HLA-presented Peptides Research Articles

ALK and RET Inhibitors Promote HLA Class I Antigen Presentation and Unmask New Antigens within the Tumor Immunopeptidome.

T-cell immunotherapies are often thwarted by the limited presentation of tumor-specific antigens abetted by the downregulation of human leukocyte antigen (HLA). We showed that drugs inhibiting ALK and RET produced dose-related increases in cell-surface HLA in tumor cells bearing these mutated kinases in vitro and in vivo, as well as elevated transcript and protein expression of HLA and other antigen-processing machinery. Subsequent analysis of HLA-presented peptides after ALK and RET inhibitor treatment identified large changes in the immunopeptidome with the appearance of hundreds of new antigens, including T-cell epitopes associated with impaired peptide processing (TEIPP) peptides. ALK inhibition additionally decreased PD-L1 levels by 75%. Therefore, these oncogenes may enhance cancer formation by allowing tumors to evade the immune system by downregulating HLA expression. Altogether, RET and ALK inhibitors could enhance T-cell-based immunotherapies by upregulating HLA, decreasing checkpoint blockade ligands, and revealing new, immunogenic, cancer-associated antigens.

Abstract 566: Neoantigens from translated unannotated open reading frames in cancer

Abstract Somatic mutations in cancer cells can generate neoantigens which can be recognized by immune cells and trigger an immune response. Patients vaccinated with neoantigen-based peptides display expanded neoantigen-specific T cells, suggesting that this could be a promising avenue for cancer treatment. Currently, neoantigen predictions are based on mutations detected by whole exome sequencing, which covers a pre-determined set of annotated exons, and often falls short for cancers with few somatic mutations. Ribosome profiling (Ribo-seq), which allows to monitor mRNA translation, has predicted a plethora of translated novel unannotated ORFs (nuORFs). Here we hypothesized that nuORFs can provide another source of neoantigens in cancer cells. In particular, we focused on nuORFs in the following categories: 1) nuORFs expressed in healthy and cancer cells, that have acquired tumor-specific somatic mutations; 2) nuORFs upregulated in or specific to cancer cells. To explore this hypothesis, we performed Ribo-seq on primary healthy and cancer cells and cell lines from melanoma, glioblastoma, colon carcinoma and chronic lymphocytic leukemia. Using this extensive dataset, we performed hierarchical ORF prediction analysis to build a database of highest confidence predicted translated nuORFs across healthy and cancer cell types. To determine if peptides from nuORFs can be a source of antigens, we searched our collection of mono-allelic MHC class I immunopeptidome mass spectrometry (MS) spectra from 94 common HLA alleles against our pan-tissue nuORF database. Additionally, we performed MHC class I immunoprecipitation followed by MS on the same cells used for Ribo-seq. We found HLA-presented unmutated peptides derived from thousands of nuORFs, found within, but out-of-frame with annotated protein-coding ORFs, within 5’ and 3’ untranslated regions of annotated protein-coding transcripts, long non-coding RNAs (lncRNAs), pseudogenes, and other RNA species. The HLA-binding motifs of peptides from nuORFs correspond to the expected motifs for given HLA types, indicating that 1) nuORFs are translated and 2) nuORF-derived peptides are presented on MHC I. To identify tumor-specific somatic mutations in nuORFs, we performed whole genome sequencing on patient-matched healthy and cancer cells and mapped somatic mutations to annotated ORFs and nuORFs. Finally, to identify nuORFs upregulated in or specific to cancer cells, we compared translation levels of nuORFs between healthy and cancer cells of the same origin. We found translated nuORFs with cancer-specific somatic mutations and nuORFs highly upregulated in and specific to cancer cells, suggesting that they can give rise to neoantigens. In conclusion, nuORFs are translated, contribute peptides to MHC I presentation, acquire somatic mutations, are expressed in tissue- and cancer-dependent manner and should be considered in the search for neoantigens in cancer. Citation Format: Tamara Ouspenskaia, Travis E. Law, Karl R. Clauser, Susan Klaeger, Derin B. Keskin, Bo Li, Elena Christian, Yuen Ting Chow, Phuong M. Le, Joshua Gould, Zhe Ji, Wandi Zhang, Pavan Bachireddy, Siranush Sarkizova, Nir Hacohen, Steven A. Carr, Catherine J. Wu, Aviv Regev. Neoantigens from translated unannotated open reading frames in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 566.

