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The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity (1-6), but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion (1,2,7). Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cell (s8,9). Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits--including durable responses--to patients with different malignancies (10-13). However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments., In this study, we used two distinct progresser 3-methylcholanthrene-induced (MCA) sarcoma cell lines (d42m1-T3 and F244) and asked whether they expressed sufficient immunogenicity to be controlled by checkpoint blockade immunotherapy. [...]

T cell receptor (TCR)–based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide–human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 (COL6A3) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors. COL-ing out TCR-Ts: Adoptive T cell therapy targeting neoantigens is a promising new therapeutic technique; however, the lack of shared neoantigens across patients limits the use of this technique. To overcome this, Kim et al. identified a shared pan-cancer collagen type VI α-3 (COL6A3) epitope that was presented on tumor stroma and was the result of an alternative splicing event. They created affinity-enhanced T cell receptor T (TCR-T) cells and treated mice in vivo to show regression in tumors that expressed physiological levels of the targeted pHLA without toxicity to normal cells. This method represents a promising treatment to target multiple cancer types and warrants further clinical investigation. [ABSTRACT FROM AUTHOR]

Recent studies suggest that CLL is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell-based immunotherapy. However, CLL is associated with a profound immune defect, which might represent a critical limitation for mounting clinically effective antitumor immune responses. As several studies have demonstrated that lenalidomide can reinforce effector T-cell responses in CLL, the combination of T-cell-based immunotherapy with the immunomodulatory drug lenalidomide represents a promising approach to overcome the immunosuppressive state in CLL. Antigen-specific immunotherapy also requires the robust presentation of tumor-associated HLA-presented antigens on target cells. We thus performed a longitudinal study of the effect of lenalidomide on the HLA ligandome of primary CLL cells in vitro. We showed that lenalidomide exposure does not affect absolute HLA class I and II surface expression levels on primary CLL cells. Importantly, semi-quantitative mass spectrometric analyses of the HLA peptidome of three CLL patient samples found only minor qualitative and quantitative effects of lenalidomide on HLA class I- and II-restricted peptide presentation. Furthermore, we confirmed stable presentation of previously described CLL-associated antigens under lenalidomide treatment. Strikingly, among the few HLA ligands showing significant modulation under lenalidomide treatment, we identified upregulated IKZF-derived peptides, which may represent a direct reflection of the cereblon-mediated effect of lenalidomide on CLL cells. Since we could not observe any relevant influence of lenalidomide on the established CLL-associated antigen targets of anticancer T-cell responses, this study validates the suitability of lenalidomide for the combination with antigen-specific T-cell-based immunotherapies. [ABSTRACT FROM AUTHOR]

The classical HLA-C and the nonclassical HLA-E and HLA-G molecules play important roles both in the innate and adaptive immune system. Starting already during embryogenesis and continuing throughout our lives, these three Ags exert major functions in immune tolerance, defense against infections, and anticancer immune responses. Despite these important roles, identification and characterization of the peptides presented by these molecules has been lacking behind the more abundant HLA-A and HLA-B gene products. In this study, we elucidated the peptide specificities of these HLA molecules using a comprehensive analysis of naturally presented peptides. To that end, the 15 most frequently expressed HLA-C alleles as well as HLA-E*01:01 and HLA-G*01:01 were transfected into lymphoblastoid C1R cells expressing low endogenous HLA. Identification of naturally presented peptides was performed by immunoprecipitation of HLA and subsequent analysis of HLA-bound peptides by liquid chromatographic tandem mass spectrometry. Peptide motifs of HLA-C unveil anchors in position 2 or 3 with high variances between allotypes, and a less variable anchor at the C-terminal end. The previously reported small ligand repertoire of HLA-E was confirmed within our analysis, and we could show that HLA-G combines a large ligand repertoire with distinct features anchoring peptides at positions 3 and 9, supported by an auxiliary anchor in position 1 and preferred residues in positions 2 and 7. The wealth of HLA ligands resulted in prediction matrices for octa-, nona-, and decamers. Matrices were validated in terms of their binding prediction and compared with the latest NetMHC prediction algorithm NetMHCpan-3.0, which demonstrated their predictive power. [ABSTRACT FROM AUTHOR]

