Molecular approaches to identify cancer T cell antigens and improve immunogenicity

Recent advances in our understanding of immunotherapeutic strategies against cancer and the development of improved analysis tools and computing power have led to a highly anticipated progression of the cancer-vaccine field. The possibilities of personalized approaches to vaccinate patients against tumor-specific antigens by the prediction of MHC-binding peptides are already in clinical studies. Currently, the field is lacking behind in antigen identification tools and optimal delivery of vaccines. The studies in this thesis are divided in two major parts which address these topics. The first part reports a novel approach for the identification of relevant MHC class I and II binding epitopes. With the combined use of exome sequencing and mass-spectrometric analysis, relevant neoantigens were characterized from MHC class I and II in murine and human models. Meanwhile, significant genomic differences were observed in the MC38 murine colorectal cancer model of different sources. The second part shows the development of a molecular approach for the tracking of ligands and peptides to improve our understanding of vaccine delivery. Therefore, bioorthogonal ‘click’ chemistry was applied for in situ ligation of fluorophores to antigen, thus providing a quantification method of processed antigens. Subsequent optimizations were explored into bioorthogonal reactions independent of copper. LUMC / Geneeskund

MC38 colorectal tumor cell lines from two different sources display substantial differences in transcriptome, mutanome and neoantigen expression

IntroductionThe cell line MC38 is a commonly used murine model for colorectal carcinoma. It has a high mutational burden, is sensitive to immune checkpoint immunotherapy and endogenous CD8+ T cell responses against neoantigens have been reported. MethodsHere, we re-sequenced exomes and transcriptomes of MC38 cells from two different sources, namely Kerafast (originating from NCI/NIH, MC38-K) and the Leiden University Medical Center cell line collection (MC38-L), comparing the cell lines on the genomic and transcriptomic level and analyzing their recognition by CD8+ T cells with known neo-epitope specificity. ResultsThe data reveals a distinct structural composition of MC38-K and MC38-L cell line genomes and different ploidies. Further, the MC38-L cell line harbored about 1.3-fold more single nucleotide variations and small insertions and deletions than the MC38-K cell line. In addition, the observed mutational signatures differed; only 35.3% of the non-synonymous variants and 5.4% of the fusion gene events were shared. Transcript expression values of both cell lines correlated strongly (p = 0.919), but we found different pathways enriched in the genes that were differentially upregulated in the MC38-L or MC38-K cells, respectively. Our data show that previously described neoantigens in the MC38 model such as Rpl18(mut) and Adpgk(mut) were absent in the MC38-K cell line resulting that such neoantigen-specific CD8+ T cells recognizing and killing MC38-L cells did not recognize or kill MC38-K cells. ConclusionThis strongly indicates that at least two sub-cell lines of MC38 exist in the field and underlines the importance of meticulous tracking of investigated cell lines to obtain reproducible results, and for correct interpretation of the immunological data without artifacts. We present our analyses as a reference for researchers to select the appropriate sub-cell line for their own studies

Approaches to Improve Chemically Defined Synthetic Peptide Vaccines

Progress made in peptide-based vaccinations to induce T-cell-dependent immune responses against cancer has invigorated the search for optimal vaccine modalities. Design of new vaccine strategies intrinsically depends on the knowledge of antigen handling and optimal epitope presentation in both major histocompatibility complex class I and -II molecules by professional antigen-presenting cells to induce robust CD8 and CD4 T-cell responses. Although there is a steady increase in the understanding of the underlying mechanisms that bridges innate and adaptive immunology, many questions remain to be answered. Moreover, we are in the early stage of exploiting this knowledge to clinical advantage. Several adaptations of peptide-based vaccines like peptide-adjuvant conjugates have been explored and showed beneficial outcomes in preclinical models; but in the clinical trials conducted so far, mixed results were obtained. A major limiting factor to unravel antigen handling mechanistically is the lack of tools to efficiently track peptide vaccines at the molecular and (sub)cellular level. In this mini-review, we will discuss options to develop molecular tools for improving, as well as studying, peptide-based vaccines.Bio-organic Synthesi

Cancer-specific T helper shared and neo-epitopes uncovered by expression of the MHC class II master regulator CIITA

We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of MHC class II-eluted natural peptides. MHC class II-presented peptide sequences are identified by introducing the MHC class II transactivator CIITA in tumor cells that were originally MHC class II-negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals many novel oncoviral-, shared and neo-epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses which helped tumor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes presented by any MHC class II allele that would be otherwise very difficult to predict and reveals  important new targets for cancer immunotherapy.NWOICI024.002.009Proteomic

A New Boson with a Mass of 125 GeV Observed with the CMS Experiment at the Large Hadron Collider

The Higgs boson was postulated nearly five decades ago within the framework of the standard model of particle physics and has been the subject of numerous searches at accelerators around the world. Its discovery would verify the existence of a complex scalar field thought to give mass to three of the carriers of the electroweak force-the W+, W-, and Z(0) bosons-as well as to the fundamental quarks and leptons. The CMS Collaboration has observed, with a statistical significance of five standard deviations, a new particle produced in proton-proton collisions at the Large Hadron Collider at CERN. The evidence is strongest in the diphoton and four-lepton (electrons and/or muons) final states, which provide the best mass resolution in the CMS detector. The probability of the observed signal being due to a random fluctuation of the background is about 1 in 3 x 10(6). The new particle is a boson with spin not equal to 1 and has a mass of about 1.25 giga-electron volts. Although its measured properties are, within the uncertainties of the present data, consistent with those expected of the Higgs boson, more data are needed to elucidate the precise nature of the new particle

Observation of a new boson at a mass of 125 GeV with the CMS experiment at the LHC

The article is the pre-print version of the final publishing paper that is available from the link below.Results are presented from searches for the standard model Higgs boson in proton–proton collisions At √s = 7 and 8 TeV in the Compact Muon Solenoid experiment at the LHC, using data samples corresponding to integrated luminosities of up to 5.1 fb−1 at 7TeV and 5.3 fb−1 at 8 TeV. The search is performed in five decay modes: γγ, ZZ, W+W−, τ+τ−, and bb. An excess of events is observed above the expected background, with a local significance of 5.0 standard deviations, at a mass near 125 GeV, signalling the production of a new particle. The expected significance for a standard model Higgs boson of that mass is 5.8 standard deviations. The excess is most significant in the two decay modes with the best mass resolution, γγ and ZZ; a fit to these signals gives a mass of 125.3±0.4(stat.)±0.5(syst.) GeV. The decay to two photons indicates that the new particle is a boson with spin different from one