ISOTOPE: ISOform-guided prediction of epiTOPEs in cancer.

Immunotherapies provide effective treatments for previously untreatable tumors and identifying tumor-specific epitopes can help elucidate the molecular determinants of therapy response. Here, we describe a pipeline, ISOTOPE (ISOform-guided prediction of epiTOPEs In Cancer), for the comprehensive identification of tumor-specific splicing-derived epitopes. Using RNA sequencing and mass spectrometry for MHC-I associated proteins, ISOTOPE identified neoepitopes from tumor-specific splicing events that are potentially presented by MHC-I complexes. Analysis of multiple samples indicates that splicing alterations may affect the production of self-epitopes and generate more candidate neoepitopes than somatic mutations. Although there was no difference in the number of splicing-derived neoepitopes between responders and non-responders to immune therapy, higher MHC-I binding affinity was associated with a positive response. Our analyses highlight the diversity of the immunogenic impacts of tumor-specific splicing alterations and the importance of studying splicing alterations to fully characterize tumors in the context of immunotherapies. ISOTOPE is available at https://github.com/comprna/ISOTOPE.

The MHC Class II Immunopeptidome of Lymph Nodes in Health and in Chemically Induced Colitis.

We recently described a mass spectrometry-based methodology that enables the confident identification of hundreds of peptides bound to murine MHC class II (MHCII) molecules. In this article, we describe its application to the characterization of MHCII-bound peptides isolated from lymph nodes (LNs) of C57BL/6 mice. More than 1000 peptides could be identified in individual analyses, allowing a direct comparison of the MHCII peptidome in different types of normal LNs or in animals with colitis. The peptide length distribution and consensus sequences in axillary, brachial, inguinal, and mesenteric LNs were virtually identical, and a substantial portion of identified peptides corresponded to proteins found in all LNs. However, skin-specific proteins Sbsn and Dmkn and intestine-specific proteins Dmbt1, Krt19, and Maoa, among others, were exclusively identified in skin-draining and mesenteric LNs, respectively. Differences in peptide-presentation patterns were also observed when comparing healthy mice and mice with dextran sodium sulfate-induced colitis. Peptides derived from a subset of proteins (including IgE, Bank1, chondroitin sulfate synthase 2, Cmip, and Fth1) were exclusively identified in mice with colitis, revealing changes in the peptidome associated with the inflammatory process, as well as activation and clonal expansion of B cells.

Membranal and Blood-Soluble HLA Class II Peptidome Analyses Using Data-Dependent and Independent Acquisition.

The interaction between HLA class II peptide complexes on antigen-presenting cells and CD4+ T cells is of fundamental importance for anticancer and antipathogen immunity as well as for the maintenance of immunological tolerance. To study CD4+ T cell reactivities, detailed knowledge of the presented peptides is necessary. In recent years, dramatic advances in the characterization of membranal and soluble HLA class I peptidomes could be observed. However, the same is not true for HLA class II peptidomes, where only few studies identify more than hundred peptides. Here we describe a MS-based workflow for the characterization of membranal and soluble HLA class II DR and DQ peptidomes. Using this workflow, we identify a total of 8595 and 3727 HLA class II peptides from Maver-1 and DOHH2 cells, respectively. Based on this data, a motif-based binding predictor is developed and compared to NetMHCIIpan 3.1. We then apply the workflow to human plasma, resulting in the identification of between 34 and 152 HLA-DR and between 100 and 180 HLA-DQ peptides, respectively. Finally, we implement a data-independent acquisition workflow to increase reproducibility and sensitivity of HLA class II peptidome characterizations.

Data-Independent Acquisition of HLA Class I Peptidomes on the Q Exactive Mass Spectrometer Platform.

The characterization of peptides presented by human leukocyte antigen (HLA) class I molecules is crucial for understanding immune processes, biomarker discovery, and the development of novel immunotherapies or vaccines. Mass spectrometry allows the direct identification of thousands of HLA-bound peptides from cell lines, blood, or tissue. In recent years, data-independent acquisition (DIA) mass spectrometry methods have evolved, promising to increase reproducibility and sensitivity over classical data-dependent acquisition (DDA) workflows. Here, we describe a DIA setup on the Q Exactive mass spectrometer, optimized regarding the unique properties of HLA class I peptides. The methodology enables sensitive and highly reproducible characterization of HLA peptidomes from individual cell lines. From up to 16 DDA analyses of 100 million human cells, more than 10 000 peptides could be confidently identified, serving as basis for the generation of spectral libraries. This knowledge enabled the subsequent interrogation of DIA data, leading to the identification of peptide sets with >90% overlap between replicate samples, a prerequisite for the comparative study of closely related specimens. Furthermore, >3000 peptides could be identified from just one million cells after DIA analysis using a library generated from 300 million cells. The reduction in sample quantity and the high reproducibility of DIA-based HLA peptidome analysis should facilitate personalized medicine applications.

