Messenger RNA Vaccines: Beckoning of a New Era in Cancer Immunotherapy
Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm of the cell. The resulting antigens are presented to the immune system cells to stimulate an immune response. Dendritic cells (DCs) can be utilized as a carrier by delivering tumor-associated antigen mRNAs or total tumor RNA to their cytoplasm; then, the mRNA-loaded DCs can be delivered to the host to elicit a specific immune response. Recently, 2 mRNA vaccines were approved for the first time for human use—to prevent COVID-19 infection—bringing excitement for the future possibilities of this approach for cancer immunotherapy as well as for preventing other infectious diseases.
Introduction
Messenger RNA (mRNA) vaccines have generated significant excitement not only for their potential to complement or even replace traditional vaccines against infectious diseases, but also for their potential use in noninfectious diseases, including cancer. Nucleic acid therapeutics have emerged as promising alternatives to conventional vaccine approaches. Exploitation of mRNA, the intermediate step between the protein-encoding DNA and the protein manufactured by ribosomes, has proven to be a favorable alternative to conventional vaccines. In this approach, a linear DNA template and a T7, a T3, or an Sp6 phage RNA polymerase are used in vitro to design the mRNA containing 5' and 3' untranslated regions (UTRs). The resulting product contains an open reading frame that encodes the protein of interest, flanking UTRs, a 5' cap, and a poly(A) tail; this tail resembles the fully processed mature mRNA molecule that naturally occurs in the cytoplasm of eukaryotic cells (FIGURE 1).1 Successful cytosolic delivery of mRNA results in synthesis of the encoded protein, which undergoes posttranslational modifications and results in a properly folded, fully functional product that is delivered to the correct cellular compartments for proper presentation or function.
In vitro transcribed mRNA is ultimately degraded by normal cellular physiological processes.2-4 Because the target for mRNA vaccines is the cytoplasm of the cell, they are easier to deliver, and cytosolic mRNA has no interaction with the genome.5 The small piece of genetic information mRNA vaccine carries is expressed only transiently, until it is degraded. mRNA can encode multiple proteins that possess very different chemical and physical properties without major changes in its physiochemical properties. mRNAs can theoretically be used to produce any protein, and doing so is a much more simple and cost-effective way of manufacturing the proteins.
While the concept of possibly using mRNA vaccines against infectious diseases, genetic disorders, and cancer has been around for decades, until now no vaccines using this technology have received FDA approval for human use. Recently, 2 phase 3 trials led to the first FDA approvals of mRNA-based vaccines, and this marks the beginning of a new era not only for the infectious disease field but also for oncology.
Background
In an early proof-of-concept study, RNA and DNA expression vectors that contained genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle; expression of all the proteins was later readily detected. The extent of expression from both the RNA and DNA constructs was comparable with that obtained from fibroblasts transfected in vitro under optimal conditions.
Despite its revolutionary promise, though, mRNA work has been hampered by several challenges. These have included the instability of mRNA molecules, leading them to degrade quickly inside the cell; the inability of mRNAs to produce high levels of proteins; and mRNA`s own immunogenicity, which triggers an immune response independent of the desired immune response, that it attempts to produce to the protein it encodes.
DNA and RNA can stimulate the mammalian innate immune system through activation of Toll-like receptors (TLRs). Observation of lack of immunostimulatory effect of DNAs containing methylated CpG motifs and selected nucleosides in naturally occurring methylated or otherwise modified RNAs led to a discovery that was reported in a seminal paper by Kariko et al. They showed that RNA creates signals through human TLR3, TLR7, and TLR8; however, the incorporation of modified nucleosides m5C, m6A, m5U, or s2U, or of pseudouridine, ablates this activity. Dendritic cells (DCs) exposed to such modified RNA express significantly fewer cytokines and activation markers than those treated with unmodified RNA. This discovery—that modified synthetic nucleosides could both increase protein production from mRNA and suppress the immune system`s recognition and degradation of mRNA—helped mRNA vaccine technology evolve further.
Under physiological conditions, RNA molecules are highly unstable and very sensitive to catalytic hydrolysis by ubiquitous ribonucleases.8 One of the main challenges for the clinical use of mRNA vaccines has been the difficulty in intracellularly delivering the mRNA. Naked mRNA, delivered by itself, is rapidly degraded after injection. Several mRNA delivery approaches have been tested, using substances including modified RNAs, RNA conjugates, viral vectors, microparticles, and, most successfully, lipid nanoparticles.9-11 Lipid nanoparticles are multicomponent lipid systems composed of an ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG)-lipid. The ionizable lipid facilitates the mRNA’s formation of a core structure, while helper lipids (phospholipid and cholesterol) envelop the lipid-mRNA complex, and the PEG-lipid protects the nanoparticle shell. Lipid nanoparticles have been the most promising innovation in this field.
