Immunofilaments Are Well Tolerated after Local or Systemic Administration in Mice.

The invention of nanosized biomaterials has paved the way for novel therapeutics that can manipulate cells on a nanoscale. Nanosized immunofilaments (IFs) are synthetic filamentous polymers consisting out of polyisocyanopeptides, which have been recently established as a powerful platform to activate specific immune cells in vivo such that they raise an antitumor immune response. However, toxicological effects or immunogenicity toward the IFs have not yet been investigated. In this study, we evaluated potential toxic or immunogenic effects in C57BL/6 mice upon intravenous or subcutaneous injection of nonfunctionalized IFs or immunostimulatory IFs over 30 days. We here present a detailed analysis of the gross pathology, hematological parameters, blood biochemistry, histology, and antibody-response against the IF backbone. Our results demonstrate that IFs do not induce severe acute or chronic toxicity in mice. After 30 days, we only found elevated IgG-titers in intravenously injected but not subcutaneously injected mice. In summary, we demonstrate that IFs can be administered into a living organism without adverse side effects, thereby establishing the safety of IFs as a therapeutic intervention.

Direct In Vivo Activation of T Cells with Nanosized Immunofilaments Inhibits Tumor Growth and Metastasis.

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.

Influence of Network Topology on the Viscoelastic Properties of Dynamically Crosslinked Hydrogels.

Biological materials combine stress relaxation and self-healing with non-linear stress-strain responses. These characteristic features are a direct result of hierarchical self-assembly, which often results in fiber-like architectures. Even though structural knowledge is rapidly increasing, it has remained a challenge to establish relationships between microscopic and macroscopic structure and function. Here, we focus on understanding how network topology determines the viscoelastic properties, i.e., stress relaxation, of biomimetic hydrogels. We have dynamically crosslinked two different synthetic polymers with one and the same crosslink. The first polymer, a polyisocyanopeptide (PIC), self-assembles into semi-flexible, fiber-like bundles, and thus displays stress-stiffening, similar to many biopolymer networks. The second polymer, 4-arm poly(ethylene glycol) (starPEG), serves as a reference network with well-characterized structural and viscoelastic properties. Using one and the same coiled coil crosslink allows us to decouple the effects of crosslink kinetics and network topology on the stress relaxation behavior of the resulting hydrogel networks. We show that the fiber-containing PIC network displays a relaxation time approximately two orders of magnitude slower than the starPEG network. This reveals that crosslink kinetics is not the only determinant for stress relaxation. Instead, we propose that the different network topologies determine the ability of elastically active network chains to relax stress. In the starPEG network, each elastically active chain contains exactly one crosslink. In the absence of entanglements, crosslink dissociation thus relaxes the entire chain. In contrast, each polymer is crosslinked to the fiber bundle in multiple positions in the PIC hydrogel. The dissociation of a single crosslink is thus not sufficient for chain relaxation. This suggests that tuning the number of crosslinks per elastically active chain in combination with crosslink kinetics is a powerful design principle for tuning stress relaxation in polymeric materials. The presence of a higher number of crosslinks per elastically active chain thus yields materials with a slow macroscopic relaxation time but fast dynamics at the microscopic level. Using this principle for the design of synthetic cell culture matrices will yield materials with excellent long-term stability combined with the ability to locally reorganize, thus facilitating cell motility, spreading, and growth.