Roel Hammink

Stress-stiffening-mediated stem-cell commitment switch in soft responsive hydrogels

Bulk matrix stiffness has emerged as a key mechanical cue in stem cell differentiation. Here, we show that the commitment and differentiation of human mesenchymal stem cells encapsulated in physiologically soft (∼0.2-0.4 kPa), fully synthetic polyisocyanopeptide-based three-dimensional (3D) matrices that mimic the stiffness of adult stem cell niches and show biopolymer-like stress stiffening, can be readily switched from adipogenesis to osteogenesis by changing only the onset of stress stiffening. This mechanical behaviour can be tuned by simply altering the materials polymer length whilst maintaining stiffness and ligand density. Our findings introduce stress stiffening as an important parameter that governs stem cell fate in a 3D microenvironment, and reveal a correlation between the onset of stiffening and the expression of the microtubule-associated protein DCAMKL1, thus implicating DCAMKL1 in a stress-stiffening-mediated, mechanotransduction pathway that involves microtubule dynamics in stem cell osteogenesis.

Polymer-based synthetic dendritic cells for tailoring robust and multifunctional t cell responses

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in T cell activation. Recent efforts in cancer immunotherapy have been directed at the development of artificial antigen presenting cells (aAPCs) loaded with tumor antigens. These aAPCs are designed to mimic DCs with the goal of triggering an efficient and specific T cell response directed against the tumor. We have designed a novel synthetic dendritic cell (sDC) that possesses the essential features of natural DCs. Our sDC is based on a semiflexible poly(isocyano peptide) polymer and carries anti-CD3 antibodies (αCD3) for triggering the T cell receptor/CD3 complex as well as anti-CD28 antibodies (αCD28) as a co-stimulatory signal. Multiple copies of both antibodies facilitate multivalent binding similar to natural DCs. The high mobility of these polymer-bound antibodies, reminiscent of protein motility in a natural plasma membrane, enables receptor rearrangements to occur during T cell activation. We show that our bifunctional αCD3/αCD28-sDC triggers T cell activation at significantly lower antibody concentrations than freely soluble antibodies. This superior performance is further demonstrated in comparison to a mixture of monofunctional αCD3-sDC and αCD28-sDC. The presence of both antibodies on the same polymer not only reduces the threshold for T cell activation but, more importantly, critically shapes the specificity of the T cell response. αCD3/αCD28-sDC is a far more efficient activator of multifunctional killer cells. These findings demonstrate the potential of multifunctional polymers for mimicking natural DCs, paving the way for their exploitation in immunotherapeutic strategies.

Controlling T-cell activation with synthetic dendritic cells using the multivalency effect

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.

Biomimetic Stress Sensitive Hydrogel Controlled by DNA Nanoswitches

One of the most intriguing and important aspects of biological supramolecular materials is its ability to adapt macroscopic properties in response to environmental cues for controlling cellular processes. Recently, bulk matrix stiffness, in particular, stress sensitivity, has been established as a key mechanical cue in cellular function and development. However, stress-stiffening capacity and the ability to control and exploit this key characteristic is relatively new to the field of biomimetic materials. In this work, DNA-responsive hydrogels, composed of semiflexible PIC polymers equipped with DNA cross-linkers, were engineered to create mimics of natural biopolymer networks that capture these essential elastic properties and can be controlled by external stimuli. We show that the elastic properties are governed by the molecular structure of the cross-linker, which can be readily varied providing access to a broad range of highly tunable soft hydrogels with diverse stress-stiffening regimes. By using cross-linkers based on DNA nanoswitches, responsive to pH or ligands, internal control elements of mechanical properties are implemented that allow for dynamic control of elastic properties with high specificity. The work broadens the current knowledge necessary for the development of user defined biomimetic materials with stress stiffening capacity.

