mRNA vaccine in cancer therapy: Current advance and future outlook

3.3.1 Based on therapeutic or prophylactic property

The therapeutic vaccines are designed to induce cell-mediated immunity to eradicate cancer cells, however, the aim of the prophylactics is to product the antibodies.⁹³ Therapeutic cancer vaccines paid more attention to autoimmune complications, as the autoimmune complication is still an unsolved problem in preventive therapies. Although HPV and HBV vaccines have been approved by FDA and made a good score in cancer prevention, clinical trials of prophylactic mRNA cancer vaccine have not yet been reported. The therapeutic and prophylactic vaccines are not quite distinct from each other, sometimes prophylactic vaccines can also be used as therapeutic vaccines because they are tested effective in the reduction of the risk of clinical relapse.⁹⁴

3.3.2 Based on types of mRNA

Based on the RNA structure, there are mainly three types of mRNA cancer vaccines: non-replicating mRNA (nrRNA) vaccine, self-amplifying mRNA (SAM) vaccine and trans-amplifying mRNA vaccine.

The nrRNA vaccine is a synthetic analogue of mature mRNA, whose constructs contain conventional mRNA vaccine sequences, usually including the universal 5ʹ Cap, 5ʹ untranslated regions (UTRs), an open reading frame (ORF), 3ʹUTRs and a 3ʹpoly(A) tail. The simple structure and relatively small size are the main advantages, but correspondingly, this also limits the activity and stability of nrRNA vaccine constructed in vivo, which is the major disadvantage. To enhance the durability of antigen expression, adjuvants can be added to optimise the structure of RNA molecules. TriMix is a kind of new potent adjuvant strategy with high safety, which demonstrated a superior T cell stimulatory capacity and enhancement of DCs’ maturation.^{95, 96} TriMix is developed by Vrije Universiteit Brussel, consisting of SAMs that encode three immune activator proteins—CD70, CD40 ligand (CD40L) and constitutively active TLR4.⁹⁷ The application of TriMix naked mRNA in AIDS patients has achieved the desired results and aroused popular concern.⁹⁸ For cancer therapy, De Keersmaecker B's clinical studies⁹⁹ indicated great anti-tumour responses in patients with terminal stage of melanoma by using TriMix electroporated together with a DC-based mRNA vaccination.

The SAM vaccines are produced through gene engineering of positive-stranded RNA viruses, including alphaviruses, picornaviruses, flaviviruses and so on, whose gene encoding structural proteins are replaced by antigen sequence.¹⁰⁰ Compared with the nrRNA, there is an extra replication component in the construction of SAM vaccine, directing the amplification of intracellular mRNA after delivery to targeted cells.¹⁰¹ As a consequence, SAM vaccines have a higher level of antigen expression and long-lasting efficacy with lower dosages, which is their obvious advantage.

Trans-amplifying mRNA vaccines are a novel type which was designed by Beissert et al.¹⁰¹ It has a replicase that can amplify the RNAs ‘in trans’, which means two genes act simultaneously on different RNAs. This special structure permits a shorter length of RNA, which could reduce the difficulty of scaled-up production and manufacturability, however, consequently adding the complexity of delivery and manufacture of two RNA drugs. Although trans-amplifying mRNA vaccines have not been reported in clinical cancer therapy, this approach has a promising future since it could be further improved by implementing new strategies benefiting from its special structure.

3.3.3 Based on route of administration

Although systematic evaluation of various administration routes in animal models or patients remains to be studied, different administration routes influence efficacy and immune-stimulation area of mRNA cancer vaccines. At present, administration strategies of mRNA cancer vaccines include subcutaneous injection,¹⁰² intradermal injection,¹⁰³ intranodular injection,¹⁰⁴ intramuscular injection,¹⁰⁵ intravenous injection,¹⁰⁶ intratumoural injection,¹⁰⁷ intrathecal injection¹⁰⁸ and so on (Figure 4), which are primary but economical methods of stimulating immune responses. Local injection is considered a highly immunocompetent method, which augments local vaccine response and triggers a distal immune reaction by lymphatic transport.¹⁰³ Intranodal or near-nodal (into soft tissue) immunizations were usually instructed by surgical exposition of a mouse lymph node. Although intranodal injection has a complicated operation, potent T cell immunity was observed compared with other administration routes (i.d./s.c./n.n.) by Kreiter et al.¹⁰⁴ Tracheal administration, targeting the lung vasculature, is usually used in pulmonary lesions, with mRNA administered as aerosol. The intrathecal injection is applied in brain lesions to help antigen presentation in cells of the central nervous system which are hard to reach. For systemic delivery, mRNA cancer vaccines are commonly administered as nanosized drug formulations and delivered intravenously, mainly targeting liver due to its abundant fenestrated capillaries.

