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.

Engineering hyaluronic acid-based cryogels for CD44-mediated breast tumor reconstruction.

Breast cancer is a major health concern worldwide and is the leading cause of cancer-related death among American women. Traditional therapies, such as surgery, chemotherapy, and radiotherapy, are usually ineffective. Furthermore, cancer recurrence following targeted therapy often results from acquired drug resistance. Therefore, more realistic tumor models than monolayer cell culture for drug screening and discovery in an in vitro setting would facilitate the development of new therapeutic strategies. Toward this goal, we first developed a simple, rapid, low-cost, and high-throughput method for generating uniform multi-cellular tumor spheroids (MCTS) with controllable size. Next, biomimetic cryogel scaffolds fabricated from hyaluronic acid (HA) were utilized as a platform to reconstruct breast tumor microtissues with aspects of the complex tumor microenvironment in three dimensions. Finally, we investigated the interactions between the HA-based cryogels and CD44-positive breast tumor cells, individually or as MCTS. We found that incorporating the adhesive RGD peptide in cryogels led to the formation of a monolayer of tumor cells on the polymer walls, whereas MCTS cultured on RGD-free HA cryogels resulted in the growth of large and dense microtumors, more similar to native tumor masses. As a result, the MCTS-laden HA cryogel system induced a highly aggressive and chemotherapy drug-resistant tumor model. RGD-free HA-based cryogels represent an effective starting point for designing tumor models for preclinical research, therapeutic drug screening, and early cancer diagnosis.

Biomaterials and Oxygen Join Forces to Shape the Immune Response and Boost COVID-19 Vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an unprecedented global health crisis, resulting in a critical need for effective vaccines that generate protective antibodies. Protein subunit vaccines represent a promising approach but often lack the immunogenicity required for strong immune stimulation. To overcome this challenge, it is first demonstrated that advanced biomaterials can be leveraged to boost the effectiveness of SARS-CoV-2 protein subunit vaccines. Additionally, it is reported that oxygen is a powerful immunological co-adjuvant and has an ability to further potentiate vaccine potency. In preclinical studies, mice immunized with an oxygen-generating coronavirus disease 2019 (COVID-19) cryogel-based vaccine (O2-CryogelVAX) exhibit a robust Th1 and Th2 immune response, leading to a sustained production of highly effective neutralizing antibodies against the virus. Even with a single immunization, O2-CryogelVAX achieves high antibody titers within 21 days, and both binding and neutralizing antibody levels are further increased after a second dose. Engineering a potent vaccine system that generates sufficient neutralizing antibodies after one dose is a preferred strategy amid vaccine shortage. The data suggest that this platform is a promising technology to reinforce vaccine-driven immunostimulation and is applicable to current and emerging infectious diseases.

Semiflexible Immunobrushes Induce Enhanced T Cell Activation and Expansion.

A variety of bioactive materials developed to expand T cells for adoptive transfer into cancer patients are currently evaluated in the clinic. In most cases, T cell activating biomolecules are attached to rigid surfaces or matrices and form a static interface between materials and the signaling receptors on the T cells. We hypothesized that a T cell activating polymer brush interface might better mimic the cell surface of a natural antigen-presenting cell, facilitating receptor movement and concomitant advantageous mechanical forces to provide enhanced T cell activating capacities. Here, as a proof of concept, we synthesized semiflexible polyisocyanopeptide (PIC) polymer-based immunobrushes equipped with T cell activating agonistic anti-CD3 (αCD3) and αCD28 antibodies placed on magnetic microbeads. We demonstrated enhanced efficiency of ex vivo expansion of activated primary human T cells even at very low numbers of stimulating antibodies compared to rigid beads. Importantly, the immunobrush architecture appeared crucial for this improved T cell activating capacity. Immunobrushes outperform current benchmarks by producing higher numbers of T cells exhibiting a combination of beneficial phenotypic characteristics, such as reduced exhaustion marker expression, high cytokine production, and robust expression of cytotoxic hallmarks. This study indicates that semiflexible immunobrushes have great potential in making T cell-based immunotherapies more effective.

Harnessing biomaterials for therapeutic strategies against COVID-19.

With the emergence of the coronavirus disease 2019 (COVID-19), the world is experiencing a profound human health crisis. The number of infections and deaths due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to increase every minute, pinpointing major shortcomings in our ability to prevent viral outbreaks. Although several COVID-19 vaccines have been recently approved for emergency use, therapeutic options remain limited, and their long-term potency has yet to be validated. Biomaterials science has a pivotal role to play in pushing the boundaries of emerging technologies for antiviral research and treatment. In this perspective, we discuss how biomaterials can be harnessed to develop accurate COVID-19 infection models, enhance antiviral drug delivery, foster new antiviral strategies, and boost vaccine efficacy. These efforts will not only contribute to stop or mitigate the current pandemic but will also provide unorthodox platforms to understand, prevent, and protect us from future viral outbreaks.

