Stimulator of Interferon Genes Pathway Activation through the Controlled Release of STINGel Mediates Analgesia and Anti-Cancer Effects in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) presents significant treatment challenges due to its poor survival and intense pain at the primary cancer site. Cancer pain is debilitating, contributes to diminished quality of life, and causes opioid tolerance. The stimulator of interferon genes (STING) agonism has been investigated as an anti-cancer strategy. We have developed STINGel, an extended-release formulation that prolongs the availability of STING agonists, which has demonstrated an enhanced anti-tumor effect in OSCC compared to STING agonist injection. This study investigates the impact of intra-tumoral STINGel on OSCC-induced pain using two separate OSCC models and nociceptive behavioral assays. Intra-tumoral STINGel significantly reduced mechanical allodynia in the orofacial cancer model and alleviated thermal and mechanical hyperalgesia in the hind paw model. To determine the cellular signaling cascade contributing to the antinociceptive effect, we performed an in-depth analysis of immune cell populations via single-cell RNA-seq. We demonstrated an increase in M1-like macrophages and N1-like neutrophils after STINGel treatment. The identified regulatory pathways controlled immune response activation, myeloid cell differentiation, and cytoplasmic translation. Functional pathway analysis demonstrated the suppression of translation at neuron synapses and the negative regulation of neuron projection development in M2-like macrophages after STINGel treatment. Importantly, STINGel treatment upregulated TGF-ß pathway signaling between various cell populations and peripheral nervous system (PNS) macrophages and enhanced TGF-ß signaling within the PNS itself. Overall, this study sheds light on the mechanisms underlying STINGel-mediated antinociception and anti-tumorigenic impact.

Local Anti-PD-1 Delivery Prevents Progression of Premalignant Lesions in a 4NQO-Oral Carcinogenesis Mouse Model.

Although the principle of systemic treatment to prevent the progression of oral premalignant lesions (OPL) has been demonstrated, there remains a lack of consensus about an optimal approach that balances clinical efficacy with toxicity concerns. Recent advances in cancer therapy using approaches targeting the tumor immune microenvironment (TIME) including immune-checkpoint inhibitors indicate that these agents have significant clinically activity against different types of cancers, including oral cancer, and therefore they may provide an effective oral cancer prevention strategy for patients with OPLs. Our past work showed that systemic delivery of a monoclonal antibody to the programmed death receptor 1 (PD-1) immune checkpoint can inhibit the progression of OPLs to oral cancer in a syngeneic murine oral carcinogenesis model. Here we report a novel approach of local delivery of a PD-1 immune-checkpoint inhibitor loaded using a hydrogel, which significantly reduces the progression of OPLs to carcinomas. In addition, we detected a significant infiltration of regulatory T cells associated with oral lesions with p53 mutation, and a severe loss of expression of STING, which correlated with a decreased infiltration of dendritic cells in the oral lesions. However, a single local dose of PD-1 inhibitor was found to restore stimulator of interferon response cGAMP interactor 1 (STING) and CD11c expression and increase the infiltration of CD8+ T cells into the TIME irrespective of the p53 mutational status. Overall, we provide evidence for the potential clinical value of local delivery of biomaterials loaded with anti-PD-1 antibodies to prevent malignant progression of OPLs. PREVENTION RELEVANCE: Oral cancer is an aggressive disease, with an overall survival rate of 50%. Preinvasive histologic abnormalities such as tongue dysplasia represent an early stage of oral cancer; however, there are no treatments to prevent oral carcinoma progression. Here, we combined biomaterials loaded with an immunotherapeutic agent preventing oral cancer progression.

Biomaterial-Facilitated Immunotherapy for Established Oral Cancers.

We evaluated a peptide-based immunotherapy termed SynerGel: an injectable, biomaterial-based platform for intratumoral drug delivery. A drug-mimicking peptide hydrogel named L-NIL-MDP was loaded with an antitumor cyclic dinucleotide (CDN) immunotherapy agonist. The biomaterial combines inducible nitric oxide synthase (iNOS) inhibition with controlled delivery of CDNs, demonstrating between 4- and 20-fold slower drug release than commercially available hydrogels. SynerGel allowed for immune-mediated elimination of established treatment-resistant oral tumors in a murine model, with a median survival of 67.5 days compared with 44 days in no-treatment control. This report details findings for a promising therapy showing improved efficacy over previous hydrogel systems.

Chemical functionality of multidomain peptide hydrogels governs early host immune response.