Abstract 5687: Integrative -omics analysis to identify drug targets for ccRCC immunotherapy

Abstract Background Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer and frequently diagnosed at advanced disease stages. Cure of advanced and metastatic ccRCC is achieved only in rare cases with currently available therapies, consisting of tyrosine kinase and mTOR inhibitors, due to intrinsic or acquired resistance mechanisms. We developed a workflow to identify potential drug targets involved in ccRCC pathogenesis for immunotherapeutic approaches and combinatorial treatment strategies. Methods We analyzed frequent ccRCC-specific peptides by HLA ligandomics of 55 ccRCC tumors, paired non-tumor tissues and 158 other benign tissues from various organs. To extract cellular pathways with enrichment in ccRCC compared to the S3 region of kidney proximal tubules, which represents the region of ccRCC origin, we performed transcriptome and gene set enrichment analyses in 51 of the 55 tumor tissues of our study cohort and in a microdissected sample of the S3 region (S3-transcriptome data taken from Cheval et al., PloS One 2012). ccRCC-specific pathway genes were intersected with the tumor-exclusive peptide source genes from the HLA ligandome analysis to retrieve a list of candidate target genes. By integrating gene expression data of an independent ccRCC cohort from the Cancer Genome Atlas (TCGA KIRC, n=452), the candidates were validated on the level of individual gene expression and pathway enrichment. Integration of DNA methylation (TCGA KIRC, n=273) and somatic mutation data (TCGA KIRC, n=392) was used to refine the candidate list. Results Of the frequent ccRCC-specific HLA class I and II peptide source genes, 203 were involved in ccRCC-enriched pathways in both cohorts. 136 of the genes passed the selection criteria of minimal expression in tumors (> 7.2 log2 FPKM-UQ) and induction compared to paired non-tumor tissues. 89 of the candidate targets were affected by somatic point mutations in one up to eight patients, potentially giving rise to patient-specific neo-epitopes. 110 of the candidate genes displayed altered DNA methylation patterns in tumors compared to non-tumor tissues, offering the possibility of epigenetic targeting. Conclusion The presented workflow, integrating HLA ligandomics, transcriptomics and genomics, yields a list of genes with frequent ccRCC-specific HLA-presented peptides that could be targeted in immunotherapeutic approaches, such as ccRCC-directed peptide vaccines. Moreover, the selected genes are involved in ccRCC-enriched cellular pathways and might therefore be important factors of ccRCC pathogenesis, potentially restraining rapid resistance development when they are used as drug targets. The list represents a pre-selection of potential drug targets for novel immunotherapeutic and combinatorial therapy approaches, which need to be further evaluated. Citation Format: Anna Reustle, Moreno Di Marco, Florian Büttner, Stefan Winter, Siarhei Kandabarau, Daniel Kowalewski, Linus Backert, Steffen Rausch, Joerg Hennenlotter, Marcus Scharpf, Falko Fend, Arnulf Stenzl, Jens Bedke, Stefan Stevanovic, Matthias Schwab, Elke Schaeffeler. Integrative -omics analysis to identify drug targets for ccRCC immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5687.