Oxygen-Generating Cryogels Restore T Cell Mediated Cytotoxicity in Hypoxic Tumors.

Solid tumors are protected from antitumor immune responses due to their hypoxic microenvironments. Weakening hypoxia-driven immunosuppression by hyperoxic breathing of 60% oxygen has shown to be effective in unleashing antitumor immune cells against solid tumors. However, efficacy of systemic oxygenation is limited against solid tumors outside of lungs and has been associated with unwanted side effects. As a result, it is essential to develop targeted oxygenation alternatives to weaken tumor hypoxia as novel approaches to restore immune responses against cancer. Herein, we report on injectable oxygen-generating cryogels (O2-cryogels) to reverse tumor-induced hypoxia. These macroporous biomaterials were designed to locally deliver oxygen, inhibit the expression of hypoxia-inducible genes in hypoxic melanoma cells, and reduce the accumulation of immunosuppressive extracellular adenosine. Our data show that O2-cryogels enhance T cell-mediated secretion of cytotoxic proteins, restoring the killing ability of tumor-specific CTLs, both in vitro and in vivo. In summary, O2-cryogels provide a unique and safe platform to supply oxygen as a co-adjuvant in hypoxic tumors and have the potential to improve cancer immunotherapies.

Supramolecular Self-Assembled Peptide-Based Vaccines: Current State and Future Perspectives.

Despite the undeniable success of vaccination programs in preventing diseases, effective vaccines against several life-threatening infectious pathogens such as human immunodeficiency virus are still unavailable. Vaccines are designed to boost the body's natural ability to protect itself against foreign pathogens. To enhance vaccine-based immunotherapies to combat infections, cancer, and other conditions, biomaterials have been harnessed to improve vaccine safety and efficacy. Recently, peptides engineered to self-assemble into specific nanoarchitectures have shown great potential as advanced biomaterials for vaccine development. These supramolecular nanostructures (i.e., composed of many peptides) can be programmed to organize into various forms, including nanofibers, nanotubes, nanoribbons, and hydrogels. Additionally, they have been designed to be responsive upon exposure to various external stimuli, providing new innovations in the development of smart materials for vaccine delivery and immunostimulation. Specifically, self-assembled peptides can provide cell adhesion sites, epitope recognition, and antigen presentation, depending on their biochemical and structural characteristics. Furthermore, they have been tailored to form exquisite nanostructures that provide improved enzymatic stability and biocompatibility, in addition to the controlled release and targeted delivery of immunomodulatory factors (e.g., adjuvants). In this mini review, we first describe the different types of self-assembled peptides and resulting nanostructures that have recently been investigated. Then, we discuss the recent progress and development trends of self-assembled peptide-based vaccines, their challenges, and clinical translatability, as well as their future perspectives.

Engineering a macroporous fibrin-based sequential interpenetrating polymer network for dermal tissue engineering.

The success of skin tissue engineering for deep wound healing relies predominantly on the design of innovative and effective biomaterials. This study reports the synthesis and characterization of a new type of naturally-derived and macroporous interpenetrating polymer network (IPN) for skin repair. These biomaterials consist of a biologically active fibrous fibrin network polymerized within a mechanically robust and macroporous construct made of polyethylene glycol and biodegradable serum albumin (PEGDM-co-SAM). First, mesoporous PEGDM-co-SAM hydrogels were synthesized and subjected to cryotreatment to introduce an interconnected macroporous network. Subsequently, fibrin precursors were incorporated within the cryotreated PEG-based network and then allowed to spontaneously polymerize and form a sequential IPN. Rheological measurements indicated that fibrin-based sequential IPN hydrogels exhibited improved and tunable mechanical properties when compared to fibrin hydrogels alone. In vitro data showed that human dermal fibroblasts adhere, infiltrate and proliferate within the IPN constructs, and were able to secrete endogenous extracellular matrix proteins, namely collagen I and fibronectin. Furthermore, a preclinical study in mice demonstrated that IPNs were stable over 1-month following subcutaneous implantation, induced a minimal host inflammatory response, and displayed a substantial cellular infiltration and tissue remodeling within the constructs. Collectively, these data suggest that macroporous and mechanically reinforced fibrin-based sequential IPN hydrogels are promising three-dimensional platforms for dermal tissue regeneration.

Vascularization strategies for skin tissue engineering.

