Cancer cells frequently display defects in their antigen-processing pathway and thereby evade CD8 T cell immunity. We described a novel category of cancer antigens, named TEIPP, that emerge on... Show moreCancer cells frequently display defects in their antigen-processing pathway and thereby evade CD8 T cell immunity. We described a novel category of cancer antigens, named TEIPP, that emerge on cancers with functional loss of the peptide pump TAP. TEIPPs are non-mutated neoantigens despite their 'self' origin by virtue of their absence on normal tissues. Here, we describe the development of a synthetic long peptide (SLP) vaccine for the most immunogenic TEIPP antigen identified thus far, derived from the TAP-independent LRPAP1 signal sequence. LRPAP1(21-30)-specific CD8 T cells were present in blood of all tested healthy donors as well as patients with non-small cell lung adenocarcinoma. SLPs with natural flanking, however, failed to be cross-presented by monocyte-derived dendritic cells. Since the C-terminus of LRPAP1(21-30) is an unconventional and weakly binding serine (S), we investigated if replacement of this anchor would result in efficient cross-presentation. Exchange into a valine (V) resulted in higher HLA-A2 binding affinity and enhanced T cell stimulation. Importantly, CD8 T cells isolated using the V-variant were able to bind tetramers with the natural S-variant and respond to TAP-deficient cancer cells. A functional screen with an array of N-terminal and C-terminal extended SLPs pointed at the 24-mer V-SLP, elongated at the N-terminus, as most optimal vaccine candidate. This SLP was efficiently cross-presented and consistently induced a strong polyclonal LRPAP1(21-30)-specific CD8 T cells from the endogenous T cell repertoire. Thus, we designed a TEIPP SLP vaccine from the LRPAP1 signal sequence ready for validation in clinical trials. Show less
In the current thesis, we provide novel insights in antigen uptake, storage, processing, and sustained cross-presentation mechanisms in dendritic cells (DCs) in vitro and in vivo. We have studied... Show moreIn the current thesis, we provide novel insights in antigen uptake, storage, processing, and sustained cross-presentation mechanisms in dendritic cells (DCs) in vitro and in vivo. We have studied antigen handling functions by dendritic cells in three different antigen delivery routes: antibody targeting involving Fcγ receptors and complement factor C1q, C-type lectin receptor targeting, and toll-like receptor ligand targeting systems. Our data highlights that antigen storage in specialized compartments in DCs, despite the chosen uptake route, is beneficial for prolonged antigen cross-presentation by DCs and sustained T cell activation. Further in vivo studies in different antigen presenting cell (APC) subsets confirmed the presence of antigen storage compartments by isolating APC subsets after in vivo antigen uptake. Besides, we revealed a dominant role of C1q in antigen-antibody immune complex uptake and cross-presentation in vivo in contrast to the crucial role of Fcγ receptors in vitro. Furthermore, we demonstrated that autophagosomes have a negative impact on the storage of antigen in those specialized compartments and thereby affecting DC cross-presentation efficiency. With the current studies, we unraveled some mechanics of antigen processing in DCs which contribute to future vaccine designs against diseases such as cancer. Show less
Dou, Y.Y.; Jansen, D.T.S.L.; Bosch, A. van den; Man, R.A. de; Montfoort, N. van; Araman, C.; ... ; Buschow, S.I. 2020
Synthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core... Show moreSynthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core protein derived SLP was capable of boosting CD4(+ )and CD8(+) T cell responses in the presence of a TLR2-ligand in chronic HBV patients ex vivo. For optimal efficacy of a therapeutic vaccine in vivo, adjuvants can be conjugated to the SLP to ensure delivery of both the antigen and the co-stimulatory signal to the same antigen-presenting cell (APC). Dendritic cells (DCs) express the receptor for the adjuvant and are optimally equipped to efficiently process and present the SLP-contained epitopes to T cells. Here, we investigated TLR2-ligand conjugation of the prototype HBV-core SLP. Results indicated that TLR2-ligand conjugation reduced cross-presentation efficiency of the SLP-contained epitope by both monocyte-derived and naturally occurring DC subsets. Importantly, cross-presentation was improved after optimization of the conjugate by either shortening the SLP or by placing a valine-citrulline linker between the TLR2-ligand and the long SLP, to facilitate endosomal dissociation of SLP and TLR2-ligand after uptake. HBV-core SLP conjugates also triggered functional patient T cell responses ex vivo. These results provide an import step forward in the design of a therapeutic SLP-based vaccine to cure chronic HBV. Show less
