As most patients still experience either primary or secondary resistance against immunotherapy, we aimed to identify what dictates immunotherapy response and searched for novel targets with the aim... Show moreAs most patients still experience either primary or secondary resistance against immunotherapy, we aimed to identify what dictates immunotherapy response and searched for novel targets with the aim to ultimately improve therapy outcome. In Part I two immunosuppressive immune cells (Treg & CD163hi macrophages) and their mechanism of action were introduced and we showed that immunotherapy resistance could be relieved by specific targeting of these cells. In Part II we then revealed that unleashing the anti-tumor potential of inflammatory myeloid cells (iNOS+ neutrophils & macrophages) in the TME can contribute to totaltumor eradication. While the immunotherapeutic treatment strategies used in these studies (ICB, vaccination, bsAb) are all T-cell based, throughout this thesis we demonstrate that the response (or resistance) is dependent on several other cells in the TME, in which the myeloid-T cell interactions take the center stage. Show less
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... Show moreRecent 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. Show less
This thesis provides novel insights in the capacity of photodynamic therapy (PDT) to disrupt the tumor and its vasculature for improved accumulation of nanosized drug carriers in the tumor.... Show moreThis thesis provides novel insights in the capacity of photodynamic therapy (PDT) to disrupt the tumor and its vasculature for improved accumulation of nanosized drug carriers in the tumor. Moreover, it has shown the importance of the origin and physiological state of extracellular vesicles used as carriers for photosensitizers. It has also expanded knowledge of PDT-induced immunogenic cell death through damage-associated molecular pattern exposure and release that can initiate specific antitumor immune responses. Furthermore, several chapters identify combinations of PDT and immunotherapy that improve the therapeutic efficacy compared to currently used treatment options. Future studies that further investigate ways to benefit from the immunostimulatory effects of PDT hold great promise and may introduce exciting new modalities for improved treatments of cancer. Show less
Immunotherapy is a highly promising treatment option for cancer. At present, only a small proportion of cancer patients benefits from immunotherapeutic interventions. There is an unmet need to... Show moreImmunotherapy is a highly promising treatment option for cancer. At present, only a small proportion of cancer patients benefits from immunotherapeutic interventions. There is an unmet need to understand which factors determine a patient’s response to immunotherapy as well as to develop novel immunotherapeutic approaches that address shortcomings of current immunotherapies.In this thesis, we have performed an unprecedented characterization of immune cell subsets participating in anti-tumor responses in colorectal cancer and pancreatic ductal adenocarcinoma with different single-cell technologies. Most cancer immunology research studied the role of cytotoxic T cells in both cancer types, while a comprehensive analysis of both innate and adaptive components of cancer immunity was largely lacking. With such an approach, we demonstrated an important involvement of understudied unconventional (γδ T cells) and innate (innate lymphoid cells (ILCs)) immune effector cells in anti-tumor immunity. These immune subsets displayed cytotoxic activity and showed potential for therapeutic exploitation. Further studies will focus on their functional characterization and potential reconversion into a therapeutic agent.This thesis resulted from the collaboration between research groups led by Noel de Miranda (Pathology, LUMC) and Frits Koning (Immunology, LUMC), and underscores the relevance of applying single-cell technologies for the study of complex biological systems. Show less
The success of checkpoint blockade therapy revolutionized cancer treatment. However, we need to increase the fraction of responding patients and overcome acquired resistance to these therapies.... Show moreThe success of checkpoint blockade therapy revolutionized cancer treatment. However, we need to increase the fraction of responding patients and overcome acquired resistance to these therapies. Recently, the inhibitory receptor NKG2A received attention as a new kid on the block of immune checkpoints. This receptor is selectively expressed on cytotoxic lymphocytes, including natural killer cells and CD8 T cells, and NKG2A+ T cells are preferentially residing in tissues, like the tumor microenvironment. Its ligand, HLA-E, is a conserved non-classical HLA class I molecule that binds a limited peptide repertoire and its expression is commonly detected in human cancer. NKG2A blockade as a standalone therapy appears poorly effective in mouse tumor models, however in the presence of activated T cells, for example induced by PD-1/PD-L1 blockade or cancer vaccines, displays strongly enhanced efficacy. Clinical trials demonstrated safety of the humanized NKG2A-blocking antibody monalizumab and first results of phase II trials demonstrate encouraging durable response rates. Further development of this axis is clearly warranted. Show less
