Inaugural lecture by Prof.dr. Sander van Kasteren On the acceptance of his position of professor of Molecular Immunology at the Universiteit Leiden on Friday December 15, 2023
Wulp, W. van der; Remst, D.F.G.; Kester, M.G.D.; Hagedoorn, R.S.; Parren, P.W.H.I.; Kasteren, S.I. van; ... ; Heemskerk, M.H.M. 2023
Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody... Show moreAntibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8+ T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo. Show less
System-level analysis of single-cell data is rapidly transforming the field of immunometabolism. Given the competitive demand for nutrients in immune microenvironments, there is a need to... Show moreSystem-level analysis of single-cell data is rapidly transforming the field of immunometabolism. Given the competitive demand for nutrients in immune microenvironments, there is a need to understand how and when immune cells access these nutrients. Here, we describe a new approach for single-cell analysis of nutrient uptake where we use in-cell biorthogonal labeling of a functionalized amino acid after transport into the cell. In this manner, the bona fide active uptake of glutamine via SLC1A5/ASCT2 could be quantified. We used this assay to interrogate the transport capacity of complex immune subpopulations, both in vitro and in vivo. Taken together, our findings provide an easy sensitive single-cell assay to assess which cells support their function via SLC1A5-mediated uptake. This is a significant addition to the single-cell metabolic toolbox required to decode the metabolic landscape of complex immune microenvironments. Show less
Bioorthogonal deprotectionsare readily used to control biologicalfunction in a cell-specific manner. To further improve the spatialresolution of these reactions, we here present a lysosome... Show moreBioorthogonal deprotectionsare readily used to control biologicalfunction in a cell-specific manner. To further improve the spatialresolution of these reactions, we here present a lysosome-targetedtetrazine for an organelle-specific deprotection reaction. We showthat trans-cyclooctene deprotection with this reagentcan be used to control the biological activity of ligands for invariantnatural killer T cells in the lysosome to shed light on the processingpathway in antigen presenting cells. We then use the lysosome-targetedtetrazine to show that long peptide antigens used for CD8(+) T cell activation do not pass through this organelle, suggestinga role for the earlier endosomal compartments for their processing. Show less
Wulp, W. van der; Gram, A.M.; Bleijlevens, B.; Hagedoorn, R.S.; Araman, C.; Kim, R.Q.; ... ; Heemskerk, M.H.M. 2023
Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects... Show moreTherapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells. Show less
Wulp, W. van der; Gram, A.M.; Bleijlevens, B.; Hagedoorn, R.S.; Araman, M.C.; Kim, R.Q.; ... ; Heemskerk, M.H.M. 2023
Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects... Show moreTherapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells. Show less
Leeuwen, T. van; Doelman, W.; Kieboom, R.W.R. van den; Florea, B.I.; Kasteren, S.I. van 2023
Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of... Show moreUptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass-spectrometry based proteomics - the ideal analysis tool in this case - requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click-antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG-based Rink amide resin, that enables capture of click-antigens via copper-catalyzed azide-alkyne [2 + 3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a-specific background material, prior to retrieval peptides by acid-mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha-labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures. Show less
Doelman, W.; Reijnen, R.C.; Dijksman, N.; Janssen, A.P.A.; Driel, N. van; Hart, B.A.; ... ; Kasteren, S.I. van 2023
The peptide spanning residues 35 to 55 of the protein myelin oligodendrocyte glycoprotein (MOG) has been studied exten-sively in its role as a key autoantigen in the neuroinflammatory autoimmune... Show moreThe peptide spanning residues 35 to 55 of the protein myelin oligodendrocyte glycoprotein (MOG) has been studied exten-sively in its role as a key autoantigen in the neuroinflammatory autoimmune disease multiple sclerosis. Rodents and nonhuman primate species immunized with this peptide develop a neuroinflammatory condition called experimental autoimmune encephalomyelitis, often used as a model for multiple sclerosis. Over the last decade, the role of citrullina-tion of this antigen in the disease onset and progression has come under increased scrutiny. We recently reported on the ability of these citrullinated MOG35-55 peptides to aggregate in an amyloid-like fashion, suggesting a new potential patho-genic mechanism underlying this disease. The immunodo-minant region of MOG is highly conserved between species, with the only difference between the murine and human pro-tein, a polymorphism on position 42, which is serine in mice and proline for humans. Here, we