Abstract 1632: Identification and analysis of EGLN3 as tumor-associated peptide in ccRCC

Abstract Introduction: Kidney cancer is the 12th most common cancer world-wide with 338.000 newly diagnosed cases in 2012, according to the World Cancer Research Fund International. With around 70-80%, clear cell renal cell carcinomas (ccRCC) make up the vast majority of kidney cancers. Advanced and metastatic ccRCCs are marked by poor survival rates despite the availability of targeted therapies. Currently several immunotherapeutic approaches, including checkpoint-blocking antibodies and dendritic cell vaccines are undergoing clinical investigations. In this study we identified tumor-associated peptides which could be used in cancer vaccines to improve treatment of patients with advanced ccRCC. Methods: To identify suitable peptides for cancer vaccines we set up a tissue collection of paired tumors and normal kidney tissues from 60 ccRCC patients. From these tissues, HLA molecules were isolated by affinity chromatography and bound peptides analyzed by mass spectrometry. HLA ligand profiles of tissues excised from various organ sites of non-ccRCC patients were used to complement the data. Based on these analyses, Egl nine homolog 3 (EGLN3) was selected as a candidate for peptide vaccination and further investigated in our own data cohort comprising the 60 ccRCC patients. Therefore, mRNA and protein expression, as well as DNA methylation data were collected and correlated with HLA ligandomics and clinical data. In addition, we integrated RNA-Seq, DNA methylation, somatic mutation and clinical data from a ccRCC patient cohort from The Cancer Genome Atlas in our analyses. Finally, EGLN3 function was analyzed in 2D and 3D kidney carcinoma cell culture systems. Results: 16 different peptides from EGLN3 were detected in 32 of the 60 analyzed tumor samples, with the most frequent peptide found in tumors of 10 different patients. Only two of the peptides were also detected in normal kidney or other non-ccRCC tissues, rendering EGLN3 peptides promising candidates to be used in ccRCC-directed vaccines. Most of our patients were HLA A*02:01 positive, however using NetMHCpan (http://www.cbs.dtu.dk/services/NetMHCpan/) only one of the peptides identified by HLA ligandomics was also predicted to be a strong binder for that particular HLA molecule. Expression of EGLN3 mRNA was not different in patients with and without HLA-presented peptides. Generally, EGLN3 mRNA expression was strongly induced in ccRCC tumors compared to normal kidney (p<10-6), whereas protein levels remained mainly unchanged in most patients. Functional investigations in different RCC cell culture models indicated involvement of EGLN3 in regulation of apoptosis and cell cycle progression. Conclusion: Advanced ccRCC is marked by poor survival rates due to ineffective therapeutic options. Peptides from EGLN3, as well as other ccRCC-exclusive peptides could represent activators of the immune system to specifically fight ccRCC tumors. The function and targetability of EGLN3 remains to be investigated. Citation Format: Anna Reustle, Moreno Di Marco, Florian Büttner, Stefan Winter, Daniel Kowalewski, Linus Backert, Steffen Rausch, Joerg Hennenlotter, Marcus Scharpf, Falko Fend, Arnulf Stenzl, Jens Bedke, Matthias Schwab, Elke Schaeffeler. Identification and analysis of EGLN3 as tumor-associated peptide in ccRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1632. doi:10.1158/1538-7445.AM2017-1632

Endogenous Immunoglobulin-Derived Neoepitopes Are Processed and Form a Sizeable Fraction of the HLA Class I Ligandome of Human Lymphoma Cells