Major achievements in the field of immune oncology have demonstrated the ability of the immune system to induce a response against cancer. The prognostic impact of pre-existing immunity in several... Show moreMajor achievements in the field of immune oncology have demonstrated the ability of the immune system to induce a response against cancer. The prognostic impact of pre-existing immunity in several cancer types, including breast and colon cancer, demonstrates the influence of the immune system on disease progression. At the same time, immunotherapeutic approaches that aim to enhance antitumor immune reactions have significantly improved the clinical outcome for a subset of patients. However, a large proportion of patients (60-80%) do not respond to immunotherapeutic treatments. To extend the benefit of immunotherapeutic strategies to a larger number of patients, it is imperative to understand the mechanisms associated with immune responsiveness. Different variables have been described to influence the development of antitumor immunity in cancer patients, including the tumor’s genetic program, the genetic makeup of the patients, and environmental factors such as the microbiome. These factors likely act in concert to modulate antitumor immune responses. This thesis aimed to dissect the molecular determinants of cancer immune responsiveness in human tumors. A systems biology approach was used to define underlying factors that shape the tumor microenvironment and reveal potential mechanisms of immune escape. Show less
Antibodies, the cardinal effector molecules of the immune system, are being leveraged to enormous success as biotherapeutic drugs. Adaptive immune responses consist of epitope-diverse polyclonal... Show moreAntibodies, the cardinal effector molecules of the immune system, are being leveraged to enormous success as biotherapeutic drugs. Adaptive immune responses consist of epitope-diverse polyclonal antibody mixtures that are capable of neutralizing their targets via binding interference and by mediating humoral and cellular effector functions. A mechanistic theme fundamental to virtually all aspects of antibody biology, including antibody-antigen binding, clonal selection and effector functions, is the utilization of avidity to drive and tune functional responses. Manipulating antibody avidity has since emerged as an important design principle for enhancing or engineering novel properties in antibody biotherapeutics. In the context of ‘classical’ effector functions, complement-dependent cytotoxicity (CDC) can be improved by single point mutations in the IgG Fc domain that increase intermolecular Fc-Fc interactions upon binding to membrane-bound targets, thereby facilitating enhanced IgG hexamer formation and C1q binding. Such engineering approaches illustrate the relevance of promoting avidity interactions such as antibody clustering to enhance effector functions. The aim of this thesis was to explore the role of antibody avidity interactions, and more specifically the importance of ‘ordered clustering’, in antibody mechanisms of action and to apply the knowledge obtained in designing novel and improved antibody-based therapeutics Show less
Arakelian, T.; Oosterhuis, K.; Tondini, E.; M. los; Vree, J.; Geldorp, M. van; ... ; Bergen, J. van 2022
Pyroptosis is a recently discovered form of inflammatory programmed necrosis characterized by caspase1-mediated and gasdermin D-dependent cell death leading to the release of pro-inflammatory... Show morePyroptosis is a recently discovered form of inflammatory programmed necrosis characterized by caspase1-mediated and gasdermin D-dependent cell death leading to the release of pro-inflammatory cytokines such as Interleukin-1 beta (IL-18). Here, we evaluated whether pyroptosis could be exploited in DNA vaccination by incorporating a constitutively active variant of caspase-1 to the antigen-expressing DNA. In vitro, transfection with constitutively active caspase-1 DNA induced pro-IL-18 maturation and IL-18 release as well as gasdermin D-dependent cell death. To test active caspase-1 as a genetic adjuvant for the induction of antigen-specific T cell responses, mice were vaccinated intradermally with a DNA vaccine consisting of the active caspase-1 plasmid together with a plasmid encoding an ovalbuminderived CD8 T cell epitope. Active caspase-1 accelerated and amplified antigen-specific CD8 T cell responses when administered simultaneously with the DNA vaccine at an equimolar dose. Moreover, upon challenge with melanoma cells expressing ovalbumin, mice vaccinated with the antigen vaccine adjuvanted with active caspase-1 showed significantly better survival compared to the non-adjuvanted group. In conclusion, we have developed a novel genetic adjuvant that for the first time employs the pyroptosis pathway to improve DNA vaccination against cancer. (c) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). Show less
In order to be able to develop effective medicine and treatments to prevent or cure autoimmune diseases or cancer we need to understand the mechanisms how they arise and what drives their course... Show moreIn order to be able to develop effective medicine and treatments to prevent or cure autoimmune diseases or cancer we need to understand the mechanisms how they arise and what drives their course.Unravelling the fundamental molecular mechanisms influencing the onset and course of diseases such as allergies, rheumatoid arthritis or cancer can be tackled using bioorthogonal antigens – chemically functionalized proteins.To tackle this challenge this thesis uses an inter-disciplinary approach. Combining standard immunological experimental methods with special, highly selective bioorthogonal chemical reactions. These reactions are bioorthogonal because, unlike normal organic chemistry, they are compatible with the physiological environment of a cell. This approach allows for following for example the location of the protein over time within a cell or alterations in the immune response due to disease related changes to the protein without disturbing the processes themself.This is a significant advantage because without changing the method used, new information can be retrieved from the same set of experiments, at any point in time during the process and a plethora of new readout options yielding additional data sets.This promises new insights into the causal relation of time, localisation and factors influencing effective anti-cancer vaccine-design and cancer immunotherapy or new biological drugs to prevent or delay onset and progression of autoimmune diseases. Show less
