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

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.

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

Blood ◽

10.1182/blood.v128.22.2169.2169 ◽

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.

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

Blood ◽

10.1182/blood.v128.22.4046.4046 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4046-4046 ◽

Cited By ~ 1

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.

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The HLA Ligandome Landscape of Chronic Myeloid Leukemia Delineates Novel T-Cell Epitopes for Immunotherapy

Blood ◽

10.1182/blood-2018-99-113376 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4243-4243

Author(s):

Tatjana Bilich ◽

Annika Nelde ◽

Leon Bichmann ◽

Helmut R. Salih ◽

Daniel Johannes Kowalewski ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Myeloid Leukemia ◽

Research Funding ◽

Hla Class I ◽

Class Ii ◽

Hla Class Ii ◽

Class I ◽

Cell Responses ◽

Tki Treatment

Abstract Chronic myeloid leukemia (CML) is characterized by the translocation t(9;22), which leads to the formation of the BCR-ABL fusion transcript. Several approved tyrosine kinase inhibitors (TKIs) target the resulting fusion protein, leading to an improved prognosis of CML patients. Currently, the main treatment goal is the achievement of a deep molecular response (MR), in which TKI therapy can be terminated. Several studies provide evidence that immunological control plays a major role for the course of CML and contributes to the achievement of deep MR in CML patients under TKI treatment (CMLTKI). This implies that reinforcing these immune responses might sustain long-term TKI-free survival or even cure for CML patients. Besides unspecific immunotherapy, such as interferon or immune checkpoint blocking antibodies, a more specific and minor side effect targeting of CML cells might be achieved by antigen-specific immunotherapy approaches. The prerequisite for such strategies is the identification of T-cell targets represented by tumor-associated human leukocyte antigen (HLA)-presented peptides on malignant cells. In this study, we used a mass spectrometry-based approach to identify naturally presented, CML-associated peptides in primary CML samples (HLA class I, n=21, 11,945 peptides, 5,478 source proteins; class II, n=20, 5,991 peptides, 1,302 proteins). Comparative HLA peptidome profiling using a comprehensive dataset of various benign tissues (e.g. blood, bone marrow, spleen, and lung) revealed frequently presented and strictly CML-associated antigens. In detail, the benign tissue dataset comprises hematological benign samples (class I, n=108, 51,233 peptides, 11,437 proteins; class II, n=88, 42,753 peptides, 4,877 proteins) and non-hematological benign tissues (28 tissues, n=166; class I, 128,590 peptides, 16,405 proteins; class II, 143,652 peptides, 13,410 proteins). We identified 50 CML-associated, HLA class I-restricted peptides with HLA allotype adjusted representation frequencies of ≥38% presented on HLA-A*02, -A*03, -A*11, and -B*07. HLA class II comparative profiling delineated 36 peptides exclusively and frequently presented in the HLA peptidome of ≥20% analyzed CML patients. For immunological characterization, we selected 8 HLA class I- and 6 class II-restricted highly CML-associated antigens. These peptides were analyzed in IFNγ ELISPOT assays using PBMCs from CMLTKI patients and healthy volunteers (HVs). Peptide-specific immune recognition was detected for 1/8 (13%) HLA class I peptides in 2/17 (12%) of CMLTKI patients. We hypothesized that this weak immune response might be due to an impaired CD8+ T cell function that reportedly is caused by TKI treatment. Thus, we compared T-cell responses against viral epitopes in IFNγ ELISPOT assays of CMLTKI patients with that of HVs and chronic lymphocytic leukemia (CLL) patients: in line with our hypothesis, we observed significantly reduced IFNγ release of T cells from CMLTKI patients compared to HVs and CLL patients (p<0.001), whereas CD8+ T-cell counts were not reduced. In contrast, no reduced IFNγ production was observed for HLA class II-restricted viral epitopes. These results were confirmed by memory T-cell responses detected for 6/6 (100%) HLA class II CML-associated peptides with frequencies up to 24% (4/17) of analyzed CMLTKI patients. To assess the immunogenicity of all HLA class I peptides, we performed in vitro artificial antigen presenting cell-based priming experiments using CD8+ T cells of HVs and CML patients. Effective priming of T cells was observed for 8/8 CML-associated peptides in ≥70% of analyzed HVs with frequencies of 0.1-33.9% (mean 2.2%) of CD8+ peptide-specific T cells. Notably, peptide-specific CD8+ T cells with frequencies of 0.1-2.2% (mean 0.4%) could also be induced in samples of CMLTKI patients that had not displayed preexisting immune responses. For 6/8 peptides, we observed multifunctionality of peptide-specific T cells by IFNγ and TNF production as well as upregulation of the degranulation marker CD107a. Cytotoxicity assays with polyclonal peptide-specific effector T cells confirmed the capacity to induce antigen-specific lysis for 3/4 analyzed peptides. Taken together, we here identified novel, naturally presented, CML-associated antigens and validated them as promising targets for tailored T cell-based immunotherapeutic approaches for CML patients. Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1. Kowalewski:Immatics Biotechnologies GmbH: Employment. Schuster:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy. Niederwieser:Miltenyi: Speakers Bureau; Novartis: Research Funding.