There are limitations in pre-clinical settings using mice as a basis for clinical development in humans. In cancer, similarities exist between humans and dogs; thus, the dog patient can be a link in the transition from laboratory research on mouse models to clinical trials in humans. Knowledge of the peptides presented on MHC molecules is fundamental for the development of highly specific T cell-based immunotherapies. This information is available for human MHC molecules but is absent for the canine MHC. In the present study, we characterized the binding motif of dog leukocyte antigen (DLA) class I allele DLA-88*50101, using human C1R and K562 transfected cells expressing the DLA-88*50101 heavy chain. MHC class I immunoaffinity-purification revealed 3720 DLA-88*50101 derived peptides, which enabled the determination of major anchor positions. The characterized binding motif of DLA-88*50101 was similar to HLA-A*02:01. Peptide binding analyses on HLA-A*02:01 and DLA-88*50101 via flow cytometry showed weak binding of DLA-88*50101 derived peptides to HLA-A*02:01, and vice versa. Our results present for the first time a detailed peptide binding motif of the canine MHC class I allelic product DLA-88*50101. These data support the goal of establishing dogs as a suitable animal model for the evaluation and development of T cell-based cancer immunotherapies, benefiting both dog and human patients. [ABSTRACT FROM AUTHOR]

Objective: Increased numbers of tumor-infiltrating lymphocytes (TILs) in high-grade serous ovarian cancer (HGSC) are associated with improved clinical outcome. Intraepithelial localization of TILs might be regulated by specific homing receptors, such as CD103, which is widely expressed by intraepithelial lymphocytes. Given the emerging role ofCD103+ TILs, we aimed to assess their contribution to the prognostic value of immunoscoring in HGSC. Methods: The density of intratumoral CD3+ and CD103+ lymphocytes was examined by immunohistochemistry on a tissue microarray of a series of 135 patients with advanced HGSC and correlated with CD4+, CD8+, CD56+, FoxP3+, and TCRγ+ T-cell counts, as well as E-cadherin staining and conventional prognostic parameters and clinical outcome. Results: Both the presence of CD103+ cells, as well as high numbers of intraepithelial CD3+ lymphocytes (CD3E), showed a significant correlation with overall survival, in the complete series, as well as in patients with optimal debulking and/or platinum sensitivity. Combining CD3 and CD103 counts improved prognostication and identified 3 major subgroups with respect to overall survival. The most pronounced effect was demonstrated for patients with optimally resected and platinum-sensitive tumors. Patients with CD3high/CD103high tumors showed a 5-year survival rate at 90%, CD3low/CD103high at 63%, and CD3low/CD103low at 0% (P < 0.001). Conclusions: These results suggest that combined assessment of CD103 and CD3 counts improves the prognostic value of TIL counts in HGSC and might identify patients with early relapse or long-term survival based on the type and extent of the immune response. [ABSTRACT FROM AUTHOR]

The article presents the study that explores on the community-based efforts in developing robust platform that will be used in the quantitative and reproducible measurement of peptides that are presented by the human leukocyte antigen (HLA) in Switzerland.

Abstract: The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity. [Copyright &y& Elsevier]

MHC class I molecules bind only those peptides with high affinity that conform to stringent length and sequence requirements. We have now investigated which peptides can aid the in vitro folding of class I molecules, and we find that the dipeptide glycyl-leucine efficiently supports the folding of HLA-A*02:01 and H-2Kb into a peptide-receptive conformation that rapidly binds high-affinity peptides. Treatment of cells with glycyl-leucine induces accumulation of peptide-receptive H-Kb and HLA-A*02:01 at the surface of cells. Other dipeptides with a hydrophobic second amino acid show similar enhancement effects. Our data suggest that the dipeptides bind into the F pocket like the C-terminal amino acids of a high-affinity peptide. [ABSTRACT FROM AUTHOR]

Valid and reproducible sampling techniques as well as processing protocols are required for the assessment of biomarkers and mediators contained in wound exudate. Moreover, the ideal technique should be easy to use even in daily clinical routine. This is challenging since wound fluid represents an inhomogeneous mixture of different exogenous and endogenous sources. Analyzing wound fluid, however, may facilitate clinical decision making. Many techniques for obtaining wound fluid have been described. There is very little validation data, and the array of different techniques appears confusing. Structuring and new standards are needed to avoid wound fluid sampling yielding an “undefined soup.” A lot of wound fluid parameters have been analyzed, although none of them have made its way into clinical practice. Nevertheless, basic principles of wound healing have been established from wound fluid analysis. With adequate techniques suitable for daily practice, basic research might foster our clinical understanding of wound healing with implications for new therapies. So far, research has mainly concentrated on analyzing available sample material with respect to either a wide variety of analytes or comparing acute with chronic wound exudate. Clinical endpoints such as healing or wound infection as well as longitudinal data may indeed be more valuable for clinical practice, enabling the discovery of meaningful biomarkers using a suitable technique. [ABSTRACT FROM PUBLISHER]