T cells recognizing solid tumors and Hodgkin's lymphoma can be released from anergy by immune checkpoint inhibitors. Its therapeutic success seems to be associated with the abundance of neoepitopes within the tumor mutanome. In neoplastic B cells, VDJ recombination and somatic hypermutation (SHM) generate unique immunoglobulin (Ig) peptide sequences that predictably contribute to the lymphoma mutanome. Efficient presentation of a neoepitope by an individual's HLA complex is an essential requirement for neoepitope-directed T-cell immunity. Presentation of Ig-derived peptides in HLA class I has hitherto been demonstrated only indirectly, and there is as yet no direct evidence for presentation of Ig-derived neoepitopes of primary human lymphoma cells.We analyzed HLA-presented Ig-derived peptides in 9 clonal B-cell populations: 3 chronic lymphocytic leukemias (CLL), 1 hairy cell leukemia, 1 follicular lymphoma (FL), 3 EBV-transformed lymphoblastoid cell lines, and the U299 myeloma cell line. Full-length B-cell receptor (BCR) VDJ and VJ sequences were obtained by unbiased ARTISAN PCR and Pacific Biosciences next generation sequencing. 1067 unique Ig-derived nonamers were predicted to bind the HLA class I alleles expressed by the respective B-cell clone by the NetCTLpan bioinformatics tool. 650 candidate epitopes (60.9%) were derived from constant (C) regions; 417 (39.1%) from variable (V) regions. 146 predicted peptides from V regions (35.0%) contained at least one amino acid change due to SHM or at least one amino acid from CDR3 and were therefore considered potential neoepitopes.To investigate experimentally which epitopes are processed intracellularly and presented by HLA class I, HLA class I-peptide complexes were immunoaffinity purified by the monoclonal antibody W6.23 and analyzed by liquid chromatography and tandem mass spectrometry. In total, 53,663 unique peptides (8-15mers) were identified by Uniprot matching with a Mascot Ion Score of >20. HLA ligandome sizes of individual B-cell populations ranged from 600 to 14,091 unique peptides per case. Within any HLA ligandome, 6 to 81 peptides were annotated as BCR-derived epitopes if they matched the individual BCR sequences. Of the total of 276 eluted BCR peptides, 203 (73.5%) where derived from C regions and 73 (26.5%) from V regions. 25 eluted peptides (range 0-10 per case) were derived from CDR3 regions or contained SHM-induced amino acid changes, thus fulfilling the definition of neoepitopes. Neoepitopes were detected in all cases with more than 109 cells as input material. Up to date, 4 neoepitopes have been synthesized and their mass spectra confirmed. Confirmation of all remaining neoepitopes is ongoing. No BCR-derived neoepitopes could be detected in an IGHV-unmutated CLL and the FL. These two cases had the lowest number of input cells and small overall ligandome sizes of only 600 and 1015 unique UniProt-matched peptides, respectively.We demonstrate for the first time that primary neoplastic B cells process and present BCR-derived neoepitopes in the context of HLA class I. Despite their origin from only two polypeptides, BCR epitopes and neoepitopes represent app. 0.5% and 0.05% of the total HLA class I ligandome, respectively. The challenging identification of BCR neoepitopes was possible by unbiased identification of BCR transcripts combined with next generation sequencing and targeted search for HLA class I-bound peptides. Matching fragmentation patterns of native and synthetic peptides suggest high specificity of this strategy. With respect to sensitivity, the ability to identify low frequency peptides appears to be strongly dependent on the amount of input cells.Our data close a gap in the mechanism underlying the evidence that highly immunogenic formulations of an idiotype vaccine are able to induce MHC class I-restricted cytotoxic T-cell responses. While phase III trials of idiotype vaccination aiming to induce anti-Ig antibodies failed to demonstrate convincing prolongation of clinical remissions achieved by chemotherapy, our data lend support for exploring idiotype-specific T-cell immunity against B-cell lymphomas. With recent advances in peptide synthesis, adjuvant formulations, and the availability of check point inhibitors to surpass regulatory activity, active immunotherapy targeting the lymphoma idiotype may regain appeal as truly personalized immune therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Carcinogenesis of renal cell carcinoma reflected in HLA ligands: A novel approach for synergistic peptide vaccination design

ABSTRACTDespite recent advances in immunotherapy of renal cell carcinoma (RCC), peptide vaccination strategies still lack an approach for personalized peptide vaccination that takes intra- and inter-tumoral heterogeneity and biological characteristics into account. In this study, we use an immunoprecipitation and mass spectrometry-based approach supplemented by network analysis of HLA ligands to target this goal. By analyzing HLA-presented peptides for HLA class I and II of 11 malignant and 6 non-malignant kidney tissue samples, more than 2,700 peptides and 1,600 corresponding source proteins were identified. A high overlap with HLA ligands derived from peripheral blood mononuclear cells (PBMCs) was detected most likely due to tumor-infiltrating inflammatory cells and therefore excluded from network analysis. Subsequent biological function analysis of HLA ligands by the GeneMANIA online platform showed enrichment for well established, but also novel, pathways and biological processes involved in carcinogenesis of RCC almost exclusively in malignant tissue samples. By exploring source proteins involved in these overrepresented pathways, we verified various known tumor-associated antigens (TAAs) for RCC (e.g., CA9, EGLN3, IGFBP3, MMP7, PAX2, VEGFA, or EGFR) but could also detect novel TAAs for RCC (e.g., PLOD2, LOX, ENPEP, or TGFBI). Some of these new TAAs had already been shown to elicit a T cell response in cancer patients. Thus, network analysis of HLA ligands provided a new platform for implementing personalized, multipeptide vaccines with potentially synergistic antitumor effects.