Purification of soluble HLA class I complexes from human serum or plasma deliver high quality immuno peptidomes required for biomarker discovery.

Soluble human leukocyte antigen class I (sHLA)-peptide complexes have been suggested to play a role in the modulation of immune responses and in immune evasion of cancer cells. The set of peptides eluted from sHLA molecules could serve as biomarker for the monitoring of patients with cancer or other conditions. Here, we describe an improved sHLA peptidomics methodology resulting in the identification of 1816 to 2761 unique peptide sequences from triplicate analyses of serum or plasma taken from three healthy donors. More than 90% of the identified peptides were 8-11mers and 74% of these sequences were predicted to bind to cognate HLA alleles, confirming the quality of the resulting immunopeptidomes. In comparison to the HLA peptidome of cultured cells, the plasma-derived peptides were predicted to have a higher stability in complex with the cognate HLA molecules and mainly derived from proteins of the plasma membrane or from the extracellular space. The sHLA peptidomes can efficiently be characterized by using the new methodology, thus serving as potential source of biomarkers in various pathological conditions.

High-resolution analysis of the murine MHC class II immunopeptidome.

The reliable identification of peptides bound to major histocompatibility complex (MHC) class II is fundamental for the study of the host immune response against pathogens and the pathogenesis of autoimmune conditions. Here, we describe an improved methodology combining immuno-affinity enrichment of MHC class II complexes, optimized elution conditions and quadrupole Orbitrap mass spectrometry-based characterization of the immunopeptidome. The methodology allowed the identification of over 1000 peptides with 1% false discovery rate from 10(8) murine A20 lymphoma cells. The study revealed the I-A(d) -specific motif in high resolution after multisequence alignment. The methodology was generally applied to the purification of MHC class II from cell lines and murine spleens. We identified 2963 peptides from BALB/c and 2712 from C57BL/6 mouse spleens. The identification of peptides bound to MHC class II in vitro and in vivo will facilitate the characterization of T-cell specificities, as well as the development of biotherapeutics and vaccines.

High-sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients' sera.

The characterization of peptides bound to human leukocyte antigen (HLA) class I is of fundamental importance for understanding CD8+ T cell-driven immunological processes and for the development of immunomodulatory therapeutic strategies. However, until now, the mass spectrometric analysis of HLA-bound peptides has typically required billions of cells, still resulting in relatively few high-confidence peptide identifications. Capitalizing on the recent developments in mass spectrometry and bioinformatics, we have implemented a methodology for the efficient recovery of acid-eluted HLA peptides after purification with the pan-reactive antibody W6/32 and have identified a total of 27 862 unique peptides with high confidence (1% false discovery rate) from five human cancer cell lines. More than 93% of the identified peptides were eight to 11 amino acids in length and contained signatures that were in excellent agreement with published HLA binding motifs. Furthermore, by purifying soluble HLA class I complexes (sHLA) from sera of melanoma patients, up to 972 high-confidence peptides could be identified, including melanoma-associated antigens already described in the literature. Knowledge of the HLA class I peptidome should facilitate multiplex tetramer technology-based characterization of T cells, and allow the development of patient selection, stratification and immunomodulatory therapeutic strategies.

Mass spectrometric analysis of the HLA class I peptidome of melanoma cell lines as a promising tool for the identification of putative tumor-associated HLA epitopes.