For efficacy, adequate in vivo delivery of exogenous mRNA is crucial (FIGURE 2). Uptake of mRNA may depend on the physicochemical properties of the mRNA complexes and the cell type. Two main strategies have been utilized for the in vivo delivery of mRNAs. One such approach involves loading mRNA into DCs ex vivo, followed by reinfusion of the transfected cells. This allows precise control of the cellular target and transfection efficiency, but it is expensive and labor intensive. The DC approach elicits a predominantly cell-mediated immune response; it has been explored primarily in cancer treatment.
The second approach is direct parenteral injection of mRNA. This is relatively quick and cost effective, but it may not be as precise and efficient in specific cell delivery. The systemic delivery of vaccine antigens can be facilitated by using RNA-lipoplexes. The lipoplexes protect RNA from extracellular ribonucleases; they mediate the efficient uptake and expression of the encoded antigen.13,14 Recently, highly efficient and nontoxic RNA carriers have been developed that allow prolonged antigen expression in vivo.15,16 Some vaccine formulations contain novel adjuvants, while others elicit potent responses in the absence of known adjuvants.
Lipid Nanoparticle Approach for mRNA Vaccines
mRNA has a number of benefits over traditional treatments, but its use as a therapeutic molecule has been challenging due to its chemical instability and susceptibility to hydrolysis, catalyzed by ubiquitous RNA nucleases. However, recent advances in nonviral delivery systems and the development of novel effective transfecting nanomaterials have brought some effective resolutions to these challenges.
The encapsulation of mRNA into a carrier is necessary to protect it from extracellular degradation and to stimulate its cellular uptake and endosomal escape.17,18 Lipid-based delivery systems of lipoplexes have some drawbacks, including high instability, relatively low transfection efficiency, and poor customizable composition, so the earlier use of these systems15,19 has been replaced by lipid nanoparticles. Lipid nanoparticles have demonstrated superior stability and structural plasticity as well as enhanced gene delivery.18,20 A lipid nanoparticle is composed of pH-responsive lipids; neutral helper lipids, such as zwitterionic lipid and/or sterol lipid (ie, cholesterol), to stabilize the lipid bilayer of the lipid nanoparticle; and a PEG-lipid to improve the colloidal stability in biological environments.18,21 The application of lipid-based nanotechnology delivery systems is now enabling the integration of mRNA-based technology with many preexisting anticancer immunotherapeutic approaches, including therapeutic vaccines, monoclonal antibodies, and chimeric antigen receptor T-cell therapy. A deeper understanding of key parameters, such as hydrophobicity and fusogenicity determining the transfection efficiency of the formulation and discoveries of how they can be modulated by varying the lipidic composition or through the introduction of novel lipids, will further enhance the development of better formulations. More research is needed to understand the reasons behind the low mRNA transfection efficiency.
mRNA Vaccines Against Cancer
While mRNA technology is a relatively new and novel approach in cancer vaccine development, vaccines in general have previously been conceptually considered in cancer immunotherapy, by utilizing viral or bacterial vaccines. fact, the concept goes as far back as 1891, when Dr. William Coley made the first attempt to stimulate the immune system to improve a patient’s condition: He used intratumoral injections of inactivated Streptococcus pyogenes and Serratia marcescens, a mixture now known as Coley’s toxin.22 The bacillus Calmette-Guérin (BCG), a bacterium that functions similarly to Coley’s toxin, is currently used to treat superficial bladder cancer.23,24
However, the clinical translation of cancer vaccines into efficacious therapies that could be used widely has been challenging for decades. The 2 very first US FDA vaccine approvals in cancer were for prophylactic use: hepatitis B virus vaccine and human papillomavirus vaccine. The first therapeutic cancer vaccine was not available until 2010, with approval of an immune cell–based vaccine, sipuleucel-T, for prostate cancer refractory to hormone therapy.25
Over the past decades, numerous diverse therapeutic cancer vaccination strategies have been under development. Recent human-use approval of the 2 mRNA vaccines for COVID-19 may mark the end of the beginning of our quest for mRNA vaccine immunotherapy of cancer.26,27 The early proof-of-concept studies that provided the evidence for the feasibility of the mRNA vaccine approach were published more than 2 decades ago.28,29 Since then, numerous preclinical and clinical research teams have been exploring the viability of this approach. Most cancer vaccines seek to stimulate cell-mediated responses, such as those from cytotoxic T lymphocytes, that are capable of clearing or reducing tumor burden.30 Cancer vaccines can be designed to target tumor-associated antigens that are preferentially expressed in cancerous cells. Growth-associated factors, or antigens that are unique to malignant cells due to somatic mutations, can be targeted in the design of mRNA cancer vaccines.31 While the simplicity of mRNA vaccines greatly reduces the difficulties generally associated with the production of biological vaccines, the route of administration and delivery format greatly influence outcomes. A variety of mRNA cancer vaccines have been studied that utilize the 2 main delivery strategies: direct injection and the DC approach.
