This thesis aims to advance anti-cancer therapy development through a comprehensive exploration.In Chapter 1, the focus is on understanding the immune system's role in breast cancer and... Show moreThis thesis aims to advance anti-cancer therapy development through a comprehensive exploration.In Chapter 1, the focus is on understanding the immune system's role in breast cancer and chemotherapy response. It examines the paradoxical roles of immune cells and macrophages, seeking novel biomarkers and immunomodulatory strategies.In Chapter 2, the impact of the adaptive immune system on HER2+ breast cancer is explored. Contrary to other breast cancer types, the adaptive immune system neither suppresses nor promotes spontaneous HER2-driven tumorigenesis or metastasis formation.Chapter 3 investigates the relationship between chemotherapy and the adaptive immune response using GEMMs. The study highlights that the endogenous adaptive immune system's role in chemotherapy response might not be as vital as previously believed when utilizing tumor cell line transplantation models. This finding leads to a reevaluation of the conventional understanding of the immune system's involvement in chemotherapy response.Chapter 4 focuses on macrophage-targeted cancer therapy. In non-immunogenic breast cancer, CSF-1R inhibition enhances chemotherapy, especially with platinum drugs. Intratumoral type I interferon activation drives their synergistic impact. Also, neutralizing immunosuppressive neutrophils is crucial for a strong anti-tumor immune response.In Chapter 5, the thesis discusses how its findings align with existing research and suggest clinical applications. Show less
Introduction: Nanoporous microneedle arrays (npMNA) are being developed as skin patches for vaccine delivery. As alternative for needle-based immunisation, they may potentially result in higher... Show moreIntroduction: Nanoporous microneedle arrays (npMNA) are being developed as skin patches for vaccine delivery. As alternative for needle-based immunisation, they may potentially result in higher vaccine acceptance, which is important for future mass vaccination campaigns to control outbreaks, such as COVID-19, and for public vaccination in general. In this study we investigated the safety and immunogenicity of needle-free intradermal delivery of a fractional third or fourth dose of mRNA-1273 vaccine by npMNA.Methods: This study was an open-label, randomised-controlled, proof-of-concept study. Healthy adults were eligible if they had received a primary immunisation series against SARS-CoV-2 with two doses of mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) mRNA vaccine. A history of a COVID-19 infection or booster vaccination with mRNA-1273 or BNT162b2 was allowed if it occurred at least three months before inclusion. Participants were randomised in a 1:1 ratio to receive 20 & mu;g mRNA-1273 vaccine, either through npMNA patch applied on the skin (ID-patch group), or through intramuscular (IM) injection (IM-control group). Primary outcomes were reactogenicity up to two weeks after vaccination, and fold-increase of SARS-CoV-2 spike S1specific IgG antibodies 14 days post-vaccination.Results: In April 2022, 20 participants were enroled. The geometric mean concentration (GMC) did not increase in the ID-patch group after vaccination, in contrast to the IM-control group (GMC was 1,006 BAU/mL (95% CI 599-1,689), 3,855 (2,800-5,306), and 3,513 (2,554-4,833) at day 1, 15 and 29, respectively). In addition, SARSCoV-2-specific T cell responses were lower after ID vaccination through npMNA.Conclusion: Needle-free delivery of 20 & mu;g mRNA-1273 vaccine by npMNA failed to induce antibody and T cell responses. As this is a potentially very useful vaccination method, it is important to determine which adjustments are needed to make this npMNA successful. Clinical trial registry (on ClinicalTrial.gov): NCT05315362. Show less
IntroductionNanoporous microneedle arrays (npMNA) are being developed as skin patches for vaccine delivery. As alternative for needle-based immunisation, they may potentially result in higher... Show moreIntroductionNanoporous microneedle arrays (npMNA) are being developed as skin patches for vaccine delivery. As alternative for needle-based immunisation, they may potentially result in higher vaccine acceptance, which is important for future mass vaccination campaigns to control outbreaks, such as COVID-19, and for public vaccination in general. In this study we investigated the safety and immunogenicity of needle-free intradermal delivery of a fractional third or fourth dose of mRNA-1273 vaccine by npMNA.MethodsThis study was an open-label, randomised-controlled, proof-of-concept study. Healthy adults were eligible if they had received a primary immunisation series against SARS-CoV-2 with two doses of mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) mRNA vaccine. A history of a COVID-19 infection or booster vaccination with mRNA-1273 or BNT162b2 was allowed if it occurred at least three months before inclusion. Participants were randomised in a 1:1 ratio to receive 20 µg mRNA-1273 vaccine, either through npMNA patch applied on the skin (ID-patch group), or through intramuscular (IM) injection (IM-control group). Primary outcomes were reactogenicity up to two weeks after vaccination, and fold-increase of SARS-CoV-2 spike S1-specific IgG antibodies 14 days post-vaccination.ResultsIn April 2022, 20 participants were enroled. The geometric mean concentration (GMC) did not increase in the ID-patch group after vaccination, in contrast to the IM-control group (GMC was 1,006 BAU/mL (95% CI 599–1,689), 3,855 (2,800–5,306), and 3,513 (2,554–4,833) at day 1, 15 and 29, respectively). In addition, SARS-CoV-2-specific T cell responses were lower after ID vaccination through npMNA.ConclusionNeedle-free delivery of 20 µg mRNA-1273 vaccine by npMNA failed to induce antibody and T cell responses. As this is a potentially very useful vaccination method, it is important to determine which adjustments are needed to make this npMNA successful. Show less
