic Leukemia ◽  
Class I ◽  
Target Cells ◽  
Apoptotic Pathway ◽  
Hla Class I Molecules ◽  
Anti Cd20

Abstract CLL cells express relatively high-levels of XIAP, a principle downstream inhibitor of procaspase activation that also is expressed in many other types of cancer. Expression of XIAP may contribute to the resistance of CLL cells (and other cancers in general) to apoptosis induced by anti-cancer drugs and immune effector mechanisms. The anti-apoptotic activity of XIAP can be circumvented by SMAC, a natural inhibitor to the inhibitors of apoptosis (IAPs) that is released from mitochondria following activation of the intrinsic apoptotic pathway. SMAC inhibits XIAP by blocking its BIR domain(s), thereby precluding XIAP from inhibiting active caspases, such as caspase 9. Using mixture-based combinatorial libraries, we identified a series of polyphenylureas that selectively target the BIR2 domain of XIAP and that do not compete with SMAC for binding to XIAP (Cancer Cell5:25–35, 2004). Structural activity studies identified analogs that had improved drug-like characteristics. We investigated whether an active (TPI 1540-14) XIAP-inhibitor or an inactive structural analog (TPI 1540-20) could influence the sensitivity of CLL cells to HLA class-I-restricted killing by allogeneic cytotoxic T lymphocytes (CTL) or to anti-CD20-directed antibody-dependent cell cytotoxicity (ADCC). For these studies we generated allogeneic CTL lines that could mediate specific killing of CLL cells in a HLA-class-I restricted manner. Moreover, the cytotoxicity of these CTL for CLL cells could be inhibited in a concentration-dependent fashion by W6/32, a mouse mAb that recognizes a framework determinant(s) common to all HLA class I molecules. Treatment of CLL cells with subsaturating amounts of W6/32 prior to the allogeneic CTL assay might mimic the situation commonly encountered by autologous CTL, which recognize cells that express relatively few class-I molecules bearing the target peptide-antigen. Treatment of CLL cells with TPI 1540-14, but not TPI 1540-20, significantly enhanced the specific killing of CLL cells by allogeneic CTL in a dose-dependent fashion. Moreover, the capacity of TPI 1540-14 to enhance CTL killing was more apparent when subsaturating concentrations of W6/32 mAb were used to treat the CLL target cells prior to the assay. With either compound, however, saturating amounts of W6/32 blocked CTL activity Similar effects were observed on the ADCC directed by the anti-CD20 mAb Rituximab using isolated allogeneic natural killer cells (NK cells) as effector cells. As noted in prior studies, NK cells failed to mediate high-level ADCC against Rituximab-treated CLL cells even at high effector:target ratios, conceivably due in part to the relatively low level expression of CD20 by CLL cells. However, treatment of CLL cells with TPI 1540-14, significantly enhanced Rituximab-directed ADCC by the allogeneic NK cells. We conclude that TPI 1540-14 can enhance CTL-mediated killing and Rituximab-directed ADCC of CLL cells in vitro. These studies suggest that inhibition of XIAP may enhance the activity of either active or passive immune therapeutic strategies in patients with this disease.

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