Direct Injection mRNA Approach
Direct injection of mRNA can be accomplished through common delivery routes, such as intradermal, subcutaneous, intranasal, and intramuscular, as well as some unconventional routes, such as intranodal, intrasplenic, intratumoral, and intravenous. Systemic administration of mRNAs has been challenging, due to concerns about aggregation with serum proteins and rapid extracellular degradation, which has led to formulations with carrier molecules. Numerous delivery formulations have been developed to facilitate mRNA uptake, increase protein translation, and protect mRNA from RNAases, as discussed above.
The intradermal route of delivery has been widely used for mRNA cancer vaccines. The presence of a variety of antigen-presenting cells in the skin makes it an ideal site for immunogen delivery.32 In several mouse tumor models of various cancers, including fibrosarcoma, melanoma, and prostate cancer, vaccines delivered intradermally have been shown to produce antitumor responses.32-34
Intranasal administration, a needle-free, noninvasive delivery model, has also been shown to enable rapid antigen uptake by DCs. In mouse tumor models, intranasal delivery of mRNA complexed with lipid nanoparticles has been shown to induce prophylactic and therapeutic antitumor immunity.35
A rather unconventional but efficient mRNA vaccine delivery approach has been the intranodal administration of unprotected mRNA, directly injected into lymphoid tissue. Intranodally injected mRNAs can stimulate potent prophylactic or therapeutic antitumor T-cell responses.36,37 Similar efficacy has been shown by intrasplenic delivery of mRNA melanoma vaccine in mouse tumor models.38 Intranodal injection of naked mRNA encoding tumor-associated antigens in patients with melanoma (NCT01684241) and with hepatocellular carcinoma (EudraCT: 2012-005572-34) is being studied in human clinical trials.
Finally, another unconventional direct delivery method that has been shown to be effective is intratumoral mRNA delivery. Here, intratumoral administration of mRNAs that do not encode tumor-associated antigens seems to stimulate tumor-specific immunity through the intrinsic immunogenic properties of mRNA.39-41
Dendritic Cell mRNA Approach
DCs are the professional antigen-presenting cells of our immune system. They can take up, process, and present antigens to T cells. mRNA is a polyvalent molecule with an attractive safety profile and the ability to induce transient but sufficiently long and high protein expression without the risk of integrating into the host genome. These characteristics make it an attractive vector to deliver tumor antigens to DCs.
The interest in using mRNA as a means of loading tumor antigens onto DCs goes back to the late 1990s, when initial attempts were based on passive pulsing. They relied on the ability of DCs to take up the mRNA, translate it into proteins, then process it into peptides and finally present it to CD8+ T cells.32,42,43 To increase the efficiency of uptake, investigators have studied several strategies, including electroporation, nucleofection, lipofection, and, more recently, sonoporation.44-47
A variety of immune-regulatory proteins in the form of mRNA-encoded adjuvants or co-stimulatory molecules have been shown to increase the potency of DC cancer vaccines. These molecules include CD83, tumor necrosis factor receptor superfamily member 4 (TNFRSF4; also known as OX40), and 4-1BB ligand. Electroporation of DCs with these mRNAs have resulted in a substantial increase in the immune stimulatory activity of DCs.48-51 Further, the use of mRNA-encoded proinflammatory cytokines, such as IL-12, or associated molecules has been shown to enhance and potentiate the efficacy of immune response.52-55 In a phase 2 trial, the combination of a mRNA DC vaccine approach with an immune checkpoint inhibitor has shown durable tumor response.56
Conclusions
Within the past 2 decades, major achievements in the field of mRNA vaccines have created a safe and attractive treatment option for a broad range of diseases, including cancer. Their simplicity, high potency, capacity for rapid development, and potential for low-cost manufacture and safe administration all make mRNA vaccines a very promising alternative to conventional vaccine approaches. Recent improvements in mRNA vaccines to increase protein translation, modulate innate and adaptive immunogenicity, and improve delivery are very encouraging. The combination of mRNA vaccination with adjunctive therapies, such as traditional chemotherapy, radiotherapy, and immune checkpoint inhibitors, may contribute further to the approach’s benefit in oncology. Challenges may include scaling up good manufacturing practice production, establishing regulations, further documenting safety, and increasing efficacy. Clinical studies in various cancer types are moving forward, and promising results with favorable clinical outcomes are eagerly awaited (TABLE). Future research should also focus on interpreting the immune pathways activated by various mRNA vaccine platforms and try to improve current approaches based on these mechanisms. Recent approval of 2 mRNA vaccines for COVID-19 for human use opens the gateway for many future applications of this novel treatment approach.
Financial Disclosure: The authors have no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.