Melanoma is one of the most immunogenic tumors, and extensive lists of potential tumor rejection antigens have been collected during the last decades. By isolating human leukocyte antigen (HLA) class I complexes from five melanoma cell lines (FM-82, FM-93/2, Mel-624, MeWo and SK-Mel-5) and sequencing HLA-eluted peptides by mass spectrometry, we identified over 10,000 unique peptides with high confidence. The majority of the peptides were 8-11 amino acids in length and were predicted to bind to the respective HLA alleles. Over 250 epitopes, corresponding to previously described tumor-associated antigens, were identified, suggesting that HLA peptidome analysis may facilitate the characterization of putative tumor rejection antigens. MeWo and SK-Mel-5 cell lines were further interrogated for neo-epitopes, revealing one peptide from MeWo cells carrying an amino acid mutation. We also observed a remarkable overlap between A*03:01 peptides eluted from Mel-624 cells and A*03:01 peptides recovered from soluble HLA complexes purified from two melanoma patients, shedding light on the similarity of the HLA peptidome in cell lines and in patient-derived material. The reliable characterization of the HLA class I peptidome in melanoma promises to facilitate the identification of tumor rejection antigens and the development of immunotherapeutic strategies.

In vivo biotinylation of the vasculature in B-cell lymphoma identifies BST-2 as a target for antibody-based therapy.

The discovery of accessible markers of lymphoma may facilitate the development of antibody-based therapeutic strategies. Here, we describe the results of a chemical proteomic study, based on the in vivo biotinylation of vascular proteins in lymphoma-bearing mice followed by mass spectrometric and bioinformatic analysis, to discover proteins expressed at the tissue-blood border of disseminated B-cell lymphoma. From a list of 58 proteins, which were more than 10-fold up-regulated in nodal and extranodal lymphoma lesions compared with their levels in the corresponding normal host organs, we validated BST-2 as a novel vascular marker of B-cell lymphoma, using immunochemical techniques and in vivo biodistribution studies. Furthermore, targeting BST-2 with 2 independent monoclonal antibodies delayed lymphoma growth in a syngeneic mouse model of the disease. The results of this study delineate a strategy for the treatment of systemic B-cell lymphoma in humans and suggest that anti-BST-2 antibodies may facilitate pharmacodelivery approaches that target the tumor-stroma interface.

Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein.

Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport.

Proteomic identification of vanin-1 as a marker of kidney damage in a rat model of type 1 diabetic nephropathy.

At present, the urinary albumin excretion rate is the best noninvasive predictor for diabetic nephropathy (DN) but major limitations are associated with this marker. Here, we used in vivo perfusion technology to establish disease progression markers in an animal model of DN. Rats were perfused with a reactive ester derivative of biotin at various times after streptozotocin treatment. Following homogenization of kidney tissue and affinity purification of biotinylated proteins, a label-free mass spectrometry-based proteomic analysis of tryptic digests identified and relatively quantified 396 proteins. Of these proteins, 24 and 11 were found to be more than 10-fold up- or downregulated, respectively, compared with the same procedure in vehicle-treated rats. Changes in the expression of selected differentially regulated proteins were validated by immunofluorescence detection in kidney tissue from control and diabetic rats. Immunoblot analysis of pooled human urine found that concentrations of vanin-1, an ectoenzyme pantetheinase, distinguished diabetic patients with macroalbuminuria from those with normal albuminuria. Uromodulin was elevated in the urine pools of the diabetic patients, regardless of the degree of albuminuria, compared with healthy controls. Thus, in vivo biotinylation facilitates the detection of disease-specific changes in the abundance of potential biomarker proteins for disease monitoring and/or pharmacodelivery applications.

DeepQuanTR: MALDI-MS-based label-free quantification of proteins in complex biological samples.

The quantification of changes in protein abundance in complex biological specimens is essential for proteomic studies in basic and applied research. Here we report on the development and validation of the DeepQuanTR software for identification and quantification of differentially expressed proteins using LC-MALDI-MS. Following enzymatic digestion, HPLC peptide separation and normalization of MALDI-MS signal intensities to the ones of internal standards, the software extracts peptide features, adjusts differences in HPLC retention times and performs a relative quantification of features. The annotation of multiple peptides to the corresponding parent protein allows the definition of a Protein Quant Value, which is related to protein abundance and which allows inter-sample comparisons. The performance of DeepQuanTR was evaluated by analyzing 24 samples deriving from human serum spiked with different amounts of four proteins and eight complex samples of vascular proteins, derived from surgically resected human kidneys with cancer following ex vivo perfusion with a reactive ester biotin derivative. The identification and experimental validation of proteins, which were differentially regulated in cancerous lesions as compared with normal kidney, was used to demonstrate the power of DeepQuanTR. This software, which can easily be used with established proteomic methodologies, facilitates the relative quantification of proteins derived from a wide variety of different samples.