Over the past decade, high HLA epitope mismatch scores have been associated with inferior transplant outcomes using several tools, of which HLAMatchmaker is most well-known. This software uses... Show moreOver the past decade, high HLA epitope mismatch scores have been associated with inferior transplant outcomes using several tools, of which HLAMatchmaker is most well-known. This software uses theoretically defined polymorphic amino acid configurations, called eplets, for HLA compatibility analysis. Although consideration of eplet mismatch loads has potential for immunological risk stratification of transplant patients, the use of eplet matching in organ allocation algorithms is hindered by lacking knowledge of the immunogenicity of individual eplets, and the possibility that single mismatched amino acids, rather than complete eplets, are responsible for HLA antibody induction.There are several approaches to define eplet immunogenicity, such as antibody verification of individual eplets, and data-driven approaches using large datasets that correlate specific eplet mismatches to donor specific antibody formation or inferior transplant outcomes. Data-driven approaches can also be used to define whether single amino acid mismatches may be more informative than eplet mismatches for predicting HLA antibody induction.When using epitope knowledge for the assignment of unacceptable antigens, it important to realize that alleles sharing an eplet to which antibodies have formed are not automatically all unacceptable since multiple contact sites determine the binding strength and thus biological function and pathogenicity of an antibody, which may differ between reactive alleles.While the future looks bright for using HLA epitopes in clinical decision making, major steps need to be taken to make this a clinical reality.(c) 2021 The Author(s). Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/). Show less
In recent years HLA epitope matching is becoming a hot topic in transplant community to prevent donor-specific antibody formation after transplantation, as such antibodies are associated with... Show moreIn recent years HLA epitope matching is becoming a hot topic in transplant community to prevent donor-specific antibody formation after transplantation, as such antibodies are associated with inferior graft survival. The number of HLA epitope mismatches between donor and recipient correlates with donor-specific antibody formation, but not every epitope mismatch will trigger an antibody response. Therefore, it is pivotal to define the immunogenic epitopes. In my thesis we describe the development of the software HLA-EMMA to determine HLA amino acid mismatches between donor and recipient to identify immunogenic amino acids. In addition, the generation of recombinant human HLA-DR monoclonal antibodies for antibody-verification of eplets/epitopes is described. These tools will contribute to the definition of immunogenic epitopes, which is required before introducing HLA epitope matching in the clinic. Show less
Background: Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in early infancy and in elderly. A pediatric vaccine against RSV would not only prevent... Show moreBackground: Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in early infancy and in elderly. A pediatric vaccine against RSV would not only prevent morbidity and mortality amongst infants and young children but could also reduce transmission to elderly. The RSVDG vaccine consists of a live-attenuated RSV that lacks the G attachment protein. RSVDG is severely impaired in binding to host cells and exhibits reduced infectivity in preclinical studies. Intranasal immunization of cotton rats with RSVDG vaccine protected against replication of wildtype RSV, without inducing enhanced disease.Methods: We performed a first-in-human trial with primary objective to evaluate safety and shedding of RSV Delta G (6.5 log(10) CCID50) after intranasal administration. Healthy adults aged between 18 and 50, with RSV neutralizing serum titers below 9.6 log(2), received a single dose of either vaccine or placebo (n = 48, ratio 3:1). In addition to safety and tolerability, nasal viral load, and systemic and humoral immune responses were assessed at selected time points until 4 weeks after immunization.Results: Intranasal administration of RSV Delta G was well tolerated with no findings of clinical concern. No infectious virus was detected in nasal wash samples. Similar to other live-attenuated RSV vaccines, neutralizing antibody response following inoculation was limited in seropositive adults.Conclusions: A single dose of 6.5 log(10) CCID50 of RSV Delta G was safe and well-tolerated in seropositive healthy adults. RSV Delta G was sufficiently attenuated but there were no signs of induction of antibodies. Safety and immunogenicity can now be explored in children and eventually in seronegative infants. (C) 2020 The Author(s). Published by Elsevier Ltd. Show less
