Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing.

Towards addressing questions related to HIV pathogenesis and vaccine design we are fortunate to have the availability of the SIV-infected rhesus macaque model. The strengths of this model which include a rapid rate of progression to AIDS and knowledge of the dose route and strain of the infecting virus complement studies in HIV-infected patients in which the reagents host genetics and access to samples are more extensive and better defined. Unfortunately there is currently still too little known about the antiviral immune responses in either system to directly and accurately compare their similarities and differences and to draw any definitive conclusions. Therefore the data and views presented herein will simply reflect what has recently been discovered in both humans and non-human primate studies. (excerpt)

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

Introduction: The epidermal growth factor receptor (EGFR) is one of the most frequently mutated oncogenes in human lung cancer. Although immune checkpoint inhibitors have been therapeutically effective in multiple cancers, patients harboring EGFR mutations have not responded well to this immunotherapy. Adoptive T cell therapy (ACT) has resulted in complete and durable regression of metastatic cancers. ACT requires that neoantigens are presented by major histocompatibility complex (MHC) class I molecules on tumor but not normal cells. The MHC binding algorithms, such as IEDB, most commonly used for antigen prediction on MHC molecules is far from perfect; thus, experimental proof of MHC-bound peptides is needed to provide direct evidence of antigen presentation. Method: We developed a mass spectrometry (MS)-based platform integrated with a whole exome sequencing (WES)-based protein database to identify tumor-specific mutant peptides presented by MHC class I. Experiments were conducted using EGFR mutant PC9 lung adenocarcinoma cell line that harbors the EGFRDel746-750mutant. MHC class I proteins and their associated peptides were immunoprecipitated, and the peptides separated from MHC proteins. The MHC proteins were subjected to trypsin digestion and HLA typing using MS. Class I-associated peptides underwent C-18 separation and tandem MS analysis. A PC9-specific database was built adding all SNVs and INDELs identified by WES to the normal human database and used to search MS data. Peptides identified by MS were subjected to IEDB algorithm search using PC9 specific HLA types. Results: The MS HLA typing identified A*02, A*24, and B*39 in PC9 cells. We identified 13,765 MHC bound peptides. 10,711 peptides were 6~15 amino acid residues in length (6~15mer), which are generally considered as class I epitopes. Using the PC9-specific protein database, 11 variant peptides (neo-peptides) were identified. These include plectin isoform 1 (PLEC_H1459R, 9mer), proteasome subunit beta type-4 (PSMB4_I234T, 9mer), prostaglandin reductase 1 (PTGR1_A27S, 8mer), cytochrome b-c1 complex subunit Rieske mitochondrial (UQCRFS1_S6A, 7mer) and obscurin-like protein 1 (OBSL1_E1365D, 10mer). The IEDB results predicted the binding of PLEC_H1459R with HLA-B*39, OBSL1_E1365D with HLA-A*02 and PSMB4_I234T with HLA-A*24; however, 8 identified variant peptides were not predicted by IEDB. Conclusion: Our MS data provided direct experimental evidence for MHC class I presentation of 11 neopeptides in PC9 cells. Neopeptides discovered by this proteogenomic platform could potentially trigger T cell response, which needs to be validated by immunological assays. Experiments are underway to utilize this workflow to identify variant neopeptides from patient tumors and patient-derived xenografts (PDXs). Citation Format: Yue Qi, Tapan K. Maity, Xu Zhang, Shaojian Gao, Nitin Roper, Meriam Bahta, Khoadang Nguyen, Constance M. Cultraro, Udayan Guha. Integrated proteogenomics utilizing mass spectrometry to identify MHC-associated neopeptides in EGFR mutant lung adenocarcinoma [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 1438.

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.