A novel reactive ester derivative of biotin with reduced membrane permeability for in vivo biotinylation experiments.

The in vivo perfusion of rodent models of disease with biotin derivatives and the subsequent comparative proteomic analysis of healthy and diseased tissues represent a promising methodology for the identification of vascular accessible biomarkers. A novel, triply charged biotinylation reagent, NHS-ß-Ala-(L-Asp)(3)-biotin, was synthesized and validated in terms of its applicability for in vivo protein biotinylation. Compared to sulfo-NHS-LC-biotin, NHS-ß-Ala-(L-Asp)(3)-biotin exhibited a reduced membrane permeability and a preferential labeling of proteins localized in compartments readily accessible in vivo from the vasculature.

Improved protein sequence coverage by on resin deglycosylation and cysteine modification for biomarker discovery.

Membrane proteins and secreted factors (soluble proteins or extracellular matrix components) are the targets of most monoclonal antibodies, which are currently in clinical development. These proteins are frequently post-translationally modified, e.g. by the formation of disulfide bonds or by glycosylation, which complicates their identification using proteomics technologies. Here, we describe a novel methodology for the on resin deglycosylation and cysteine modification of proteins after in vitro, in vivo or ex vivo biotinylation. Biotinylated proteins are captured on streptavidin resin and all subsequent modifications, as well as the proteolytic digestion, which yields peptides for MS analysis, are performed on resin. Using biotinylated bovine fetuin-A as a test protein, an improvement in sequence coverage from 7.9 to 58.7% could be shown, including the identification of all three glycosylation sites. Furthermore, a complex mixture derived from the ex vivo biotinylation of vascular structures in human kidney with cancer obtained by perfusion after surgical resection revealed almost a doubling of sequence coverage for all checked proteins when analyzed by LC-MALDI TOF/TOF.

Antibody-based Delivery of TNF to the Tumor Neovasculature Potentiates the Therapeutic Activity of a Peptide Anticancer Vaccine.

PURPOSE: There is a growing interest in the use of tumor antigens for therapeutic vaccination strategies. Unfortunately, in most cases, the use of peptide vaccines in patients does not mediate shrinkage of solid tumor masses.Experimental Design: Here, we studied the opportunity to boost peptide vaccination with F8-TNF, an antibody fusion protein that selectively delivers TNF to the tumor extracellular matrix. AH1, a model antigen to investigate CD8+ T-cell immunity in BALB/c mice, was used as vaccine. RESULTS: Peptide antigens alone exhibited only a modest tumor growth inhibition. However, anticancer activity could be substantially increased by combination with F8-TNF. Analysis of T cells in tumors and in draining lymph nodes revealed a dramatic expansion of AH1-specific CD8+ T cells, which were strongly positive for PD-1, LAG-3, and TIM-3. The synergistic anticancer activity, observed in the combined use of peptide vaccination and F8-TNF, was largely due to the ability of the fusion protein to induce a rapid hemorrhagic necrosis in the tumor mass, thus leaving few residual tumor cells. While the cell surface phenotype of tumor-infiltrating CD8+ T cells did not substantially change upon treatment, the proportion of AH1-specific T cells was strongly increased in the combination therapy group, reaching more than 50% of the CD8+ T cells within the tumor mass. CONCLUSIONS: Because both peptide vaccination strategies and tumor-homing TNF fusion proteins are currently being studied in clinical trials, our study provides a rationale for the combination of these 2 regimens for the treatment of patients with cancer.

Antibody-Based Delivery of Cytokine Payloads to Carbonic Anhydrase IX Leads to Cancer Cures in Immunocompetent Tumor-Bearing Mice.