Multidomain Peptide (MDP) hydrogels are nanofibrous materials with many potential biomedical applications. The peptide sequence design of these materials offers high versatility and allows for the incorporation of various chemical functionalities into the nanofibrous scaffold. It is known that host response to biomaterials is strongly affected by factors such as size, shape, stiffness, and chemistry. However, there is a lack of fundamental understanding of the host response to different MDP hydrogels. In particular, it is unknown what effect the chemical functionality displayed on the nanofiber has on biological activity. Here we evaluated the early inflammatory host response to four MDP hydrogels displaying amines, guanidinium ions, and carboxylates in a subcutaneous injection model. While all the studied peptide materials possess similar nanostructure and physical properties, they trigger markedly different inflammatory responses. These were characterized by immunophenotyping of the cellular infiltrate using multi-color flow cytometry. The negatively-charged peptides elicit minimal inflammation characterized by tissue-resident macrophage infiltration, fast remodeling, and no collagen deposition or blood vessel formation within the implants. In contrast, the positively-charged peptides are highly infiltrated by immune cells, are remodeled at a slower rate, promote angiogenesis, and result in a high degree of collagen deposition. The presence of dynamic cell phenotypes characterizes the inflammation caused by the lysine-based peptide, including inflammatory monocytes, macrophages, and lymphoid cells, which is seen to be resolving over time. The arginine-based hydrogel shows higher inflammatory response with a persistent and significant infiltration of polymorphonuclear myeloid-derived cells, even ten days after implantation. This understanding of the immune response to peptide biomaterials improves our ability to design effective materials and to tailor their use for specific biomedical applications.

Self-Assembling Multidomain Peptides: Design and Characterization of Neutral Peptide-Based Materials with pH and Ionic Strength Independent Self-Assembly.

Self-assembly of peptides is a powerful method of preparing nanostructured materials. These peptides frequently utilize charged groups as a convenient switch for controlling self-assembly in which pH or ionic strength determines the assembly state. Multidomain peptides have been previously designed with charged domains of amino acids, which create molecular frustration between electrostatic repulsion and a combination of supramolecular forces including hydrogen bonding and hydrophobic packing. This frustration is eliminated by the addition of multivalent ions or pH adjustment, resulting in a self-assembled hydrogel. However, these charged functionalities can have profound, unintended effects on the properties of the resulting material. Access to neutral self-assembled nanostructured hydrogels may allow for distinct biological properties that are not available to highly charged analogues. Here, we designed a series of peptides to determine if self-assembly could be mediated by the steric interactions created by neutral hydroxyproline (O) domains, eliminating the need for charged residues and creating a neutral peptide hydrogel. The series of peptides, O n (SL)6O n , was studied to determine the effect of oligo-hydroxyproline on peptide self-assembly and nanostructure. We show that peptide solubility and nanofiber length increase with a higher number of hydroxyproline residues. Within this series, O5(SL)6O5 displayed the optimal properties for self-assembly and hydrogelation. In vitro, this hydrogel supports cell viability of fibroblasts, while in vivo it is infiltrated with cells and easily degraded over time without promoting a strong inflammatory response. This neutral self-assembling peptide hydrogel shows promising properties for biomedical, cell preservation, and tissue regeneration applications.

Advances in immunotherapy delivery from implantable and injectable biomaterials.

Macroscale biomaterials, such as preformed implantable scaffolds and injectable soft materials, possess powerful synergies with anti-cancer immunotherapies. Immunotherapies on their own typically have poor delivery properties, and often require repeated high-dose injections that result in serious off-tumor effects and/or limited efficacy. Rationally designed biomaterials allow for discrete localization and controlled release of immunotherapeutic agents, and have been shown in a large number of applications to improve outcomes in the treatment of cancers via immunotherapy. Among various strategies, macroscale biomaterial delivery systems can take the form of robust tablet-like scaffolds that are surgically implanted into a tumor resection site, releasing programmed immune cells or immunoregulatory agents. Alternatively they can be developed as soft gel-like materials that are injected into solid tumors or sites of resection to stimulate a potent anti-tumor immune response. Biomaterials synthesized from diverse components such as polymers and peptides can be combined with any immunotherapy in the modern toolbox, from checkpoint inhibitors and stimulatory adjuvants, to cancer antigens and adoptive T cells, resulting in unique synergies and improved therapeutic efficacy. The field is growing rapidly in size as publications continue to appear in the literature, and biomaterial-based immunotherapies are entering clinical trials and human patients. It is unarguably an exciting time for cancer immunotherapy and biomaterial researchers, and further work seeks to understand the most critical design considerations in the development of the next-generation of immunotherapeutic biomaterials. This review will discuss recent advances in the delivery of immunotherapies from localized biomaterials, focusing on macroscale implantable and injectable systems. STATEMENT OF SIGNIFICANCE: Anti-cancer immunotherapies have shown exciting clinical results in the past few decades, yet they suffer from a few distinct limitations, such as poor delivery kinetics, narrow patient response profiles, and systemic side effects. Biomaterial systems are now being developed that can overcome many of these problems, allowing for localized adjuvant delivery, focused dose concentrations, and extended therapy presentation. The field of biocompatible carrier materials is uniquely suited to be combined with immunotherapy, promising to yield significant improvements in treatment outcomes and clinical care. In this review, the first pioneering efforts and most recent advances in biomaterials for immunotherapeutic applications are explored, with a specific focus on implantable and injectable biomaterials such as porous scaffolds, cryogels, and hydrogels.

Drug-Mimicking Nanofibrous Peptide Hydrogel for Inhibition of Inducible Nitric Oxide Synthase.