Background: We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Accumulation of somatic mutations is one characteristic feature of malignanT-cells. These single-nucleotide variants (SNVs) can lead to altered amino acid sequences of the translated proteins, which in turn can be presented by malignant cells as antigenic peptides on HLA molecules. A peptide vaccination to induce neoantigen-specific T-cell responses, therefore, is a promising and versatile immunotherapeutic approach for the treatment of malignant diseases. Such approaches were predominantly applied in malignancies with high mutational load in adult patients until now. We developed a vaccine design platform based on sequencing data generated from a cohort of acute lymphoblastic leukemia (ALL) patients and tested individualized peptide vaccines in pediatric patients suffering from a variety of tumors with low mutational profiles. Methods: Nonsynonymous mutations were identified by whole-exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. HLA binding peptides harboring the altered amino acids were subsequently predicted in silico by algorithms SYFPEITHI, NetMHC and NetMHCpan for the patients’ individual HLA type. Individual peptides for treatment attempts were produced by chemical synthesis and vaccination cocktails were formulated. The vaccination schedule was 16 vaccinations over 33 weeks using GM-CSF and Imiquimod as adjuvants. Some patients received simultaneous checkpoint blockade treatment with pembrolizumab or nivolumab. Response to the vaccination was monitored by detection of T-cells recognizing the vaccinated peptides occurring over time in peripheral blood of the patients. Monitoring was performed for each vaccination time point by pre-stimulation with the peptides and subsequent intracellular cytokine staining (ICS) of T-cells and FACS analysis. Results: Whole-exome sequencing was performed for 25 patients to identify ALL-specific SNVs using a comparative bioinformatics pipeline. We found an average of 39.2 mutations per patient on DNA level, with an average validation rate of 47% by RNA sequencing. Based on these data, an average of 35.1 HLA binders could be predicted per patient. We applied our platform for 6 patients with various malignancies based on compassionate need and designed individual peptide vaccines. In all cases validated mutations could be identified and epitope prediction was performed for HLA Class I and II binders. In 6/6 patients a de novo induced T-cell response against the vaccinated mutated HLA-binding peptides was detectable. Combination therapy with PD-1 blockade and peptide vaccination was well tolerated. T-cell responses were predominantly, but not exclusively, CD4+-restricted. Encouraged by these findings, we started a phase I/II clinical trial in patients with primary/relapsed ALL with the aim to prevent relapse and show safety and immunogenicity of the personalized peptide vaccine. Conclusions: We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Whole-exome sequencing of pediatric ALL patients is feasible and yields a small amount of tumor-specific mutations per patient. However, these few mutations are sufficient to predict HLA-binding peptides that are immunogenic when vaccinated and elicit specific T-cell responses in patients. The universal character of individualized peptide vaccination allows for application in virtually any malignant disease, as well as combination therapy approaches. The concept is now translated to clinical application in a phase I/II clinical trial in ALL, started in 2016 (NCT03559413). Citation Format: Armin Rabsteyn, Christina Kyzirakos, Christopher Schroeder, Marc Sturm, Christopher Mohr, Jakob Matthes, Magdalena Feldhahn, Nicolas Casadei, Martin Ebinger, Stefan Stevanovic, Peter Bauer, Oliver Kohlbacher, Cecile Gouttefangeas, Juergen Schaefer, Hans-Georg Rammensee, Rupert Handgretinger, Peter Lang. Personalized peptide vaccination based on patient-individual tumor-specific variants induces T-cell responses in pediatric patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B124.

Immunotherapy is a treatment option that has had limited success with prostate cancer patients. The major histocompatibility complex (MHC) Class I plays a pivotal role in the adaptive immune response by presenting neo-antigens on the surface of cancer cells to CD8+ T-cells. Prostate cancer cells have markedly lower expression of MHC Class I genes compared to immune-responsive cancers. Loss of MHC Class I is also associated with more aggressive disease and immune evasion in prostate cancer. However, the molecular mechanisms that control MHC Class I downregulation in prostate cancer are still unknown. We hypothesize that increasing MHC Class I expression in prostate cancer cells will increase antigen presentation and improve the efficacy of immunotherapy agents. To investigate the mechanism of MHC Class I regulation in prostate cancer cells, we conducted a whole-genome CRISPRi flow cytometry screen. In this screen, C42B cells containing a non-catalytic Cas9 (dCas9) were infected with a lentivirus that contained a sgRNA library of 100,000 guides targeting 20,000 genes. Infected cells were selected and stained with a pan-Class I MHC antibody, sorted, and the highest and lowest 25-30% of MHC Class I expressing cells were collected for analysis. As expected, knockdown of MHC Class I genes such as B2M, HLA-A, and TAP1 decreased MHC Class I expression. Surprisingly, knockdown of AR and AR co-factors GRHL2 and FOXA1 resulted in a dramatic increase of MHC Class I surface expression. Additional AR inhibition experiments using enzalutamide, an AR degrader, or charcoal-stripped serum also showed increased MHC Class I expression over time. These in vitro results were substantiated with RNA expression analyses of patient biopsy samples taken before or after neoadjuvant enzalutamide treatment, which showed significantly increased MHC Class I expression post-treatment. Results from a small library CRISPR screen, showed that elimination of androgen response elements upstream of MHC Class I genes increased MHC expression, providing a mechanism by which AR regulates MHC Class I. Importantly, AR knockdown was shown to increase antigen-specific T-cell response in co-culture and transgenic mouse models. These observations were further validated in a mCRPC phase II clinical trial. Bulk RNA expression analyses comparing responders and non-responders to anti-PD1 therapy in enzalutamide treated patients, showed decreased expression of AR and increased expression of MHC Class I in patients who responded to immunotherapy. Overall, our data show that AR suppresses MHC Class I expression in prostate cancer, and that androgen-targeted therapies can increase antigen presentation and improve T-cell response. By understanding how AR regulates MHC Class I expression, we can identify new combination treatments utilizing AR signaling inhibition and immunotherapy that lead to a improved anti-cancer response. Citation Format: Lisa Chesner, Julie Graff, Fanny Polesso, Alexis Smith, Arian Lundberg, Martin Sjoestroem, Zheng Xia, Simon Linder, Andries Bergman, Alan Ashworth, David Quigley, Wilbert Zwart, Luke Gilbert, Amy Moran, Felix Feng. AR suppresses MHC Class I expression and T-cell response in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B041.

