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Contains fulltext : 294765.pdf (Publisher’s version ) (Open Access), Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. We propose a radically different approach aimed at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), with a soluble semiflexible polyisocyanopeptide backbone that presents peptide-loaded major histocompatibility complexes and costimulatory molecules multivalently. IFs readily activated and expanded antigen-specific T cells like natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach the spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display strong antitumor efficacy resulting in inhibition of the formation of melanoma metastases and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.

Item does not contain fulltext, T cells require a co-stimulatory signal in addition to T-cell receptor (TCR) stimulation to achieve full activation. While most studies focus on the co-stimulatory receptor CD28, little is known about the role of the other co-stimulatory receptors in T-cell signaling. A deeper understanding of how co-stimulatory receptor signaling cooperates with TCR signaling could improve the ability to control T-cell function and benefit the design of T-cell based immunotherapies. Artificial antigen presenting cells (aAPCs) enable tight control over the signals given to T cells. In this study, filamentous polyisocyanopeptide (PIC) polymers (immunofilaments) are used as nanosized aAPCs to study the role of the engagement of six distinct co-stimulatory molecules on human T-cell phenotype, function, and fate in the context of TCR signaling. The immunofilaments highlight important roles for CD28 and CD2 signaling in T-cell priming, proliferation, cytokine production, and multifunctionality. Taken together, this work provides insight into the role of combined TCR and co-stimulation on T-cell phenotype, function, and fate using immunofilaments. Notably, the findings on the roles of co-stimulatory molecule function can be used for the rational design of future cancer immunotherapies.

Contains fulltext : 233607.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 206170.pdf (publisher's version ) (Open Access)

Radboud University, 19 september 2018, Promotor : Figdor, C.G. Co-promotores : Tel, J., Hammink, R., Contains fulltext : 194280.pdf (publisher's version ) (Open Access)

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Contains fulltext : 199026.pdf (publisher's version ) (Open Access)

Contains fulltext : 174408.pdf (publisher's version ) (Open Access), Artificial antigen-presenting cells (aAPCs) have recently gained a lot of attention. They efficiently activate T cells and serve as powerful replacements for dendritic cells in cancer immunotherapy. Focusing on a specific class of polymer-based aAPCs, so-called synthetic dendritic cells (sDCs), we have investigated the importance of multivalent binding on T-cell activation. Using antibody-functionalized sDCs, we have tested the influence of polymer length and antibody density. Increasing the multivalent character of the antibody-functionalized polymer lowered the effective concentration required for T-cell activation. This was evidenced for both early and late stages of activation. The most important effect observed was the significantly prolonged activation of the stimulated T cells, indicating that multivalent sDCs sustain T-cell signaling. Our results highlight the importance of multivalency for the design of aAPCs and will ultimately allow for better mimics of natural dendritic cells that can be used as vaccines in cancer treatment.

Contains fulltext : 177944.pdf (publisher's version ) (Closed access), Water-soluble polyisocyanopeptides (PICs) are a new class of synthetic polymers that mimic natural protein-based filaments. Their unique semiflexible properties combined with a length of several hundred nanometers have recently enabled a number of biomedical applications ranging from tissue engineering to cancer immunotherapy. One crucial step toward the further development of PICs for these applications is the efficient and controlled synthesis and purification of PIC-biomolecule conjugates. Considering the large size of PICs and the biomolecules to be conjugated, conjugation reactions do usually not proceed to completion due to steric effects. As a consequence, purification of the reaction mixture is necessary to separate the obtained bioconjugates from unreacted biomolecules. As a direct result of the semiflexible nature of PICs, standard polymer and protein purification methods based on molecular weight have not been successful. Here, we introduce a new affinity-based purification method utilizing biotin as an affinity tag. PICs decorated with a controlled and tunable density of biotin molecules (biotinPICs) were efficiently bound to and eluted from a monoavidin resin in buffered aqueous solution. Using these biotinPICs, two different protein conjugates were synthesized, one carrying the enzyme alkaline phosphatase (PhoA) and the other T-cell activating anti-CD3 antibodies. The resulting biotinPIC-protein conjugates were successfully obtained in high purity (>90%) and without any loss of protein activity. The high purity greatly simplifies the analysis of biotinPIC bioconjugates, such as the determination of the average number of biomolecules conjugated per biotinPIC chain. Most importantly, it allows for the direct and straightforward application of the obtained bioconjugates in the desired applications. The new method developed may further be adapted for the purification of other advanced bioconjugates that are difficult to obtain in high purity with the avail

Contains fulltext : 137111.pdf (publisher's version ) (Open Access), Active anti-cancer immune responses depend on efficient presentation of tumor antigens and co-stimulatory signals by antigen-presenting cells (APCs). Therapy with autologous natural APCs is costly and time-consuming and results in variable outcomes in clinical trials. Therefore, development of artificial APCs (aAPCs) has attracted significant interest as an alternative. We discuss the characteristics of various types of acellular aAPCs, and their clinical potential in cancer immunotherapy. The size, shape, and ligand mobility of aAPCs and their presentation of different immunological signals can all have significant effects on cytotoxic T cell activation. Novel optimized aAPCs, combining carefully tuned properties, may lead to efficient immunomodulation and improved clinical responses in cancer immunotherapy.

Item does not contain fulltext, Strain-promoted alkyne-nitrone cycloadditon (SPANC) was optimized as a versatile strategy for dual functionalization of peptides and proteins. The usefulness of the dual labeling protocol is first exemplified by the simultaneous introduction of a chloroquine and a stearyl moiety, two endosomal escape-improving functional groups, into the cell-penetrating peptide hLF (human lactoferrin). Additionally, we demonstrate that dual labeling of proteins is feasible by combining metal-free and copper-catalyzed click chemistry. First, SPANC is applied to enhanced green fluorescent protein to introduce both biotin and a terminal alkyne. The terminal acetylene then serves as a convenient anchor point for the CuAAC reaction with azido-containing fluorescein, thereby demonstrating the potential of combined SPANC and CuAAC for the straightforward, dual functionalization of proteins.