T Cell Receptor Gene Therapy Targeting the Intracellular Transcription Factor Bob1 for the Treatment of Multiple Myeloma and Other B Cell Malignancies

Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. In contrast to mAbs and CARs, T cell receptors (TCRs) recognize antigen-derived peptides that are bound to human leukocyte antigen (HLA) molecules on the cell surface. Since HLA molecules constantly sample the entire endogenous proteome of a cell, extracellular and intracellular antigens are presented and can thus be recognized by a TCR.Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to TCRs and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a HLA-A2/B7-negative healthy individual we isolated T cell clone 4G11 demonstrating high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. No harmful toxicities of clone 4G11 were observed against a wide panel of Bob1-negative stimulator cells including HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Furthermore, stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. Clone 4G11 demonstrated clinical applicability by efficiently recognizing HLA-B7+ primary ALL, CLL and MCL. Furthermore, reproducible strong recognition of purified primary HLA-B7+ MM could be demonstrated.Therefore, the TCR of clone 4G11 may be used for immunotherapy by administering TCR-transduced T cells to patients suffering from B cell malignancies including multiple myeloma. Retroviral gene transfer of TCR 4G11 led to efficient cell surface expression demonstrated by binding of TCR-transduced CD8+ T cells to pMHC-tetramer composed of peptide Bob144 bound to HLA-B7. TCR-modified CD8+ T cells strongly recognized Bob1-expressing HLA-B7+ multiple myeloma cell lines U266 and UM9, and ALL cell lines. TCR-modified T cells efficiently lysed HLA-B7+ primary ALL, CLL and MCL at very low effector-to-target ratios. In addition, highly purified primary multiple myeloma samples were also readily lysed. Furthermore, TCR-transduced T cells strongly proliferated in an antigen-specific manner when stimulated with primary malignant cell samples including ALL, CLL, and MCL or MM cell lines. As expected, TCR-transduced T cells also lysed autologous primary and CD40L-stimulated B cells since these targets cells also express Bob1. In contrast, no lysis of Bob1-negative autologous primary and activated T cells, or monocytes was observed when co-cultured with TCR-transduced T cells.In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. Gene transfer of TCR of clone 4G11 installed Bob1-reactivity and specificity onto recipient T cells shown here by cytolytic capacity and proliferation upon antigen encounter. TCR gene transfer approaches using this Bob1-specific TCR can bring novel treatment modalities and possibly curative therapy to patients with B cell malignancies including multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

ImmTACs: bi-specific TCR-based reagents for targeted cancer immunotherapy (VAC7P.1037)

Abstract ImmTACs (immune mobilizing monoclonal T cell receptors against cancer) are a new class of soluble bi-specific reagent that exploit the natural antigen recognition pathway mediated by the T cell receptor (TCR). As TCRs recognise HLA-presented peptides, ImmTACs can access the larger pool of intracellular tumor antigens unavailable to antibody based therapies. ImmTACs target tumors via a soluble monocloncal TCR, engineered to recognise a given tumor associated peptide-HLA complex with exceptionally high sensitivity and specificity. Once bound to their target, ImmTACs redirect host polyclonal T cells via an anti-CD3 antibody fragment. In this way ImmTACs facilitate targeted T cell recognition of tumors leading to potent T cell activation and tumor cell killing, with the potential to break T cell tolerance. ImmTACs are specific for humans, thus efficacy, specificity and off target effects cannot be assessed in animal models. Using antigen positive tumor cells and HLA appropriate primary human cell lines representing a range of tissues, the in vitro pre-clinical package can be predictive of in vivo clinical observations. A first ImmTAC, IMCgp100, has entered phase I/IIa clinical trials for patients with advanced melanoma. The drug is well tolerated and many patients show tumor shrinkage after the first dose of ImmTAC with tumor shrinkage improving with each dose. Emerging data demonstrate durable partial responses in three patients and a complete response in a fourth patient.

