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.

pH and ROS Responsiveness of Polymersome Nanovaccines for Antigen and Adjuvant Codelivery: An In Vitro and In Vivo Comparison.

The antitumor immunity can be enhanced through the synchronized codelivery of antigens and immunostimulatory adjuvants to antigen-presenting cells, particularly dendritic cells (DCs), using nanovaccines (NVs). To study the influence of intracellular vaccine cargo release kinetics on the T cell activating capacities of DCs, we compared stimuli-responsive to nonresponsive polymersome NVs. To do so, we employed "AND gate" multiresponsive (MR) amphiphilic block copolymers that decompose only in response to the combination of chemical cues present in the environment of the intracellular compartments in antigen cross-presenting DCs: low pH and high reactive oxygen species (ROS) levels. After being unmasked by ROS, pH-responsive side chains are exposed and can undergo a charge shift within a relevant pH window of the intracellular compartments in antigen cross-presenting DCs. NVs containing the model antigen Ovalbumin (OVA) and the iNKT cell activating adjuvant α-Galactosylceramide (α-Galcer) were fabricated using microfluidics self-assembly. The MR NVs outperformed the nonresponsive NV in vitro, inducing enhanced classical- and cross-presentation of the OVA by DCs, effectively activating CD8+, CD4+ T cells, and iNKT cells. Interestingly, in vivo, the nonresponsive NVs outperformed the responsive vaccines. These differences in polymersome vaccine performance are likely linked to the kinetics of cargo release, highlighting the crucial chemical requirements for successful cancer nanovaccines.

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.

Robust Antigen-Specific T Cell Activation within Injectable 3D Synthetic Nanovaccine Depots.

Synthetic cancer vaccines may boost anticancer immune responses by co-delivering tumor antigens and adjuvants to dendritic cells (DCs). The accessibility of cancer vaccines to DCs and thereby the delivery efficiency of antigenic material greatly depends on the vaccine platform that is used. Three-dimensional scaffolds have been developed to deliver antigens and adjuvants locally in an immunostimulatory environment to DCs to enable sustained availability. However, current systems have little control over the release profiles of the cargo that is incorporated and are often characterized by an initial high-burst release. Here, an alternative system is designed that co-delivers antigens and adjuvants to DCs through cargo-loaded nanoparticles (NPs) incorporated within biomaterial-based scaffolds. This creates a programmable system with the potential for controlled delivery of their cargo to DCs. Cargo-loaded poly(d,l-lactic-co-glycolic acid) NPs are entrapped within the polymer walls of alginate cryogels with high efficiency while retaining the favorable physical properties of cryogels, including syringe injection. DCs cultured within these NP-loaded scaffolds acquire strong antigen-specific T cell-activating capabilities. These findings demonstrate that introduction of NPs into the walls of macroporous alginate cryogels creates a fully synthetic immunostimulatory niche that stimulates DCs and evokes strong antigen-specific T cell responses.

PLGA Nanoparticles Co-encapsulating NY-ESO-1 Peptides and IMM60 Induce Robust CD8 and CD4 T Cell and B Cell Responses.

Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo. Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.

Nanovaccine administration route is critical to obtain pertinent iNKt cell help for robust anti-tumor T and B cell responses.

Nanovaccines, co-delivering antigen and invariant natural killer T (iNKT) cell agonists, proved to be very effective in inducing anti-tumor T cell responses due to their exceptional helper function. However, it is known that iNKT cells are not equally present in all lymphoid organs and nanoparticles do not get evenly distributed to all immune compartments. In this study, we evaluated the effect of the vaccination route on iNKT cell help to T and B cell responses for the first time in an antigen and agonist co-delivery setting. Intravenous administration of PLGA nanoparticles was mainly targeting liver and spleen where iNKT1 cells are abundant and induced the highest serum IFN-y levels, T cell cytotoxicity, and Th-1 type antibody responses. In comparison, after subcutaneous or intranodal injections, nanoparticles mostly drained or remained in regional lymph nodes where iNKT17 cells were abundant. After subcutaneous and intranodal injections, antigen-specific IgG2 c production was hampered and IFN-y production, as well as cytotoxic T cell responses, depended on sporadic systemic drainage. Therapeutic anti-tumor experiments also demonstrated a clear advantage of intravenous injection over intranodal or subcutaneous vaccinations. Moreover, tumor control could be further improved by PD-1 immune checkpoint blockade after intravenous vaccination, but not by intranodal vaccination. Anti PD-1 antibody combination mainly exerts its effect by prolonging the cytotoxicity of T cells. Nanovaccines also demonstrated synergism with anti-4-1BB agonistic antibody treatment in controlling tumor growth. We conclude that nanovaccines containing iNKT cell agonists shall be preferentially administered intravenously, to optimally reach cellular partners for inducing effective anti-tumor immune responses.

Co-delivery of PLGA encapsulated invariant NKT cell agonist with antigenic protein induce strong T cell-mediated antitumor immune responses.

Antitumor immunity can be enhanced by the coordinated release and delivery of antigens and immune-stimulating agents to antigen-presenting cells via biodegradable vaccine carriers. So far, encapsulation of TLR ligands and tumor-associated antigens augmented cytotoxic T cell (CTLs) responses. Here, we compared the efficacy of the invariant NKT (iNKT) cell agonist α-galactosylceramide (α-GalCer) and TLR ligands (R848 and poly I:C) as an adjuvant for the full length ovalbumin (OVA) in PLGA nanoparticles. We observed that OVA+α-GalCer nanoparticles (NP) are superior over OVA+TLR-L NP in generating and stimulating antigen-specific cytotoxic T lymphocytes without the need for CD4+ T cell help. Not only a 4-fold higher induction of antigen-specific T cells was observed, but also a more profound IFN-γ secretion was obtained by the addition α-GalCer. Surprisingly, we observed that mixtures of OVA containing NP with α-GalCer were ineffective, demonstrating that co-encapsulation of both α-GalCer and antigen within the same nanoparticle is essential for the observed T cell responses. Moreover, a single immunization with OVA+α-GalCer NP provided substantial protection from tumor formation and even delayed the growth of already established tumors, which coincided with a prominent and enhanced antigen-specific CD8+ T cell infiltration. The provided evidence on the advantage of antigen and α-GalCer coencapsulation should be considered in the design of future nanoparticle vaccines for therapeutic purposes.

Targeted delivery of a sialic acid-blocking glycomimetic to cancer cells inhibits metastatic spread.

Sialic acid sugars are overexpressed by cancer cells and contribute to the metastatic cascade at multiple levels. Therapeutic interference of sialic acids, however, has been difficult to pursue because of the absence of dedicated tools. Here we show that a rationally designed sialic acid-blocking glycomimetic (P-3F(ax)-Neu5Ac) successfully prevents cancer metastasis. Formulation of P-3F(ax)--Neu5Ac into poly(lactic-co-glycolic acid nanoparticles coated with antityrosinase-related protein-1 antibodies allowed targeted delivery of P-3F(ax)--Neu5Ac into melanoma cells, slow release, and long-term sialic acid blockade. Most importantly, intravenous injections of melanoma-targeting P-3F(ax)--Neu5Ac nanoparticles prevented metastasis formation in a murine lung metastasis model. These findings stress the importance of sialoglycans in cancer metastasis and advocate that sialic acid blockade using rationally designed glycomimetics targeted to cancer cells can effectively prevent cancer metastases. This targeting strategy to interfere with sialic acid-dependent processes is broadly applicable not only for different types of cancer but also in infection and inflammation.