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-speciflc CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF56-64 and CD59106-114, were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY311-319, an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY311-319 specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides. [ABSTRACT FROM AUTHOR]

Genome sequencing has uncovered an array of recurring somatic mutations in different non-Hodgkin lymphoma (NHL) subtypes. If affecting protein-coding regions, such mutations may yield mutation-derived peptides that may be presented by HLA class I proteins and recognized by cytotoxic T cells. A recurring somatic and oncogenic driver mutation of the Toll-like receptor adaptor proteinMYD88, Leu265Pro (L265P) was identified in up to 90% of different NHL subtype patients. We therefore screened the potential ofMYD88L265P-derived peptides to elicit cytotoxic T cell responses as tumor-specific neoantigens. Based onin silicopredictions, we identified potentialMYD88L265P-containing HLA ligands for several HLA class I restrictions. A set of HLA class IMYD88L265P-derived ligands elicited specific cytotoxic T cell responses for HLA-B*07 and -B*15. These data highlight the potential ofMYD88L265Pmutation-specific peptide-based immunotherapy as a novel personalized treatment approach for patients withMYD88L265P+NHLs that may complement pharmacological approaches targeting oncogenic MyD88 L265P signaling. [ABSTRACT FROM AUTHOR]

Despite 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. [ABSTRACT FROM PUBLISHER]

The recent approval of clincially effective immune checkpoint inhibitors illustrates the potential of cancer immunotherapy. A challenging task remains the identification of specific targets guiding immunotherapy. Facilitated by technical advances, the direct identification of physiologically relevant targets is enabled by analyzing the HLA ligandome of cancer cells. Since recent publications demonstrate the immunogenicity of ovarian cancer (OvCa), immunotherapies, including peptide-based cancer vaccines, represent a promising treatment approach. To identify vaccine peptides, we employed a combined strategy of HLA ligandomics in high-grade serous OvCa samples and immunogenicity analysis. Only few proteins were naturally presented as HLA ligands on all samples analyzed, including histone deacetylase (HDAC) 1 and 2.In vitropriming of CD8+T cells demonstrated that two HDAC1/2-derived HLA ligands can induce T-cell responses, capable of killing HLA-matched tumor cells. High HDAC1 expression shown by immunohistochemistry in 136 high-grade serous OvCa patients associated with significantly reduced overall survival (OS), whereas patients with high numbers of CD3+tumor-infiltrating lymphocytes (TILs) in the tumor epithelium and CD8+TILs in the tumor stroma showed improved OS. However, correlating HDAC1 expression with TILs, high levels of TILs abrogated the impact of HDAC1 on OS. This study strengthens the role of HDAC1/2 as an important tumor antigen in OvCa, demonstrating its impact on OS in a large cohort of OvCa patients. We further identified two immunogenic HDAC1-derived peptides, which frequently induce multi-functional T-cell responses in many donors, suitable for future multi-peptide vaccine trials in OvCa patients. [ABSTRACT FROM AUTHOR]

Background & Aims We report a novel experimental immunotherapeutic approach in a patient with metastatic intrahepatic cholangiocarcinoma. In the 5 year course of the disease, the initial tumor mass, two local recurrences and a lung metastasis were surgically removed. Lacking alternative treatment options, aiming at the induction of anti-tumor T cells responses, we initiated a personalized multi-peptide vaccination, based on in-depth analysis of tumor antigens (immunopeptidome) and sequencing. Methods Tumors were characterized by immunohistochemistry, next-generation sequencing and mass spectrometry of HLA ligands. Results Although several tumor-specific neo-epitopes were predicted in silico , none could be validated by mass spectrometry. Instead, a personalized multi-peptide vaccine containing non-mutated tumor-associated epitopes was designed and applied. Immunomonitoring showed vaccine-induced T cell responses to three out of seven peptides administered. The pulmonary metastasis resected after start of vaccination showed strong immune cell infiltration and perforin positivity, in contrast to the previous lesions. The patient remains clinically healthy, without any radiologically detectable tumors since March 2013 and the vaccination is continued. Conclusions This remarkable clinical course encourages formal clinical studies on adjuvant personalized peptide vaccination in cholangiocarcinoma. Lay Summary Metastatic cholangiocarcinomas, cancers that originate from the liver bile ducts, have very limited treatment options and a fatal prognosis. We describe a novel therapeutic approach in such a patient using a personalized multi-peptide vaccine. This vaccine, developed based on the characterization of the patient’s tumor, evoked detectable anti-tumor immune responses, associating with long-term tumor-free survival. [ABSTRACT FROM AUTHOR]