T Cell Receptors Specific for the Intracellular Transcription Factor Bob1 Allow Efficient Targeting of Human B Cell Leukemia and Multiple Myeloma

Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. However, loss of CD20 and CD19 expression or absence of these molecules on other malignancies such as multiple myeloma restricts their application.Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to T cell receptors (TCRs) and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a total of 3 x 109 peripheral blood mononuclear cells from 6 different HLA-A2/B7-negative healthy donors, we isolated and clonally expanded more than 1000 CD8+ T cells binding to peptide-MHC-tetramers composed of the Bob1-derived peptides bound to HLA-A2 or HLA-B7. The T cell clones were tested for stringent peptide-specificity by stimulation with Bob1-negative K562 cells expressing either HLA-A2 or B7 unloaded or pulsed with Bob1-derived peptides. This resulted in the selection of 15 T cell clones highly specific for Bob1. To identify the T cell clones of highest avidity, T cell clones were compared for peptide-sensitivity by testing the recognition of stimulator cells loaded with titrated amounts of Bob1-derived peptides and of Bob1-expressing HLA-A2/B7-positive EBV-transformed B cells. T cell clone 4G11 was selected because of high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7 and T cell clone 3C10 specifically recognized peptide Bob1245 bound to HLA-A2. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. To investigate whether harmful toxicities could be caused by these T cell clones, we tested their reactivity against a wide panel of Bob1-negative stimulator cells demonstrating absence of recognition of HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. These data illustrate a safe reactivity profile with little chance of off-target toxicity. To test their clinical applicability, clone 4G11 and 3C10 were tested for recognition of various primary B cell malignancies. Clone 4G11 efficiently recognized HLA-B7-positive primary ALL, CLL and mantle cell lymphoma while clone 3C10 recognized HLA-A2-positive primary B cell malignancies albeit to a lesser degree. Furthermore, reproducible strong recognition of purified primary HLA-B7-positive multiple myeloma could be demonstrated for clone 4G11. Therefore, T cell clone 4G11’s TCR may be used for immunotherapy by administering TCR-transduced T cells to multiple myeloma patients. To test whether introduction of 4G11’s TCR confers Bob1-reactivity onto recipient cells, the TCR was cloned into a retroviral vector. Highly specific reactivity against HLA-B7-positive Bob1-expressing target cells could be installed to TCR-transduced recipient T cells.In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies and multiple myeloma. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. TCR gene transfer approaches using Bob1-specific TCRs can bring novel treatment modalities for patients with B cell malignancies or multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

HLA Ligandome Profiling Identifies a Novel Category of Pathophysiologically Relevant CLL Associated Antigens