The presence of protein aggregates is commonly believed to be an important risk factor for immunogenicity of therapeutic proteins. Among all types of aggregates, dimers are relatively abundant in... Show moreThe presence of protein aggregates is commonly believed to be an important risk factor for immunogenicity of therapeutic proteins. Among all types of aggregates, dimers are relatively abundant in most commercialized monoclonal antibody (mAb) products. The aim of this study was to investigate the immunogenicity of artificially created mAb dimers relative to that of unstressed and stressed mAb monomers. A monoclonal murine IgG1 (mIgG1) antibody was exposed to low pH, elevated temperature, or UV irradiation to induce dimerization. Dimers and monomers were purified via size-exclusion chromatography. Physicochemical analysis revealed that upon all stress conditions, new deamidation or oxidation or both of amino acids occurred. Nevertheless, the secondary and tertiary structures of all obtained dimers were similar to those of unstressed mIgG1. Isolated dimers were administered subcutaneously in Balb/c mice, and development of antidrug antibodies and accumulation of follicular T helper cells in draining lymph nodes and spleens were determined. None of the tested dimers or stressed monomers were found to be more immunogenic than the unstressed control in our mouse model. In conclusion, both dimers and monomers generated by using 3 different stress factors have a low immunogenicity similar to that of the unstressed monomers. Show less
Transfusion of red blood cells (RBCs) causes exposure to foreign antigens and, consequently, may induce alloimmunization. This research focused on identifying clinical determinants of RBC... Show moreTransfusion of red blood cells (RBCs) causes exposure to foreign antigens and, consequently, may induce alloimmunization. This research focused on identifying clinical determinants of RBC alloimmunization, eventually aiming to prevent alloimmunization by pre-emptively select extended matched blood for the predicted responder patient. Both RBC antigen intrinsic characteristics and patient-related factors were studied. Regarding antigen immunogenicity, K was confirmed to be the most potent antigen, followed by E, Cw, e, Jka and c. Of importance, anti-Jka is known to easily induce complement-mediated hemolysis. Inflammation due to severe bacterial and viral infections was associated to increased RBC alloimmunization incidences. Remarkably, although in line with murine models, Gram-negative bacteremia coincided with a twofold reduction of alloimmunization risk. In a non-hemoglobinopathy population, alloimmunization post-splenectomy was a highly unlikely event. Consequently, the Caucasian splenectomized patient does not benefit from RBC products matched beyond ABO/RhD. Patients with acute (either myeloid of lymphoblastic) leukemia, mature lymphomas, myelodysplastic syndrome, or patients post-autologous or -allogeneic stem cell transplantation demonstrated strongly reduced incidences of RBC alloimmunization, primarily explained by the intense immunosuppressive nature of treatments. Consequently, matching for the MDS population deserves renewed focus and should be based on the cumulative transfusion burden rather than on the diagnosis itself. Show less
Therapeutic proteins have become very successful in the treatment of various chronic and life-threatening diseases. However, besides their benefits, therapeutic proteins seem to have a common... Show moreTherapeutic proteins have become very successful in the treatment of various chronic and life-threatening diseases. However, besides their benefits, therapeutic proteins seem to have a common problem - the response of a patient’s immune system against the protein. This means that the immune system of the patient actively removes the drug from the body, thereby potentially decreasing or reversing the effect of the therapy. By now there is strong consensus that damaged and aggregated proteins are important risk factors. Protein aggregates are, due to their heterogeneity and often low quantity, challanging to characterize. Further, there is a large academic interest in understanding the mechanisms of aggregation and the role of non-proteinaceous particles in the process of protein aggregation and unwanted immunogenicity in order to design more effective and safe protein-based medicines. This PhD thesis supported that research effort by developing and improving analytical methodologies to detect the size, quantity and other properties of protein aggregates and particles, especially in the relevant nano- and micrometer size range. These techniques were then applied to study a so far unknown nanoparticulate impurity in pharmaceutical-grade sugars. Further, the results shown in this thesis revealed that these nanoparticulate impurities pose a threat to protein stability. Show less
The aim of the research described in this thesis is to study the chemical mechanisms responsible for protein aggregation induced by metal catalyzed oxidation and to investigate the relationship... Show moreThe aim of the research described in this thesis is to study the chemical mechanisms responsible for protein aggregation induced by metal catalyzed oxidation and to investigate the relationship between protein oxidation, aggregation and immunogenicity. To this end, recombinant human insulin rhIFN_-1a and rhIFN_-2a are used in conjunction with transgenic mice immune tolerant for the respective human endogenous counterparts. The impact of PEGylation on aggregate formation and excipients to prevent metal catalyzed oxidation are also evaluated. Show less