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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 ◽

10.1182/blood.v124.21.4581.4581 ◽

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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Identification of Four New HLA Class II Restricted Minor Histocompatibility Antigens Contributing to Graft Versus Leukemia Reactivity.

Blood ◽

10.1182/blood.v112.11.3247.3247 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3247-3247

Author(s):

Anita N. Stumpf ◽

Edith D. van der Meijden ◽

Cornelis A.M. van Bergen ◽

Roelof Willemze ◽

J.H. Frederik Falkenburg ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Hla Class I ◽

Class Ii ◽

Hla Class Ii ◽

Cd4 T Cell ◽

Class I ◽

T Cell Clones ◽

Cell Clones

Abstract Patients with relapsed hematological malignancies after HLA-matched hematopoietic stem cell transplantation (HSCT) can be effectively treated with donor lymphocyte infusion (DLI). Donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effect but may also induce detrimental graft-versus-host disease (GvHD). These T cell responses are directed against polymorphic peptides which differ between patient and donor due to single nucleotide polymorphisms (SNPs). These so called minor histocompatibility antigens (mHag) are presented by HLA class I or II, thereby activating CD8+ and CD4+ T cells, respectively. Although a broad range of different HLA class I restricted mHags have been identified, we only recently characterized the first autosomal HLA class II restricted mHag phosphatidylinositol 4-kinase type 2 beta (LB-PI4K2B-1S; PNAS, 2008, 105 (10), p.3837). As HLA class II is predominantly expressed on hematopoietic cells, CD4+ T cells may selectively confer GvL effect without GvHD. Here, we present the molecular identification of four new autosomal HLA class II restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLAmatched HSCT who experienced long-term complete remission after DLI with only mild GvHD of the skin. By sorting activated CD4+ T cells from bone marrow mononuclear cells obtained 5 weeks after DLI, 17 highly reactive mHag specific CD4+ T cell clones were isolated. Nine of these T cell clones recognized the previously described HLADQ restricted mHag LB-PI4K2B-1S. The eight remaining T cell clones were shown to exhibit five different new specificities. To determine the recognized T cell epitopes, we used our recently described recombinant bacteria cDNA library. This method proved to be extremely efficient, since four out of five different specificities could be identified as new HLA-class II restricted autosomal mHags. The newly identified mHags were restricted by different HLA-DR molecules of the patient. Two mHags were restricted by HLA-DRB1 and were found to be encoded by the methylene-tetrahydrofolate dehydrogenase 1 (LBMTHFD1- 1Q; DRB1*0301) and lymphocyte antigen 75 (LB-LY75-1K; DRB1*1301) genes. An HLA-DRB3*0101 restricted mHag was identified as LB-PTK2B-1T, which is encoded by the protein tyrosine kinase 2 beta gene. The fourth mHag LB-MR1-1R was restricted by HLA-DRB3*0202 and encoded by the major histocompatibility complex, class I related gene. All newly identified HLA class II restricted mHags exhibit high population frequencies of 25% (LB-MR1-1R), 33% (LB-LY75-1K), 68% (LB-MTHFD1- 1Q), and 70% (LB-PTK2B-1T) and the genes encoding these mHags show selective (LY- 75) or predominant (MR1, MTHFD1, PTK2B) expression in cells of hematopoietic origin as determined by public microarray databases. All T cell clones directed against the newly identified mHags recognized high HLA class II-expressing B-cells, mature dendritic cells (DC) and in vitro cultured leukemic cells with antigen-presenting phenotype. The clone recognizing LB-MTHFD1-1Q also showed direct recognition of CD34+ CML precursor cells from the patient. In conclusion, we molecularly characterized the specificity of the CD4+ T cell response in a patient with CML after HLA-matched HSCT who went into long-term complete remission after DLI. By screening a recombinant bacteria cDNA library, four new different CD4+ T cell specificities were characterized. Our screening method and results open the possibility to identify the role of CD4+ T cells in human GvL and GvHD, and to explore the use of hematopoiesis- and HLA class II-restricted mHag specific T cells in the treatment of hematological malignancies.