Pathogenic Role for Virus-Specific CD4 T Cells in Mice with Coronavirus-Induced Acute Encephalitis

Activation of CFTR-specific T Cells in Cystic Fibrosis Mice Following Gene Transfer

Human macrophages infected with Mycobacterium tuberculosis present peptides derived from substrates of type VII secretion systems on MHC class I via a pathway dependent on the ESX-1 secretion system and independent of antigen processing by the proteasome and cathepsins.

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.

Hierarchy of multiple viral CD8+ T-cell epitope mutations in sequential selection in simian immunodeficiency infection

A more effective vaccine against Mycobacterium tuberculosis is needed, and a number of M. tuberculosis vaccine candidates are currently in preclinical or clinical phase I and II studies. One of the strategies to select M. tuberculosis (protein) targets to elicit a CD8(+) or CD4(+) T-cell response is to gauge the binding of candidate peptides to major histocompatibility complex (MHC) class I or class II molecules, a prerequisite for successful peptide presentation and to expand antigen-specific T cells. We scanned 61 proteins from the M. tuberculosis proteome for potential MHC class II-presented epitopes that could serve as targets for CD4(+) T-cell responses. We constructed a peptide microarray consisting of 7,466 unique peptides derived from 61 M. tuberculosis proteins. The peptides were 15-mers overlapping by 12 amino acids. Soluble recombinant DRB1*0101 (DR1), DRB1*1501 (DR2), and DRB1*0401 (DR4) monomers were used to gauge binding to individual peptide species. Out of 7,466 peptides, 1,282, 674, and 1,854 peptides formed stable complexes with HLA-DR1, -DR2, and -DR4, respectively. Five hundred forty-four peptides bound to all three MHC class II molecules, 609 bound to only two, and 756 bound to only a single MHC class II molecule. This allowed us to rank M. tuberculosis proteins by epitope density. M. tuberculosis proteins contained "hot spots," i.e., regions with enriched MHC class II binding epitopes. Two hundred twenty-two peptides that formed MHC class II-peptide complexes had previously been described as exclusively recognized by IgG in sera from patients with active pulmonary tuberculosis, but not in sera from healthy individuals, suggesting that these peptides serve as B-cell and CD4(+) T-cell epitopes. This work helps to identify not only M. tuberculosis peptides with immunogenic potential, but also the most immunogenic proteins. This information is useful for vaccine design and the development of future tools to explore immune responses to M. tuberculosis.

Little is known about the quantitative modulation of major histocompatibility complex (MHC) Class I expression on human gliomas that is effected by interferons; even less is known about the immunogenic peptides that are accommodated in the peptide-binding motifs of MHC Class I alleles in these brain tumors. In this article the authors investigated the ability of interferon (IFN)alpha and IFNgamma to upregulate MHC Class I expression and to modulate acid-eluted Class I-bound peptides on human glioblastoma multiforme (GBM) cells in vitro. Early-passage primary human GBM cell cultures and U87MG GBM cells were incubated with varying doses of INFalpha or IFNgamma ranging between 0 and 2000 U/ml. Upregulation of MHC Class I expression was assayed by immunocytochemical analysis, flow cytometry, and Western blot analysis. Modulation of acid-eluted MHC Class I-bound peptides from the IFN-treated GBM cells was examined with the aid of mass spectroscopy. The in vitro expression of the MHC Class I molecule was upregulated by both IFNalpha and IFNgamma in a dose-dependent manner. Interferon-gamma exhibited a more potent effect on MHC Class I upregulation, peaking at 10 U/ml; whereas the effect of IFNalpha was less marked, reaching a plateau at 500 U/ml. In addition, a native peptide eluted from MHC Class I molecules of human GBM cells was identified and found to be consistently upregulated by IFN treatment. Interferon-alpha and IFN-gamma can significantly upregulate the MHC Class I molecules that are expressed on the cell surface of human GBM cells as well as the potentially immunogenic peptides bound to the MHC. These results may help explain the molecular basis for increased immunogenicity with IFN treatment of human GBMs and might provide added insight into the design of future antitumor vaccines for human brain tumors.

Toward an effective AIDS vaccine development

DENDRITIC CELL BASED VACCINES: PROGRESS IN IMMUNOTHERAPY STUDIES FOR PROSTATE CANCER