The breakthrough clinical success of immune checkpoint inhibition with PD-1 and CTLA-4 antibodies illustrates the potential of T cell based immunotherapy to effectively treat malignancies. However, a remaining major challenge is to increase the specificity and frequency of anti-cancer immune responses. A rational approach to achieve this goal is therapeutic vaccination, as highlighted by a recent phase II vaccination study using naturally presented HLA ligands, which induced vaccine-specific immune responses that were associated with improved clinical outcome (Walter et al., Nat. Med. 2012). This underscores the potential of identifying patho-physiologically relevant targets by direct differential analysis of the entire landscape of HLA-presented peptides, termed the HLA ligandome.Here we applied this approach to chronic lymphocytic leukemia (CLL) with the aim of developing a CLL-specific multi-peptide vaccine. Using cohorts of 30 patients and 30 healthy volunteers, we comparatively and extensively mapped the HLA ligandome landscape of CLL and identified more than 20,000 different HLA class I and II ligands representing 7,377 source proteins, attaining >95% of the maximum attainable coverage (as extrapolated from saturation analysis). A set of 49 HLA ligand source proteins (225 different HLA ligands) showed CLL-exclusive representation in >20% of CLL patients, thereby constituting the novel category of ligandome-derived tumor associated antigens (LiTAAs). These LiTAAs were validated to be broadly and frequently represented across different stages and mutational subtypes of CLL and found to be robustly represented in HLA ligandomes of patients undergoing standard chemo-/immunotherapy. Functional characterization by IFNγ-ELISPOT revealed peptide-specific immune recognition of 14/15 (93.3%) corresponding HLA ligands (LiTAPs) exclusively in CLL patients. These immune responses were strictly CLL (LiTAP-) directed and mediated by functional patient-derived CD8+ T cells. Notably, for immunoreactive LITAPs, a linear correlation between frequencies of representation in the CLL ligandomes and immune recognition by CLL patient PBMC was observed (R²=0.59), suggesting that LiTAP presentation on cancer cells is a direct prerequisite for the presence of anti-LiTAP immune responses. Moreover, we investigated the prognostic relevance of LiTAP-specific immune responses: Retrospective survival analysis of 45 CLL patients analyzed by IFNγ-ELISPOT assays comparing cases with T cell responses specific for 0-1 LiTAPs (n=32) versus >1 LiTAPs (n=13) suggests a prolonged overall survival in the multiple-response cohort as compared to the single/non-responders (P=0.0695, log-rank test, Fig. 1). Taken together these data provide clear evidence for tumor-dependent priming of LiTAP-specific T cells in CLL patients and indicate their involvement in tumor control in vivo. This directly implies these non-mutant self-peptides as pathophysiologically relevant tumor antigens in CLL thereby validating our target identification approach and encouraging future evaluation in clinical vaccination trials. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells.

Identification of CD8(+) cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4(+) SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)-mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4(+) T cells into strategies designed to enhance T cell immunity. HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4(+) T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.

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

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.

HLA Ligandome Analysis of Acute Myeloid Leukemia (AML) Revealed Novel Tumor-Associated Antigens for Immunotherapy.

AbstractAbstract 3012 Introduction:Eradication of minimal residual disease (MRD) by antigen-specific immunotherapy after conventional chemotherapy and also after allogeneic stem cell transplantation may improve overall survival in acute myeloid leukemia (AML). To achieve this goal there is a need for novel leukemia-associated HLA-presented peptides, which are able to induce a tumor-specific T cell response. Accordingly we here aimed to identify novel leukemia associated antigens employing, for the first time, the approach of direct elution and analysis of naturally processed and presented HLA ligands from the surface of primary AML cells. In particular we focused on peptides derived from AML specific mutated proteins, which are produced only by leukemia cells, only.Methods: HLA class I ligands were isolated from MACS-purified PBMCs of AML patients using immunoprecipitation. Liquid chromatography tandem mass spectrometry (LC-MS/MS) based peptide sequencing was performed to identify HLA presented peptides. The obtained data were mined for leukemia associated peptides by comparison of HLA ligandomes of AML cells with that of PMBCs and granulocytes from healthy donors, investigation of gene expression databases and literature research. All acquired peptide sequences were blasted against the COSMIC database (http://www.sanger.ac.uk/genetics/CGP/cosmic/), to identify sequences from AML specific mutated proteins. Identified mutations were confirmed by PCR. In addition, HLA quantification experiments on the cell surface of AML cells and autologous healthy monocytes and granulocytes were performed using a flow cytometric indirect immunofluorescence assay. Results:Quantitative preliminary results showed similar amounts of HLA class I molecules on AML cells as compared to autologous healthy monocytes and granulocytes lymphocytes (p=0.56, unpaired t-test). For HLA class II we could show a clear upregulation on AML cells compared to autologous healthy leukocytes, not reaching level of significance due to the small cohort examined till now (p= 0.12, unpaired t-test).We were able to identify a total of more than 10.000 peptides from 6 AML patients, including 2 NPM1 mutated AMLs, 1 FLT3-ITD mutated AML, 1 JAK2 mutated AML from osteomyelofibrosis and two AMLs from myelodysplastic syndrome without any cytogenetical aberrations. Using a new approach of spectral counting the obtained sequences could be compared semi-quantitatively to the same amount of peptides obtained from HLA matched healthy volunteers. Thus several new naturally processed and presented leukemia-associated peptides (LEUMAPs) were identified. This comprised, amongst others, ligands of p53, c-myc oncogene, PRAME, RHAMM and NPM1.The sequences of identified LEUMAPs were all verified by LC-MS/MS-based peptide sequencing of their synthetic counterparts. Testing for immunogenicity by ELISPOTs-assay and an in vitro T cell priming approach is presently ongoing. Conclusions:Our analysis revealed several new leukemia associated antigens, which were, for the first time, directly obtained from the HLA ligandomes of AML patients. Quantitative HLA analysis showed no loss or downregulation of HLA molecules on the surface of AML cells, thereby excluding a potential major obstacle for successful immunotherapy. Our results may pave the way for future peptide based immunotherapy approaches to eradicate MRD after obtaining a remission by presently available conventional therapies in AML patients. Disclosures:No relevant conflicts of interest to declare.