In this work, we develop a drug-mimicking nanofibrous peptide hydrogel that shows long-term bioactivity comparable to a small-molecule inhibitor of inducible nitric oxide synthase (iNOS). The iNOS inhibitor, N 6-(1-iminoethyl)-l-lysine (l-NIL), is a positively charged amino acid whose structure could be readily integrated into the framework of a positively charged multidomain peptide (MDP) through the modification of lysine side chains. This new l-NIL-MDP maintains the self-assembling properties of the base peptide, forming ß-sheet nanofibers, which entangle into a thixotropic hydrogel. The l-NIL-MDP hydrogel supports cell growth in vitro and allows syringe-directed delivery that persists in a targeted location in vivo for several weeks. Multiple characterization assays demonstrate the bioactivity of the l-NIL-MDP hydrogel to be comparable to the l-NIL small molecule. This includes iNOS inhibition of macrophages in vitro, reduced nitrotyrosine immunostaining in murine subcutaneous histology, and reduced serum levels of vascular endothelial growth factor in vivo. This study expands the toolbox of available peptide hydrogel scaffold designs that can modify biological activity without the need for any additional small-molecule drugs, proteins, or cells.

Multidomain Peptide Hydrogel Accelerates Healing of Full-Thickness Wounds in Diabetic Mice.

In vivo, multidomain peptide (MDP) hydrogels undergo rapid cell infiltration and elicit a mild inflammatory response which promotes angiogenesis. Over time, the nanofibers are degraded and a natural collagen-based extracellular matrix is produced remodeling the artificial material into natural tissue. These properties make MDPs particularly well suited for applications in regeneration. In this work, we test the regenerative potential of MDP hydrogels in a diabetic wound healing model. When applied to full-thickness dermal wounds in genetically diabetic mice, the MDP hydrogel resulted in significantly accelerated wound healing compared to a clinically used hydrogel, as well as a control buffer. Treatment with the MDP hydrogel resulted in wound closure in 14 days, formation of thick granulation tissue including dense vascularization, innervation, and hair follicle regeneration. This suggests the MDP hydrogel could be an attractive choice for treatment of wounds in diabetic patients.

STINGel: Controlled release of a cyclic dinucleotide for enhanced cancer immunotherapy.

Recent advancements in the field of immunotherapy have yielded encouraging results for the treatment of advanced cancers. Cyclic dinucleotides (CDNs) are a powerful new class of immunotherapy drugs known as STING (Stimulator of Interferon Genes) agonists, currently in clinical trials. However, previous studies of CDNs in murine cancer models have required multiple injections, and improve survival only in relatively nonaggressive tumor models. Therefore, we sought to improve the efficacy of CDN immunotherapy by developing a novel biomaterial we call "STINGel." STINGel is an injectable peptide hydrogel that localizes and provides controlled release of CDN delivery, showing an 8-fold slower release rate compared to a standard collagen hydrogel. The carrier hydrogel is a positively charged, MultiDomain Peptide (MDP) which self-assembles to form a nanofibrous matrix and is easily delivered by syringe. The highly localized delivery of CDN from this nanostructured biomaterial affects the local histological response in a subcutaneous model, and dramatically improves overall survival in a challenging murine model of head and neck cancer compared to CDN alone or CDN delivered from a collagen hydrogel. This study demonstrates the feasibility of biomaterial-based immunotherapy platforms like STINGel as strategies for increasing the efficacy of CDN immunotherapies.

Sequence-specific, nanomolar peptide binding via cucurbit[8]uril-induced folding and inclusion of neighboring side chains.

This paper describes the molecular recognition of the tripeptide Tyr-Leu-Ala by the synthetic receptor cucurbit[8]uril (Q8) in aqueous buffer with nanomolar affinity and exceptional specificity. This combination of characteristics, which also applies to antibodies, is desirable for applications in biochemistry and biotechnology but has eluded supramolecular chemists for decades. Building on prior knowledge that Q8 binds to peptides with N-terminal aromatic residues, a library screen of 105 peptides was designed to test the effects of residues adjacent to N-terminal Trp, Phe, or Tyr. The screen used tetramethylbenzobis(imidazolium) (MBBI) as a fluorescent indicator and resulted in the unexpected discovery that MBBI can serve not only as a turn-off sensor via the simultaneous inclusion of a Trp residue but also as a turn-on sensor via the competitive displacement of MBBI upon binding of Phe- or Tyr-terminated peptides. The unusual fluorescence response of the Tyr series prompted further investigation by (1)H NMR spectroscopy, electrospray ionization mass spectrometry, and isothermal titration calorimetry. From these studies, a novel binding motif was discovered in which only 1 equiv of peptide binds to Q8, and the side chains of both the N-terminal Tyr residue and its immediate neighbor bind within the Q8 cavity. For the peptide Tyr-Leu-Ala, the equilibrium dissociation constant value is 7.2 nM, whereas that of its sequence isomer Tyr-Ala-Leu is 34 µM. The high stability, recyclability, and low cost of Q8 combined with the straightforward incorporation of Tyr-Leu-Ala into recombinant proteins should make this system attractive for the development of biological applications.