show that the biophysical and biochemical behavior we previously observed for citrulli-nated murine MOG35-55 is fundamentally different for hu-man and mouse MOG35-55. The citrullinated human peptides do not show amyloid-like behavior under the conditions where the murine peptides do. Moreover, we tested the ability of these peptides to stimulate lymphocytes derived from MOG immu-nized marmoset monkeys. While the citrullinated murine peptides did not produce a proliferative response, one of the citrullinated human peptides did. We postulate that this un-expected difference is caused by disparate antigen processing. Taken together, our results suggest that further study on the role of citrullination in MOG-induced experimental autoim-mune encephalomyelitis is necessary. Show less
Fan, B.W.; Torres Garcia, D.; Salehi, M.; Webber, M.J.; Kasteren, S.I. van; Eelkema, R. 2023
Dextran-based hydrogels are promising therapeutic materials for drug delivery, tissue regeneration devices, and cell therapy vectors, due to their high biocompatibility, along with their ability to... Show moreDextran-based hydrogels are promising therapeutic materials for drug delivery, tissue regeneration devices, and cell therapy vectors, due to their high biocompatibility, along with their ability to protect and release active therapeutic agents. This report describes the synthesis, characterization, and application of a new dynamic covalent dextran hydrogel as an injectable depot for peptide vaccines. Dynamic covalent crosslinks based on double Michael addition of thiols to alkynones impart the dextran hydrogel with shear-thinning and self-healing capabilities, enabling hydrogel injection. These injectable, non-toxic hydrogels show adjuvant potential and have predictable sub-millimolar loading and release of the peptide antigen SIINFEKL, which after its release is able to activate T-cells, demonstrating that the hydrogels deliver peptides without modifying their immunogenicity. This work demonstrates the potential of dynamic covalent dextran hydrogels as a sustained-release material for the delivery of peptide vaccines. Show less
Technological advances have largely driven the revolution in our understanding of the structure and function of microbial communities. Culturing, long the primary tool to probe microbial life, was... Show moreTechnological advances have largely driven the revolution in our understanding of the structure and function of microbial communities. Culturing, long the primary tool to probe microbial life, was supplanted by sequencing and other -omics approaches, which allowed detailed quantitative insights into species composition, metabolic potential, transcriptional activity, secretory responses and more. Although the ability to characterize "who's there" has never been easier or cheaper, it remains technically challenging and expensive to understand what the diverse species and strains that comprise microbial communities are doing in situ, and how these behaviors change through time. Our aim in this brief review is to introduce a developing toolkit based on click chemistry that can accelerate and reduce the expense of functional analyses of the ecology and evolution of microbial communities. After first outlining the history of technological development in this field, we will discuss key applications to date using diverse labels, including BONCAT, and then end with a selective (biased) view of areas where click-chemistry and BONCAT-based approaches stand to have a significant impact on our understanding of microbial communities. Show less
Torres-Garcia, D.; Plassche, M.A.T. van de; Boven, E. van; Leeuwen, T. van; Groenewold, M.G.J.; Sarris, A.J.C.; ... ; Kasteren, S.I. van 2022
Bioorthogonal chemistry combines well with activity-based protein profiling, as it allows for the introduction of detection tags without significantly influencing the physiochemical and biological... Show moreBioorthogonal chemistry combines well with activity-based protein profiling, as it allows for the introduction of detection tags without significantly influencing the physiochemical and biological functions of the probe. In this work, we introduced methyltetrazinylalanine (MeTz-Ala), a close mimic of phenylalanine, into a dipeptide fluoromethylketone cysteine protease inhibitor. Following covalent and irreversible inhibition, the tetrazine allows vizualisation of the captured cathepsin activity by means of inverse electron demand Diels Alder ligation in cell lysates and live cells, demonstrating that tetrazines can be used as live cell compatible, minimal bioorthogonal tags in activity-based protein profiling. Show less