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Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-003404 ◽

2021 ◽

Vol 9 (10) ◽

pp. e003404

Author(s):

Ana Marcu ◽

Andreas Schlosser ◽

Anne Keupp ◽

Nico Trautwein ◽

Pascal Johann ◽

...

Keyword(s):

T Cells ◽

Tumor Cell ◽

Human Leukocyte ◽

Hla Class I ◽

Class Ii ◽

Class I ◽

Atypical Teratoid ◽

Atypical Teratoid Rhabdoid Tumors ◽

Rhabdoid Tumors ◽

Hla Ligandome

BackgroundAtypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells.MethodsHere, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM.ResultsComparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80% of AT/RT exclusive peptides were able to successfully prime CD8+ T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors.ConclusionsThese findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion.

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NATURAL AND CRYPTIC PEPTIDES DOMINATE THE IMMUNOPEPTIDOME OF ATYPICAL TERATOID RHABDOID TUMORS

10.1101/2021.06.11.21258679 ◽

2021 ◽

Author(s):

Ana Marcu ◽

Andreas Schlosser ◽

Anne Keupp ◽

Nico Trautwein ◽

Pascal Johann ◽

...

Keyword(s):

T Cells ◽

Tumor Cell ◽

Hla Class I ◽

Class Ii ◽

Class I ◽

Therapeutic Vaccines ◽

Atypical Teratoid ◽

Atypical Teratoid Rhabdoid Tumors ◽

Rhabdoid Tumors ◽

Hla Ligandome

Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS-tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear, which epitopes T-cells might recognize on AT/RT cells. Here, we report a comprehensive MS-based analysis of naturally presented HLA-class-I and class-II ligands on 23 AT/RTs. Comparative HLA ligandome analysis of the HLA-ligandome revealed 55 class-I and 139 class-II tumor-exclusive peptides. No peptide originated from the SMARCB1-region. In addition, 61 HLA-class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas but no concordance was found with extracranial tumors. More than 80% of AT/RT-exclusive peptides were able to successfully prime CD8+ T-cells, whereas naturally occurring memory responses in AT/RT-patients could only be detected for class-II epitopes. Interestingly, >50% of AT/RT-exclusive class-II ligands were also recognized by T-cells from glioblastoma patients but not from healthy donors. These findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA-class-I and class-II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion.

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

Blood ◽

10.1182/blood.v128.22.4232.4232 ◽

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.

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Engineered Antigen- Presenting Cells Expressing Human HLA Class-II DR Alleles Support Efficient Enrichment and Expansion of Antigen Specific Th1 CD4[+] T-Cells Which Are Functionally Augmented by IL-15Rα/IL-15 Complexes

Blood ◽

10.1182/blood.v120.21.1905.1905 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1905-1905

Author(s):

Aisha N. Hasan ◽

Rosanna J. Ricafort ◽

Annamalai Selvakumar ◽

Ekaterina Doubrovina ◽

Isabelle Riviere ◽

...

