PLGA-particle vaccine carrying TLR3/RIG-I ligand Riboxxim synergizes with immune checkpoint blockade for effective anti-cancer immunotherapy

Abstract

With emerging supremacy, cancer immunotherapy has evolved as a promising therapeutic modality compared to conventional antitumor therapies. Cancer immunotherapy composed of biodegradable poly(lactic-co-glycolic acid) (PLGA) particles containing antigens and toll-like receptor ligands induces vigorous antitumor immune responses in vivo. Here, we demonstrate the supreme adjuvant effect of the recently developed and pharmaceutically defined double-stranded (ds)RNA adjuvant Riboxxim especially when incorporated into PLGA particles. Encapsulation of Riboxxim together with antigens potently activates murine and human dendritic cells, and elevated tumor-specific CD8⁺ T cell responses are superior to those obtained using classical dsRNA analogues. This PLGA particle vaccine affords primary tumor growth retardation, prevention of metastases, and prolonged survival in preclinical tumor models. Its advantageous therapeutic potency was further enhanced by immune checkpoint blockade that resulted in reinvigoration of cytotoxic T lymphocyte responses and tumor ablation. Thus, combining immune checkpoint blockade with immunotherapy based on Riboxxim-bearing PLGA particles strongly increases its efficacy.

Introduction

Cancer still represents one of the most prevalent and challenging malignancies worldwide as a failure of standard therapies results in tumor relapse and metastasis formation. Hence, there is an urgent need for the development of alternative therapies such as immunotherapy with cancer vaccines¹. Due to the unique ability of dendritic cells (DCs) to prime and activate naive T cells, effective antigen charging and stimulation of DCs are key goals in immunotherapy. We and others have established the use of PLGA-MP (microparticles) and NP (nanoparticles) as efficient antigen delivery systems for targeting DCs in situ². The aliphatic copolymer PLGA is extensively used for optimizing controlled and joint release of immune stimulants and antigens in cancer immunotherapy in preclinical settings³. Due to its biodegradability and proven safety profile, PLGA and its formulations have been licensed by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) for pharmaceutical applications via parenteral and mucosal routes⁴. Especially, PLGA-MP exhibit ideal properties for facilitated uptake by antigen-presenting cells (APCs). Concomitant delivery of antigens and adjuvants to the same APC leads to efficient DC or macrophage activation and simultaneous activation of both, CD4⁺ T helper cells and CD8⁺ cytotoxic T lymphocytes (CTL), via cross-presentation⁵.

Successful immunotherapy against cancer also requires an ideal adjuvant. Multiple immunostimulatory RNA duplexes with agonistic activity for pattern-recognition receptors (PRR) have been reported as candidates for adjuvants in cancer immunotherapy. Double-stranded RNA molecules classically trigger endosomal TLR3 (Toll-like receptor 3). Depending on the structural characteristics and length of the viral RNA mimetic, it may also activate cytosolic RIG-I-like receptors (RLRs), including retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5). TLR3 and RIG-I downstream signaling results in nuclear translocation of the key transcription factors nuclear factor κ/light-chain enhancer of activated B cells (NF-κB) and interferon regulatory factor (IRF)3/7 culminating in secretion pro-inflammatory cytokines and of type I interferons (IFN), respectively. Co-encapsulation of TLR3 ligands with antigen greatly enhanced PLGA particle-mediated cancer vaccine efficacy by inducing reliable maturation of DCs⁶. The dsRNA analog poly(I:C) (polyinosinic:polycytidylic acid) is the most widely used TLR3 agonist but also engages RIG-I and preferably MDA5⁷. Its adjuvant properties result from induction of type I interferons and pro-inflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF), and interleukin (IL)−1β⁸ thus enabling an improved efficacy of cancer immunotherapy in murine tumor models^9,10,11. However, its ill-defined macromolecular structure and heterogeneous composition prevented polyI:C from extensive clinical use due to unpredictable pharmacokinetics and toxicity issues. The TLR3/RIG-I ligand Riboxxim™, in contrast, is characterized by its well-defined chemical structure as double-stranded RNA and nucleotide composition of 100 bp for effective TLR3 triggering. An uncapped triphosphate moiety at the 5′ end of Riboxxim also enables the activation of RIG-I¹². Riboxxim exhibits very good solubility in water, prolonged stability in solution at 4 °C and in serum….