In this thesis we describe our studies aimed at optimizing the efficacy of synthetic long peptide (SLP) vaccines via the encapsulation in Poly-(lactic-co-glycolic acid) (PLGA)particles.... Show moreIn this thesis we describe our studies aimed at optimizing the efficacy of synthetic long peptide (SLP) vaccines via the encapsulation in Poly-(lactic-co-glycolic acid) (PLGA)particles. Immunotherapy based on SLP-vaccines has resulted in strong tumor specific immune response and importantly, improved clinical benefit in patients with pre-malignant lesions. One important drawback associated with SLP-vaccines is their current form of administration in Montanide, a clinical grade water-in-oil emulsion. The aim of this Ph.D project was to device an alternative method of delivery which overcomes the drawbacks associated with the use of Montanide. For this purpose we explored the use of PLGA (nano)particles (NP) as a delivery vehicle for SLP. Several important aspects for vaccination were assessed in this thesis; from the pharmaceutical formulation to the immunological characterization of different PLGA-SLP preparations. Together, the data presented in this thesis show that PLGA-NP mediated delivery of SLP is a very efficient method to target, load and mature Dendritic cells (DCs) as immune stimulatory compounds can be co-encapsulated with the vaccine Ag Show less
Lalwani, P.; Raftery, M.J.; Kobak, L.; Rang, A.; Giese, T.; Matthaei, M.; ... ; Schonrich, G. 2013
Major advancements in the understanding of the immune system have provided us with the opportunity for rational design of therapeutic immunological interventions. The notion that dendritic cells ... Show moreMajor advancements in the understanding of the immune system have provided us with the opportunity for rational design of therapeutic immunological interventions. The notion that dendritic cells (DC) play a crucial role in the activation of T lymphocytes has made DC biology of central importance for vaccine development. Accordingly, efficient delivery of antigen to DCs is one of main objectives in vaccine development. In this thesis, antibody-mediated antigen targeting is evaluated as a potential antigen delivery strategy for therapeutic vaccination. Complexes of protein antigen and antigen-specific antibodies are natural formulations that bind to Fc__ receptors. Fc__R ligation on DCs leads to efficient uptake, DC maturation and presentation of the antigen to T lymphocytes. Interaction of Ag-Ab complexes with Fc__Rs on DCs provides a link between the humoral and cellular arms of the immune response. This thesis contains an extensive evaluation of Fc__R-mediated antigen delivery to dendritic cells in the context of T lymphocyte-mediated immunotherapy. In addition, it contains a detailed analysis of Fc__R function on DCs and addresses the kinetics of cross-presentation of antigen after Fc__R-mediated uptake. Show less
Surgery is the most effective cancer therapy, followed by radiotherapy. These techniques usually target tumour specific tissue only, unlike most forms of chemotherapy as is best illustrated by the... Show moreSurgery is the most effective cancer therapy, followed by radiotherapy. These techniques usually target tumour specific tissue only, unlike most forms of chemotherapy as is best illustrated by the relatively moderate side effects of such treatments. When the immune system could find and destroy tumour cells, they (and their metastases) would be selectively destroyed without to many side effects as well. But then tumour cells have to be recognized and this requires presentation of tumour specific proteins to the immune system. This process called antigen presentation by the MHC class I molecules is studied here. Chapter 1 and 2 form an introduction to the ubiquitin proteasome system and the MHC class I antigen presentation route, which is operational in most cell types and is involved in presentation of antigens derived from degraded intracellular proteins (of self, tumour or viral origin). Proteins are not randomly degraded, but targeted for degradation by ubiquitin or ubiquitin-like post-translational modifications and subsequently degraded by the major cellular protease, the proteasome. Proteins are not only targeted for degradation because they are old, they may also be targeted for example in a cell cycle specific way or just because they have not been folded correctly during protein synthesis. Further trimming to free amino acids by other proteases follows degradation of cellular proteins by the proteasome. Only a minor pool of peptides that meets the requirements for antigen presentation may circumvent further degradation by binding to proteins involved in MHC class I presentation, like the transporter associated with antigen presentation (TAP), and MHC class I itself. Once the peptide is loaded onto MHC class I, the MHC class I-peptide complex can be transported to the plasma membrane. Here, the peptide is presented to cytotoxic T-cells (CTLs), which can in this way examine the intracellular protein content in their search for foreign content. The first step in antigen presentation by MHC class I is the decoration of target proteins with a degradation signal. The first discovered and best-studied degradation signal is a polymer of ubiquitin proteins. A ubiquitin polymer of more than four ubiquitin proteins can be recognized by the proteasome and subsequently unfolded, de-ubiquitylated and degraded by the proteasome. Free ubiquitin and mono- ubiquitylated proteins are not targets for degradation, but serve other functions. Most studies on ubiquitin have been of biochemical nature, but the introduction of the green fluorescent protein (GFP) allowed the study of ubiquitin behaviour in