Keyword(s):

T Cells ◽

Peptide Pool ◽

Antigen Presenting Cells ◽

Hla Class I ◽

Class Ii ◽

Hla Class Ii ◽

Class I ◽

Invariant Chain ◽

Antigen Presenting ◽

Nih 3T3

Abstract Abstract 1905 Previous studies have affirmed the therapeutic efficacy of adoptively transferred antigen specific CD8+ and CD4+ T-cells (TC) against viral infections and tumors. A major challenge in optimizing this approach is to develop strategies to permit generation of CD4+ and long lived CD8+ TC of defined antigen specificity. We previously described a panel of NIH 3T3 based artifical antigen presenting cells (AAPC) for the immediate generation of HLA class-I restricted CMVpp65 specific CD8+ TC. We now describe a panel of NIH 3T3 based AAPC, each transduced to express a shared HLA DRA 0101 alpha chain and one of the following β chains of the human HLA class-II alleles DRB1 0301, 0701, 1501, 0401 and 1101. At least one of these alleles is inherited by 61% and 59% of caucasians and blacks respectively. These AAPCs were also transduced to co-express the human TC costimulatory molecules B7.1, ICAM-1 and LFA-3. Sensitization of TC from seropositive donors in the presence of IL-2 with AAPCs sharing one of these alleles, either loaded with a CMVpp65 peptide pool or transduced to express the CMV pp65 protein, resulted in 33–71 fold expansion of CMVpp65 specific CD4+ TC that exhibited a Th1 cytokine profile, producing TNF-α and IFNγ in response to the same CMVpp65 epitopes. These TC were also cytotoxic against peptide loaded HLA class-II sharing targets. Epitope mapping demonstrated that the HLA DRB1 0301 restricted TC responded to a CMVpp65 epitope known to be presented by this allele QEFFWDANDIY (aa 509–527) and to an unreported epitope DVEEDLTMTRN (aa 245–263). The DRB1 0701 restricted CD4+ TC responded to 4 different epitopes. Two of these also included nonamer peptide sequences previously reported to be presented by HLA class-I alleles; Q IFLEV QAIRE and PQYSEH PTFTS presented by HLA B44, and a third AGILARNLVPM, contained an epitope presented by HLA B0801. The fourth epitope, KYQEFFWDANDIY is known to be also presented by HLA DRB1 0301. The DRB1 1501 restricted CD4+ TC were also responsive to a known class-II epitope LLQTGIHVRVS (aa 37–55) as well as a new epitope LVSQYTPDSTP (aa 53–71). CD4+ TC from 3 donors also responded to CMVpp65 when sensitized with autologous DCs loaded with CMVpp65 peptide pool, and each recognized the same epitopes as TC sensitized with the class-II AAPCs. Supplementation of TC cultures with soluble IL-15Rα/ IL-15 complexes markedly augmented the proportion of IFNg+ CD4+ TC, while increasing concentrations of IL-2 resulted in generation of Th2 type CD4+ TC generating IL-4, IL-5 and IL-2 in response to re-stimulation with CMVpp65 peptides. This system can therefore be harnessed by cytokine modulation to selectively generate CD4+ TC with a Th1, or Th2 cytokine profile. The fact that the class-II AAPC transduced to express the full sequence of CMVpp65 are able to process and present antigenic epitopes on the surface of the expressed HLA class-II allele in the absence of the human invariant chain and HLA-DM suggests that the mouse 3T3 cells contain sufficiently homologous proteins to permit the transfer of processed peptides to human Class-II alleles for presentation. Alternatively, invariant chain independent pathways could permit delivery of certain immunogenic epitopes to the expressed class II HLA alleles. The repertoire of epitopes presented by the Class-II AAPCs with or without the invariant chain is currently under study. Our results suggest that the panel of AAPCs expressing these HLA DRB1 alleles provides a novel and rapid approach for the generation of Th1 CD4+ virus-specific TC of desired HLA class-II restriction for adoptive therapy of CMV disease to foster lasting immunity with co-infused CMVpp65-specific CD8+ TC. Class-II AAPCs used with different concentrations or types of cytokines may also be useful to generate other functional subsets of CD4+ TC to promote tolerance or enhance tumor- specific immunity. Disclosures: No relevant conflicts of interest to declare.

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