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

Abstract

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.

Introduction

Essential to modern medicine is the effective delivery of drug therapies. Injectable biomaterials have been established as powerful methods of therapy administration due to their ability to control the release profile of loaded agents and influence the local biological reponse [[1], [2], [3]]. Such biomaterial-based delivery systems have been used for a host of useful applications, including tissue engineering, growth factor release, DNA delivery, and vaccine incorporation [[4], [5], [6], [7]]. Recent interest has focused on biomaterials as platforms for cancer immunotherapy, which can provide extended and localized drug release, and also allow for the intelligent modulation of immune cells in situ.

Cancer immunotherapy has arisen as an exciting treatment modality for several advanced-stage cancers, with the potential to generate specific and durable anti-tumor responses through the use of the host's natural immune reponse [[8], [9], [10], [11]]. Significant progress has been made in this field, generating encouraging results in the treatment of cancers such as metastatic melanoma, for which the 5-year overall survival rate has increased from less than 10%, to almost 40% [[12], [13], [14]]. However, in the case of other types of cancer such as head and neck squamous cell carcinoma (HNSCC) survival rates have failed to improve at a comparable rate, remaining relatively stagnant for the past few decades and emphasizing the need for new treatment options [15].

HNSCC is the sixth most common cancer worldwide, with more than 60,000 men and women in the U.S. expected to develop HNSCC this year [16,17]. Etiologic risk factors include exposure to carcinogens such as tobacco and alcohol, and viral infections such as high-risk types of human papilloma viruses (HPVs) [[18], [19], [20]]. Given the well-known co-morbidities and recurrence rates associated with conventional treatments such as surgery, radiation therapy, and chemotherapy, there is a real need for innovative approaches to treat HNSCC. While some HNSCC patients undergoing immune checkpoint inhibitor antibody therapy experience durable complete remissions, the majority (80–85%) of patients derive no benefit [21]. Improving the rate of success in HNSCC immunotherapy is therefore a highly desirable goal.

Recently, a novel class of immunotherapeutics based on synthetic cyclic dinucleotides (CDNs) have been found to induce strong anti-tumor responses in preclinical models through the Stimulator of Interferon Genes (STING) pathway [[22], [23], [24]]. The STING pathway has emerged as a key mechanism linking the detection of cytosolic tumor DNA to downstream activation of innate immune cells [25,26]. The rationally-designed synthetic Cyclic Dinucleotide dithio-(R_P,R_P)-[cyclic[A(2′,5′)pA(3′,5′)p]] (abbreviated as ML RR-S2 CDA or just CDN, see Fig. 1A) is a promising candidate molecule in clinical trials (see NCT02675439) that has shown efficacy in murine cancer models, promoting the specific rejection of several types of tumors [[22], [27]]. However, to date CDN monotherapy has shown poor efficacy in preclinical models of HNSCC, requiring multiple injections and concomitant administration of immune checkpoint antibodies [28]. Current clinical trials are evaluating intratumoral injections of CDN as monotherapy, a strategy that may prove to be insufficient [27]. Thus, novel approaches to improve the efficacy of CDN in challenging, treatment-refractory tumor models are warranted.

In response to this challenge, we have developed a novel peptide hydrogel-based platform for intratumoral CDN delivery, which we call “STINGel.” This localizable drug delivery vehicle, utilizing the power of immunotherapy, is based on prior work in our laboratory studying the utility of multidomain peptides (MDPs) as unique supramolecular biomaterials. These self-assembling peptides mimic the extracellular matrix of cells through the formation of a nanofibrous network, and can act as biofunctional delivery platforms that allow for an immense diversity of functionality to be introduced [29,30]. Rationally engineered MDPs have been shown to form anti-parallel β-sheets of peptide nanofibers in solution, which when electrostatically crosslinked with multivalent ions form extended nanofiber networks to create robust hydrogels (see Fig. 1B–D) [30].

MDPs possess a number of attractive characteristics as biomaterials. 1) The peptide hydrogels are thixotropic, allowing them to be easily delivered by syringe and yet remain localized for applications such as intratumoral injections. 2) The design of the peptide sequence can allow for the incorporation of charged small molecule drugs by nanofiber crosslinking to achieve extended release, or can allow for the encapsulation and gradual release of small hydrophobic drugs such as daunorubicin, etodolac, levofloxacin, and others [29,31,32]. 3) The hydrogels undergo complete cellular infiltration within three days post injection in vivo and are not fibrously encapsulated, maximizing matrix-tissue interaction.

In this study, we sought to use the MDP hydrogel with sequence K₂(SL)₆K₂ to deliver a promising STING agonist CDN, by taking advantage of favorable electrostatic interactions between the CDN's negative thiophosphate linkages and the positive lysine residues at the peptide termini (Fig. 1). We hypothesized that a combination of controlled release and favorable local environment would result in improved tumor treatment efficacy in vivo.