Using a variety of relevant preclinical mouse models, this thesis not only showed several ways to improve antibody-based cancer immunotherapy but also demonstrates the importance of the choice of... Show moreUsing a variety of relevant preclinical mouse models, this thesis not only showed several ways to improve antibody-based cancer immunotherapy but also demonstrates the importance of the choice of the model for the translational relevance of the preclinical observations. Show less
Bulk, J. van den; Verdegaal, E.M.E.; Ruano, D.; Ijsselsteijn, M.E.; Visser, M.; Breggen, R. van der; ... ; Miranda, N.F.C.C. de 2019
Background: The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high... Show moreBackground: The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high mutation burden. However, this observation does not exclude the existence of neoantigen-specific T cells in colorectal cancers with low mutation burden and the exploitation of their anti-cancer potential for immunotherapy. Therefore, we investigated whether autologous neoantigen-specific T cell responses could also be observed in patients diagnosed with mismatch repair-proficient colorectal cancers.Methods: Whole-exome and transcriptome sequencing were performed on cancer and normal tissues from seven colorectal cancer patients diagnosed with mismatch repair-proficient tumors to detect putative neoantigens. Corresponding neo-epitopes were synthesized and tested for recognition by in vitro expanded T cells that were isolated from tumor tissues (tumor-infiltrating lymphocytes) and from peripheral mononuclear blood cells stimulated with tumor material.Results: Neoantigen-specific T cell reactivity was detected to several neo-epitopes in the tumor-infiltrating lymphocytes of three patients while their respective cancers expressed 15, 21, and 30 non-synonymous variants. Cell sorting of tumor-infiltrating lymphocytes based on the co-expression of CD39 and CD103 pinpointed the presence of neoantigen-specific T cells in the CD39(+)CD103(+) T cell subset. Strikingly, the tumors containing neoantigen-reactive TIL were classified as consensus molecular subtype 4 (CMS4), which is associated with TGF-beta pathway activation and worse clinical outcome.Conclusions: We have detected neoantigen-targeted reactivity by autologous T cells in mismatch repair-proficient colorectal cancers of the CMS4 subtype. These findings warrant the development of specific immunotherapeutic strategies that selectively boost the activity of neoantigen-specific T cells and target the TGF-beta pathway to reinforce T cell reactivity in this patient group. Show less
Infusions of T cells engineered with TCRs directed against tumor-expressed antigens have demonstrated clinical efficacy in the treatment of cancer. To broaden the applicability of this... Show moreInfusions of T cells engineered with TCRs directed against tumor-expressed antigens have demonstrated clinical efficacy in the treatment of cancer. To broaden the applicability of this approach a broad repertoire of clinically relevant TCRs is required. This thesis describes a high throughput methodology for the isolation of TCRs useful in the treatment of patients suffering from B-cell malignancies. To induce strong immune responses against tumor-expressed antigens, the immunogencity of allogeneic (non-self) HLA molecules is exploited. Around this fundamental knowledge about T-cell reactivity a pipeline is developed that consists of the selection of targetable tumor-expressed antigens by mining gene expression databases, discovery of HLA-presented peptides from peptide elution studies describing the HLA ligandome of B lymphocytes, a peptide-MHC tetramer based high throughput methodology for the isolation and characterization of T cells expressing TCRs specific for tumor-expressed antigens. Several TCRs were isolated targeting antigens that are expressed on several B-cell malignancies including multiple myeloma. This is of particular interest since novel immunotherapies for multiple myeloma are currently lacking. The work described in this thesis can significantly broaden the applicability of immunotherapy by providing novel tools and reagents for strategies relying on tumor recognition through antigen presentation in the context of HLA. Show less
Cancer immunotherapy has taken up its place in oncological practice and it is likely there to stay. Although the field has come from far, much is still to learn as oncologists observe that not... Show moreCancer immunotherapy has taken up its place in oncological practice and it is likely there to stay. Although the field has come from far, much is still to learn as oncologists observe that not all patients with the same disease type respond to cancer immunotherapy, Furthermore, in some cancer types, no clinical benefit is observed at all, despite the presence of an abundant local immune infiltrate, indicating that our understanding of cancer – immune interaction is still incomplete. The overall goal of this thesis is to help address these matters in three ways. First, we have retrospectively analyzed a cohort of melanoma patients treated with ipilimumab, an anti-CTLA-4 antibody, for markers of responsiveness and review the literature to explore the limitations and curative potential of treatment strategies currently used in the clinic or under development. Second, we have developed several technology platforms that allow us to characterize and compare different types of T-cell responses that are directed against tumor-specific antigens in patient tumor material. Third, we aim to increase our understanding of the potential of cancer immunotherapy in ovarian and colorectal cancer, by analysis of the tumor-reactive compartment within these diseases. Show less