A number of challenges in skin grafting for wound healing have drawn researchers to focus on skin tissue engineering as an alternative solution. The core idea of tissue engineering is to use scaffolds, cells, and/or bioactive molecules to help the skin to properly recover from injuries. Over the past decades, the field has significantly evolved, developing various strategies to accelerate and improve skin regeneration. However, there are still several concerns that should be addressed. Among these limitations, vascularization is known as a critical challenge that needs thorough consideration. Delayed wound healing of large defects results in an insufficient vascular network and ultimately ischemia. Recent advances in the field of tissue engineering paved the way to improve vascularization of skin substitutes. Broadly, these solutions can be classified into two categories as (1) use of growth factors, reactive oxygen species-inducing nanoparticles, and stem cells to promote angiogenesis, and (2) in vitro or in vivo prevascularization of skin grafts. This review summarizes the state-of-the-art approaches, their limitations, and highlights the latest advances in therapeutic vascularization strategies for skin tissue engineering.

Injectable Cryogels for Biomedical Applications.

To prevent postoperative complications, there has been a substantial interest in designing syringe-injectable hydrogels. To date, cryogels remain the only viable option for preformed and large-scale hydrogels to be delivered through a conventional needle-syringe injection. Cryogels, a type of hydrogel with exceptional features, are fabricated at subzero temperatures. This process typically results in a biomaterial with a unique macroporous network, shape-memory properties, and exceptional flexibility allowing syringe injectability. These advanced biomaterials have been used for a number of biomedical applications, including tissue engineering, drug delivery, and more recently, immunotherapy. This review summarizes the recent progress on the design of injectable cryogels, their current limitations, and strategies to further improve their properties for translatability into the clinic.

Synthetic Semiflexible and Bioactive Brushes.

Polymer brushes are extensively used for the preparation of bioactive surfaces. They form a platform to attach functional (bio)molecules and control the physicochemical properties of the surface. These brushes are nearly exclusively prepared from flexible polymers, even though much stiffer brushes from semiflexible polymers are frequently found in nature, which exert bioactive functions that are out of reach for flexible brushes. Synthetic semiflexible polymers, however, are very rare. Here, we use polyisocyanopeptides (PICs) to prepare high-density semiflexible brushes on different substrate geometries. For bioconjugation, we developed routes with two orthogonal click reactions, based on the strain-promoted azide-alkyne cycloaddition reaction and the (photoactivated) tetrazole-ene cycloaddition reaction. We found that for high brush densities, multiple bonds between the polymer and the substrate are necessary, which was achieved in a block copolymer strategy. Whether the desired biomolecules are conjugated to the PIC polymer before or after brush formation depends on the dimensions and required densities of the biomolecules and the curvature of the substrate. In either case, we provide mild, aqueous, and highly modular reaction strategies, which make PICs a versatile addition to the toolbox for generating semiflexible bioactive polymer brush surfaces.

Injectable Biomimetic Hydrogels as Tools for Efficient T Cell Expansion and Delivery.

Biomaterial-based scaffolds are promising tools for controlled immunomodulation. They can be applied as three dimensional (3D) culture systems in vitro, whereas in vivo they may be used to dictate cellular localization and exert spatiotemporal control over cues presented to the immune system. As such, scaffolds can be exploited to enhance the efficacy of cancer immunotherapies such as adoptive T cell transfer, in which localization and persistence of tumor-specific T cells dictates treatment outcome. Biomimetic polyisocyanopeptide (PIC) hydrogels are polymeric scaffolds with beneficial characteristics as they display reversible thermally-induced gelation at temperatures above 16°C, which allows for their minimally invasive delivery via injection. Moreover, incorporation of azide-terminated monomers introduces functional handles that can be exploited to include immune cell-modulating cues. Here, we explore the potential of synthetic PIC hydrogels to promote the in vitro expansion and in vivo local delivery of pre-activated T cells. We found that PIC hydrogels support the survival and vigorous expansion of pre-stimulated T cells in vitro even at high cell densities, highlighting their potential as 3D culture systems for efficient expansion of T cells for their adoptive transfer. In particular, the reversible thermo-sensitive behavior of the PIC scaffolds favors straightforward recovery of cells. PIC hydrogels that were injected subcutaneously gelated instantly in vivo, after which a confined 3D structure was formed that remained localized for at least 4 weeks. Importantly, we noticed no signs of inflammation, indicating that PIC hydrogels are non-immunogenic. Cells co-delivered with PIC polymers were encapsulated within the scaffold in vivo. Cells egressed gradually from the PIC gel and migrated into distant organs. This confirms that PIC hydrogels can be used to locally deliver cells within a supportive environment. These results demonstrate that PIC hydrogels are highly promising for both the in vitro expansion and in vivo delivery of pre-activated T cells. Covalent attachment of biomolecules onto azide-functionalized PIC polymers provides the opportunity to steer the phenotype, survival or functional response of the adoptively transferred cells. As such, PIC hydrogels can be used as valuable tools to improve current adoptive T cell therapy strategies.

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.

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.

Towards efficient cancer immunotherapy: advances in developing artificial antigen-presenting cells.

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.

N-Terminal dual protein functionalization by strain-promoted alkyne-nitrone cycloaddition.

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.