Protein glycosylation is a key post-translational modification important to many facets of biology. Glycosylation can have critical effects on protein conformation, uptake and intracellular routing... Show moreProtein glycosylation is a key post-translational modification important to many facets of biology. Glycosylation can have critical effects on protein conformation, uptake and intracellular routing. In immunology, glycosylation of antigens has been shown to play a role in self/non-self distinction and the effective uptake of antigens. Improperly glycosylated proteins and peptide fragments, for instance those produced by cancerous cells, are also prime candidates for vaccine design. To study these processes, access to peptides bearing well-defined glycans is of critical importance. In this review, the key approaches towards synthetic, well-defined glycopeptides, are described, with a focus on peptides useful for and used in immunological studies. Special attention is given to the glycoconjugation approaches that have been developed in recent years, as these enable rapid synthesis of various (unnatural) glycopeptides, enabling powerful carbohydrate structure/activity studies. These techniques, combined with more traditional total synthesis and chemoenzymatic methods for the production of glycopeptides, should help unravel some of the complexities of glycobiology in the near future. Show less
Bertheussen, K.; Plassche, M.A.T. van de; Bakkum, T.; Gagestein, B.; Ttofi, I.; Sarris, A.J.C.; ... ; Kasteren, S.I. van 2022
In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable... Show moreIn the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene-containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron-demand Diels-Alder reaction with quenched tetrazine-fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging. Show less
Most lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT)... Show moreMost lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT) super-resolution microscopy method to capture weak glycan-lectin interactions at the single-molecule level in living cells (Glyco-PAINT). Glyco-PAINT exploits weak and reversible sugar binding to directly achieve single-molecule detection and quantification in cells and is used to establish the relative kon and koff rates of a synthesized library of carbohydrate-based probes, as well as the diffusion coefficient of the receptor-sugar complex. Uptake of ligands correlates with their binding affinity and residence time to establish structure-function relations for various synthetic glycans. We reveal how sugar multivalency and presentation geometry can be optimized for binding and internalization. Overall, Glyco-PAINT represents a powerful approach to study weak glycan-lectin interactions on the surface of living cells, one that can be potentially extended to a variety of lectin-sugar interactions. Show less
Leun, A.M. van der; Hoekstra, M.E.; Reinalda, L.; Scheele, C.L.G.J.; Toebes, M.; Graaff, M.J. van de; ... ; Schumacher, T.N. 2021
The functional activity and differentiation potential of cells are determined by their interactions with surrounding cells. Approaches that allow unbiased characterization of cell states while at... Show moreThe functional activity and differentiation potential of cells are determined by their interactions with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a tradeoff between spatial resolution and cell profiling depth. Here, we develop a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran) cage coupled to a set of nanobodies allows high-resolution photo-uncaging of different cell types in areas of interest. Single-cell RNA-sequencing of spatially defined CD8+ T cells is used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples. Show less
Leeuwen, T. van; Araman, C.; Pournara, L.P.; Kampstra, A.S.B.; Bakkum, T.; Marqvorsen, M.H.S.; ... ; Kasteren, S.I. van 2021
Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and... Show moreProteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation. Show less
Bacterial infections are still one of the leading causes of death worldwide; despite the near-ubiquitous availability of antibiotics. With antibiotic resistance on the rise, there is an urgent need... Show moreBacterial infections are still one of the leading causes of death worldwide; despite the near-ubiquitous availability of antibiotics. With antibiotic resistance on the rise, there is an urgent need for novel classes of antibiotic drugs. One particularly troublesome class of bacteria are those that have evolved highly efficacious mechanisms for surviving inside the host. These contribute to their virulence by immune evasion, and make them harder to treat with antibiotics due to their residence inside intracellular membrane-limited compartments. This has sparked the development of new chemical reporter molecules and bioorthogonal probes that can be metabolically incorporated into bacteria to provide insights into their activity status. In this review, we provide an overview of several classes of metabolic labeling probes capable of targeting either the peptidoglycan cell wall, the mycomembrane of mycobacteria and corynebacteria, or specific bacterial proteins. In addition, we highlight several important insights that have been made using these metabolic labeling probes. Show less
Leeuwen, T. van; Araman, C.; Pournara, L.P.; Kampstra, A.S.B.; Bakkum, T.; Marqvorsen, M.H.S.; ... ; Kasteren, S.I. van 2021
Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and... Show moreProteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation. Show less
