Immunotherapy is a type of cancer treatment that helps your immune system fight cancer. In the past decade, cancer immunotherapy has flourished, including immunostimulatory small molecules, immune checkpoint inhibitors (ICIs) targeting immune cells, autologous T cells expressing chimeric antigen receptors (CARs), or natural killers (NK) cells and mRNAs expressing tumor antigens or CARs for cancer immunotherapy. Among them, small molecule, ICIs, and mRNA therapies are used as stand-alone treatments for many solid tumors, such as melanoma, non-small cell lung cancer (NSCLC), and urothelial cancer.
However, despite the promise of cancer immunotherapy, these immunotherapies also have significant limitations: poor water solubility, immune-mediated adverse events (IRAEs), and loss of biological activity after long-term administration limit the immunostimulatory efficacy of small molecule therapies. Therefore, the major challenges facing cancer immunotherapy can be attributed to the lack of delivery systems that can bring therapeutic drugs closer to their targets. Lipid-based nanoparticles (NPs), including liposomes, lipid nanoparticles (LNPs), and nanoemulsions (NEs), have been developed as platforms for the delivery of multiple therapeutic agents. Compared with other nanoscale delivery systems, LNPs maintain high solubility in aqueous phase while minimizing systemic toxicity, which neither polymeric NPs nor inorganic NPs can overcome in clinical applications. These advantages making LNPs the most common type of nanomedicine approved by the FDA.
Recent advances in LNP development make it possible to deliver not only small molecules but also mRNAs for effective anticancer immunotherapy through cytotoxic immune cell activation, checkpoint blockade and CAR-T cell therapy.
Components of LNPs
Lipid-based nanoparticles exhibit various types of structures. Most LNPs are nearly spherical with one or more lipid outer layers.
Although liposomes, LNPs, and NEs may exhibit different internal structures, typical lipid-based NPs consist of cationic lipids or ionizable lipids with tertiary or quaternary amines to encapsulate anionic payloads. Auxiliary lipids are also used to stabilize lipid layers and promote membrane fusion. Polyethylene glycol (PEG) lipids or surfactants are added to improve colloidal stability for long-term storage and prevent rapid degradation of the payload as it enters the bloodstream throughout the body. In addition, NEs includes an oil phase, which can be triacylglycerol, diacylglycerol or monoacylglycerol, vegetable oils, mineral oils, free fatty acids, etc.
LNPs used for immune activation of small molecules
There is increasing evidence that the host immune system also plays an important role in the process of chemotherapy, ultimately leading to antitumor responses. LNPs was first used to encapsulate chemotherapy drugs as anticancer agents. The chemotherapeutic drug oxaliplatin led to upregulation of tumor MHCI and reduction of immunosuppressive cells (Treg, MDSC, and TAM) in a mouse model of colorectal cancer. Interestingly, the liposomal formulation of oxaliplatin exhibited better antitumor immunity compared to free oxaliplatin, suggesting that precise delivery of chemotherapeutic drugs to the TME would trigger better antitumor immunity.
Immune system agonists (small molecules, nucleic acids or peptides)
Antigen-presenting cells (APCs) sense tumor-associated antigens or pathogen/damage-associated molecular patterns (PAMPs/DAMPs) via pattern recognition receptors (PRRs). Activated APC triggers proinflammatory cytokines and chemokines to activate the adaptive immune system to kill tumor cells.
Agonists of TLRs, NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) were developed to induce proinflammatory immune responses that favor antitumor activity. Pam3Csk4 is a TLR1/2 agonist using LNP with OVA mRNA. TLR7/8/9 agonists have also been extensively studied, and encapsulation of imidazoquinoline TLR 7/8 agonists, such as imiquimod and resiquimod, loaded into liposomal preparations have been shown to prolong their retention in the circulation. Stimulator of interferon genes (STING) is another PRR located in the endoplasmic reticulum, and activated STING will enhance the synergy of type I interferons with other pro-inflammatory cytokines, thereby enhancing antitumor immunity. Cyclic dinucleotides (CDNs) are potent STING receptor agonists that can be encapsulated into LNPs to enhance systemic antitumor immunity.
RNA interference (RNAi) technology
RNAi technology (siRNA, shRNA, miRNA, ASO, etc.) can induce specific gene regulation and become a new therapeutic area for infectious diseases, neurodegenerative diseases, cancer, and other rare diseases. In addition to directly targeting specific oncogenes, RNAi can enhance antitumor immune responses by downregulating immunosuppressive proteins.
From tumor antigens to mRNA-based therapy
Neoantigens expressed in the mutated tumor microenvironment will allow the development of personalized cancer vaccines with patient-specific neoepitopes. Anti-tumor tumor antigens mainly depend on the delivery of tumor antigen peptides or encoding mRNAs. The use of liposomes to deliver TAAs/TSA long synthetic peptides can greatly protect them from degradation while making APCs more accessible.
The first personalized IVAC mutant group using LNPs is an RNA vaccine encoding multiple re-epitopes, whose safety, immunogenicity, and tolerability have been evaluated in a phase I clinical trial in melanoma patients (NCT02035956). Strong immune responses against vaccine antigens were observed with no adverse drug reactions and were well tolerated. In addition, many other personalized mRNA cancer vaccines encoding different antigens use lipid nanosystems and have reached the clinical stage (NCT03897881, NCT02316457, NCT03313778, NCT03480152, NCT03303398).
Lipid Nanoparticles (LNPs) for cell therapy
Recently, personalized adoptive cell therapy has shown great promise in clinical trials of hematologic neoplasms. Adoptive cell therapy includes TIL therapy, engineered T cell receptor therapy (TCR-T), CAR-T cell therapy and NK cell therapy. Despite the great potential of adoptive cell therapy, concerns about immune side effects and insertional mutagenesis have also been raised due to the use of viral vectors for in vitro cell engineering. In addition, complex manufacturing and high cost also hinder the application of CAR-T in a wider patient population. Therefore, new in vitro transfection techniques are needed to enable safer and more economical adoptive cell therapy.
In preclinical studies, LNPs containing encoding DNA or mRNA showed excellent efficacy in transient transfection. LNP-based encapsulated mRNAs can be formulated by simple and rapid mixing, therefore, the selection of LNPs for cell engineering mainly focuses on the transfection efficiency of their payloads. In addition, LNPs are generally considered to have low cytotoxicity, thus, the processes of gene transfection and T/NK cell activation can be performed simultaneously.
A study reported the development of ionizable LNP-encapsulated CAR for in vitro T cell engineering, demonstrating for the first time that LNP-engineered CAR-T in vitro has similar tumor-killing activity to lentiviral-engineered CAR-T. LNPs/mRNA transfection strategies are also used in NK cell engineering. The development of liposome-engineered super NK cells containing TRAIL was first reported by Chandrasekaran et al. TRAIL-engineered NK cells showed potent tumor-killing activity by inducing apoptosis in tumor-draining lymph nodes in vivo.
Lipid-based NPs represent the most advanced and widely used delivery vehicles for small molecules and nucleic acids. In cancer immunotherapy, lipid-based NPs can not only deliver small molecules and mRNAs in vivo for enormous antitumor activity, but also enable in vitro engineered cell therapy with efficiencies comparable to other non-viral or viral vectors. It is believed that with the emergence of more immunotherapy methods, artificial immune cells and new nanomaterials, their combination will profoundly affect the field of cancer immunotherapy.
1. Application of lipid-based nanoparticles in cancer immunotherapy. Front Immunol.2022; 13: 967505.
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