Antibody-cytokine fusion proteins can have the potential to increase the density and activity of subsets of leukocytes within the tumor mass. Here, we describe the design, production, and characterization of four novel antibody-cytokine fusion proteins directed against human carbonic anhydrase IX, a highly validated marker of hypoxia that is overexpressed in clear cell renal cell carcinoma and other malignancies. As immunomodulatory payloads we used TNF, IL2, IFNα2 (corresponding to products that are in clinical use), and IL12 (as this cytokine potently activates T cells and NK cells). Therapy experiments were performed in BALB/c mice, bearing CT26 tumors transfected with human carbonic anhydrase IX, in order to assess the performance of the fusion proteins in an immunocompetent setting. The biopharmaceuticals featuring TNF, IL2, or IL12 as payloads cured all mice in their therapy groups, whereas only a subset of mice was cured by the antibody-based delivery of IFNα2. Although the antibody fusion with TNF mediated a rapid hemorrhagic necrosis of the tumor mass, a slower regression of the neoplastic lesions (which continued after the last injection) was observed with the other fusion proteins, and treated mice acquired protective anticancer immunity. A high proportion of tumor-infiltrating CD8+ T cells was specific to the retroviral antigen AH1; however, the LGPGREYRAL peptide derived from human carbonic anhydrase IX was also present on tumor cells. The results described herein provide a rationale for the clinical use of fully human antibody-cytokine fusions specific to carbonic anhydrase IX.

T cell recognition of novel shared breast cancer antigens is frequently observed in peripheral blood of breast cancer patients.

Advances within cancer immunotherapy have fueled a paradigm shift in cancer treatment, resulting in increasing numbers of cancer types benefitting from novel treatment options. Despite originally being considered an immunologically silent malignancy, recent studies encourage the research of breast cancer immunogenicity to evaluate immunotherapy as a treatment strategy. However, the epitope landscape in breast cancer is minimally described, limiting the options for antigen-specific, targeted strategies. Aromatase, never in mitosis A-related kinase 3 (NEK3), protein inhibitor of activated STAT3 (PIAS3), and prolactin are known as upregulated proteins in breast cancer. In the present study, these four proteins are identified as novel T cell targets in breast cancer. From the four proteins, 147 peptides were determined to bind HLA-A*0201 and -B*0702 using a combined in silico/in vitro affinity screening. T cell recognition of all 147 peptide-HLA-A*0201/-B*0702 combinations was assessed through the use of a novel high-throughput method utilizing DNA barcode labeled multimers. T cell recognition of sequences within all four proteins was demonstrated in peripheral blood of patients, and significantly more T cell responses were detected in patients compared to healthy donors for both HLA-A*0201 and -B*0702. Notably, several of the identified responses were directed toward peptides, with a predicted low or intermediate binding affinity. This demonstrates the importance of including low-affinity binders in the search for epitopes within shared tumor associated antigens (TAAs), as these might be less subject to immune tolerance mechanisms. The study presents four novel TAAs containing multiple possible targets for immunotherapy of breast cancer.

Intron retention is a source of neoepitopes in cancer.

We present an in silico approach to identifying neoepitopes derived from intron retention events in tumor transcriptomes. Using mass spectrometry immunopeptidome analysis, we show that retained intron neoepitopes are processed and presented on MHC I on the surface of cancer cell lines. RNA-derived neoepitopes should be considered for prospective personalized cancer vaccine development.

Sarcoma Eradication by Doxorubicin and Targeted TNF Relies upon CD8+ T-cell Recognition of a Retroviral Antigen.

Antibody-cytokine complexes may offer new tools to treat cancer. Here, we show how TNF-linked antibodies, which recognize tumor-selective splice isoforms of fibronectin (F8-TNF), can be exploited to eradicate sarcomas in immunocompetent mice. We treated mice bearing WEHI-164 fibrosarcoma with a combination of F8-TNF and doxorubicin, curing the majority of treated animals (29/37). Notably, cured mice were resistant to rechallenge not only by WEHI-164 cells but also heterologous C51 or CT26 colorectal tumor cells in a CD8+ T-cell-dependent process. Mechanistic analyses revealed that each tumor cell line presented AH1, a common endogenous retroviral peptide. Numbers of AH1-specific CD8+ T cells exhibiting cytotoxic capacity were increased by F8-TNF plus doxorubicin treatment, arguing that cognate CD8+ T cells contributed to tumor eradication. Sequence analysis of T-cell receptors of CD8+ T cells revealed the presence of H-2Ld/AH1-specific T cells and an expansion of sequence diversity in treated mice. Overall, our findings provide evidence that retroviral genes contribute to tumoral immunosurveillance in a process that can be generally boosted by F8-TNF and doxorubicin treatment. Cancer Res; 77(13); 3644-54. ©2017 AACR.

Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA.

The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.