living cells. It was shown before that a GFP-ubiquitin construct could be stably expressed in human cells. In chapter 4, we have used this chimeric protein to study ubiquitin in living cells under normal cell culture conditions and during proteotoxic cell stress as the result of proteasome inhibition, and heat shock. In untreated cells we were able to confirm previous biochemical experiments showing that a large pool of ubiquitin molecules is coupled to histone 2A and 2B in the nucleus, whereas a small pool of ubiquitin is present as free monomers in both nucleus and cytosol. A third pool of ubiquitin was present in the form of ubiquitin polymers in both the nucleus and the cytosol. Manipulation of the cells with different proteotoxic stress conditions revealed a rapid de-ubiquitylation of the histone-bound ubiquitin pool in favour of poly-ubiquitin chains, which may even reach a size similar to the proteasome complex, which is at least one hundred times bigger as a single ubiquitin molecule. These rapid changes in the ubiquitin equilibrium do not only affect proteasomal degradation, but also induce chromatin condensation and altered gene transcription, thus establishing cross talk between these, at first sight unrelated, cellular processes. Alterations in the UPS are correlated with a variety of human pathologies, like cancer, immunological disorders, inflammation and neurodegenerative diseases. The exact role of the UPS in the pathophysiology of these diseases however, remains poorly understood. Because ubiquitin and the ubiquitin proteasome system are involved in several neurodegenerative diseases like Parkinson__s disease, Alzheimer__s disease and polyglutamine diseases like Huntington__s disease we set out our hypothesis of a sensitive ubiquitin equilibrium in the cell in chapter 5. Besides surgery, radiotherapy is one of the most effective ways of anticancer treatment. The main effects of radiotherapy on cells are induction of double-stranded DNA breaks and the formation of reactive radical species, which may lead to protein modifications like amino acid side-chain oxidation and breakage of di-sulphide bonds. These modifications will hopefully lead to DNA and protein damage, sufficient for cells to enter apoptosis or cell arrest. In chapter 6 we have shown that following exposure to g-irradiation, cell surface MHC class I-peptide complex expression is dose dependently upregulated in two phases. In the first phase of upregulation, proteins are degraded and presented that were directly damaged by the radiation and subsequent radical formation. The second phase is caused by a radiation driven activation of the mTOR pathway, which results in enhanced protein synthesis. This leads to the formation of malformed proteins called rapidly degraded proteins (RDPs) or defective ribosomal products (DRiPs) that are subsequently degraded by the proteasome and presented by MHC class I. The second phase does not only quantitatively alter MHC class I expression, but because of the mTOR pathway-specific protein expression also qualitatively. In addition, proteins may be upregulated to g-irradiation especially DNA repair proteins, resulting in more specific peptides. CTLs directed against these radiation-specific peptides were found in peripheral blood, but appeared in an anergic state. The existence of these CTLs and the expression of radiation-specific peptides may explain the inhibition of distant tumours after local radiotherapy if these CTLs could be activated. This effect is known as the abscopal effect of local radiotherapy. If these CTLs could be activated prior to irradiation in a combination therapy, these could induce a potent immune response against the irradiated cells. We show that prior radiation of a local tumour strongly improves the response to immunotherapy (adoptively transferred CTLs), showing the feasibility of a novel combination therapy: radio-immuno therapy. The majority of MHC class I loaded peptides is derived from cytosolic proteins. But it has been shown that MHC class I also presents peptides derived from extracellular sources like bacteria and proteins from neighbouring cells. This phenomenon is called cross-presentation and many pathways have been postulated to explain how proteins from extracellular sources may intersect with the MHC class I loading machinery. Examples are endosome to cytosol relocation, intercellular peptide transport through gap-junctions, exosomes and ER-phagosome fusion. In chapter 3, we have evaluated the evidence for and against the ER-phagosome theory and concluded that cross-presentation via fusion of phagosomes with the ER is very inefficient if at all possible. Our evaluation of the ER-phagosome theory was a commentary on a study by Touret et al, 2005. This study attempted to validate previous results leading to the ER-phagosome fusion theory, but failed to do so. We have also tried to show ER-phagosomal fusion in dendritic cells, but the best near-fusion event of the ER we could find was a close encounter of ribosome containing ER membranes with a mitochondrion. Also our calculations on the odds of presentation of phagosome-derived peptides were not in favour of antigen presentation via ER-phagosome fusion events. We conclude that cross-presentation to support vaccination should find a different route. Show less