Quantification of HLA class I molecules on renal cell carcinoma using Edman degradation

BackgroundUnimpaired HLA class I antigen presentation is a prerequisite for the recognition of tumor cells by cytotoxic T lymphocytes and thus essential for the success of anticancer immunotherapeutic concepts. Several approaches have been taken in the immunotherapy of metastatic renal cell carcinoma (RCC), however of limited success. HLA loss or down-regulation have often been reported and might interfere with immunotherapeutic approaches aimed at the recognition of HLA-presented peptides.MethodsWe employed a quantitative method of molecular analysis for the comparison of HLA amounts on primary tumor, normal kidney and metastases of RCC, using Edman degradation. We analyzed a series of 47 RCC samples including corresponding renal parenchyma, local lymph node metastases and distant metastases.ResultsResults of quantitative Edman degradation revealed significantly higher HLA yields on primary tumor and metastases compared to normal kidney tissue. This effect was shown not to result from infiltrating immune cells, since tumor-infiltrating lymphocytes had no influence on the overall HLA recovery from tumor tissue. Unexpectedly, we found a higher amount of HLA class I molecules on distant metastases compared to local lymph node metastases.ConclusionEdman degradation allows the direct quantitative comparison of HLA class I protein expression by tumor or normal tissue and metastases of RCC patients. Our results raise hopes for improving the success and effectiveness of future immunotherapeutic concepts for metastatic RCC.

HLA ligand profiles of primary renal cell carcinoma maintained in metastases.

In recent years, several approaches have been taken in the peptide-based immunotherapy of metastatic renal cell carcinoma (RCC), although little is known about HLA presentation on metastases compared to primary tumor and normal tissue of RCC. In this study we compared primary tumor, normal tissue and metastases with the aim of identifying similarities and differences between these tissues. We performed this comparison for two RCC patients on the level of the HLA ligandome using mass spectrometry and for three patients on the level of the transcriptome using oligonucleotide microarrays. The quantitative results show that primary tumor is more similar to metastasis than to normal tissue, both on the level of HLA ligand presentation and mRNA. We were able to characterize a total of 142 peptides in the qualitative analysis of HLA-presented peptides. Six of them were significantly overpresented on metastasis, among them a peptide derived from CD151; fourteen were overpresented on both primary tumor and metastasis compared to normal tissue, among them an HLA ligand derived from tumor protein p53. Thus, we could demonstrate that peptide-based immunotherapy might affect tumor as well as metastasis of RCC, but not healthy kidney tissue. Furthermore we were able to identify several peptides derived from tumor-associated antigens that are suitable for vaccination of metastatic RCC.