Towards synthetic immune cells for cancer immunotherapy

Most cellular therapies targeting cancer require culture of autologous cells. The dedicated facilities and personnel needed, the variability in quality of the product depending on the condition of the patient and state of disease, hamper widespread use. In an attempt to generate an of the shelf product to target cancer we exploited the ever expanding possibilities to build supramolecular structures offered by chemistry to mimic nature including the construction of artificial immune cells or functions thereof. Effective immunotherapy critically depends on efficient production of antigen-specific cytotoxic T-cells. Herein lies an opportunity for both chemists and immunologists to design and synthesize so-called artificial antigen presenting cells (aAPCs) that can generate in vivo T-cell expansion. We have designed a novel synthetic dendritic cell (sDC) that possesses essential features of natural DCs. Our sDC is based on a semi-flexible poly(isocyano peptide) polymer and, as proof of principle, carries both anti-CD3 antibodies for triggering the T cell receptor/CD3 complex as well as anti-CD28 antibodies as a co-stimulatory signal. Multiple copies of both antibodies facilitate multivalent binding similar to natural DCs. The high mobility of these polymer-bound antibodies, reminiscent of protein motility in a natural plasma membrane, enables receptor rearrangements to occur during T cell activation. We observed effective T cell activation at significantly lower antibody concentrations than freely soluble antibodies. We also demonstrate antigen specific T cell activation when MHC/peptide complexes were bound. These findings demonstrate the potential of multifunctional polymers for mimicking functions of natural DCs or other immune cells, paving the way for their exploitation in immunotherapeutic strategies. Citation Format: Carl G. Figdor, Subhra Mandal, Roel Hammink, Loek Eggermont, Jorieke Weiden, Dion Voerman, Jurjen Tel, Zaskia H. Eksteen-Akeroyd, Kerstin Blank, Alan E. Rowan. Towards synthetic immune cells for cancer immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA29.

Strategies To Increase the Thermal Stability of Truly Biomimetic Hydrogels: Combining Hydrophobicity and Directed Hydrogen Bonding

Enhancing the thermal stability of proteins is an important task for protein engineering. There are several ways to increase the thermal stability of proteins in biology, such as greater hydrophobic interactions, increased helical content, decreased occurrence of thermolabile residues, or stable hydrogen bonds. Here, we describe a well-defined polymer based on β-helical polyisocyanotripeptides (TriPIC) that uses biological approaches, including hydrogen bonding and hydrophobic interactions for its exceptional thermal stability in aqueous solutions. The multiple hydrogen bonding arrays along the polymer backbone shield the hydrophobic core from water. Variable temperature CD and FTIR studies indicate that, on heating, a better packed polymer conformation further stiffens the backbone. Driven by hydrophobic interactions, TriPIC solutions give fully reversible hydrogels that can withstand high temperatures (80 °C) for extended times. Cryo-scanning electron microscopy (cryo-SEM), small-angle X-ray scattering (SAXS), and thorough rheological analysis show that the hydrogel has a bundled architecture, which gives rise to strain stiffening effects on deformation of the gel, analogous to many biological hydrogels.

Affinity-Based Purification of Polyisocyanopeptide Bioconjugates

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 available standard methods.

Cytokine-Functionalized Synthetic Dendritic Cells for T Cell Targeted Immunotherapies

Widespread application of cytokine‐based therapies is hampered by severe toxic side effects, resulting from off‐target (immune) cell stimulation. This emphasizes the need for more precise targeting of cytokines to immune cells. While cytokines are generally active as soluble proteins, it is demonstrated here that synthetic mimics of immune cells based on polyisocyanopeptides (PICs), called synthetic dendritic cells (sDCs) can efficiently present immobilized cytokines to T cells, when targeted by anti‐CD3 antibodies. Anti‐CD3/IL‐2‐functionalized PICs induce strong T cell activation and proliferation, when compared to PICs functionalized with interleukin‐2 (IL‐2) alone. In contrast to the semi‐flexible PICs, immobilization of IL‐2 on a rigid micro‐sized scaffold results in significant loss of IL‐2 activity, signifying the importance of molecular flexibility on the PIC polymers to maintain function. Similarly, anti‐CD3/IFNα‐functionalized PICs support long‐term proliferation of T cells. Interferon‐α containing sDCs additionally promote the development of cytotoxic effector functions of T cells with limited upregulation of the inhibitory immune checkpoint PD‐1. This high cytolytic activity and low PD‐1 expression are essential to maintain effective anti‐cancer activity. Together, these results demonstrate that PICs form unique nano‐sized scaffolds that efficiently present immobilized cytokines to T cells, thus creating a powerful tool to improve cytokine‐based immunotherapies without concomitant off‐target toxicity.