3.4.1 Modification of mRNA structural elements

Given that the basic structure of mRNA plays a pivotal role in the rate of translation and half-life of the transcript, structural improvement is of interest to mRNA vaccine design¹²⁰ (Figure 5A). One crucial step for translation is the binding of the 5′ cap to eukaryotic initiation factor 4E (eif4e) which is the rate-limiting step of mRNA translation. mRNA is capped enzymatically by recombinant vaccinia virus.¹²¹ Decapping enzymes render the mRNA molecule inactive. Synthetic cap analogues are also available, but these are not very effective. Reverse capping is a technique to increase the translational efficacy of mRNA and half-life.^{122, 123} A recent study used the integration of endogenous UTRs with further de novo design to rationally engineer the UTRs of mRNA to increase protein production. Through bioinformatics analysis of endogenous gene expression and de novo design of UTRs, the most effective combination of 5′ and 3′ UTR are detected as NCA-7d as the 5′ UTR and S27a plus a functional motif R3U as the 3′ UTR (termed NASAR UTR). The injection of TT3-formulated receptor-binding domain (RBD)-encoding NASAR mRNA presented effective vaccination in mice, and intramuscular injection was five-fold more potent than subcutaneous injection in inducing antigen-specific antibodies.

In addition, strong vaccine antigen expression and immune response can be witnessed when using a novel RNA vaccine approach which is based on a trans-amplifying RNA split-vector system derived from an alphaviral self-amplifying RNA. Then the replicase from alphaviral self-amplifying RNA can be deleted to form a transreplicon, and transreplicon has the dose-free properties of SAM, as a very low dose (50 ng) can effectively induce protective immune responses in mice, even when delivered as unformulated mRNA.^{118, 124} Since the poly (A) tail is an essential determinant for efficient translation and the lifespan of mRNA molecules, it is necessary to ensure the optimal length of the poly (A) tail,^{125, 126} and the incorporation of poly (A) tails at about 100 nt is ideal for mRNA therapeutics production.¹²⁷ Convenient and stable methods of polyadenylation tend to be critical for mRNA therapeutic application. A study described a simple approach that applies type IIS restriction enzymes to generate and maintain poly (A)-encoding DNA sequences required for IVT of mRNA. The simple approach entails repeated asymmetric cleavage with type IIS restriction enzymes, then ligation and propagation to extend the homopolymeric sequence up to approximately 100 bp in circular plasmids, which can serve as a template for mRNA transcription. Moreover, the poly (A) tail of the in vitro transcribed transcript functions in vivo as well¹²⁸ (Figure 5B).

3.4.2 Optimisation of mRNA manufacturing platform

A series of manufacturing processes should be implemented to produce therapeutic quality mRNA. Although production of the mRNA with specific quality attributes is not particularly challenging at present, a well-established manufacturing platform is still lacking. The steps of mRNA production can be classified into upstream processing, including the enzymatic generation of mRNA, and downstream processing, involving mRNA product purification.¹²⁹ In upstream processing, IVT enzymatic reaction used to generate mRNA is less time-consuming than the conventional processes,^{130, 131} and the capping method performed during the IVT reaction shows high efficiency¹³² (Figure 5A). However, cap analogues affect the cost of production, the high price has hindered this method from being popularized, particularly during large-scale manufacturing.¹³³ Alternatively, a co-transcriptional copping strategy termed CleanCap^®, which does not compete with guanosine triphosphate and can add a natural 5′cap 1 construct to a specific transcription start sequence during IVT reaction, thus simplifying and reducing the cost of mRNA production.¹³⁴ In downstream processing, removing the impurities plays a crucial role in mRNA performance. The conventional lab-scale purification approaches comprise DNase digestion and lithium chloride (LiCl) precipitation, which cannot remove abnormal mRNA species like dsRNA and truncated RNA fragments.^{118, 135}

The chromatography purification process is popular in pharmaceutical manufacturing. For example, size exclusion chromatography (SEC) separates molecules based on their size, but similar size impurities, such as dsRNA may be missed.¹³⁶ The ion-pair reverse-phase chromatography (IPC) method can effectively remove dsRNA impurities without interfering with the process's yield. However, IPC is complicated and expensive to scale, and the use of toxic reagents prevented its generalization.^{117, 137, 138} Compared to IPC, ion-exchange chromatography (IEC) exhibits higher binding abilities, and its scalable and cost-effective properties make it more suitable for use in large-scale manufacturing. Nevertheless, IEC requires a more sophisticated process.^{139, 140}

A new cellulose-based chromatography process, which is useful at laboratory and industrial scales, is based on the selective binding of dsRNA to cellulose in the presence of ethanol. This research claimed that more than 90% of the dsRNA contaminants can be removed from IVT mRNA samples, despite the length, coding sequence or nucleoside composition.¹⁴¹ The combination of mRNA precipitation and tangential flow filtration (TFF) technique serves as a large-scale adaptation of general laboratory-scale mRNA purification method. The author declared that this method can effectively remove reactants, enzymes and byproducts including prematurely aborted RNA sequences while maintaining the integrity of mRNA. Furthermore, using merely aqueous buffers as solvents rather than any caustic or flammable solvents, this method is able to be successfully carried out, indicating its effectiveness, reliability and safety in purifying mRNA¹⁴² (Figure 5B).

3.4.3 Development of mRNA delivery systems

mRNA needs to cross the cell membrane to enter the cytosol to express specific antigens to maintain function. This is challenging due to the negative charge of both the mRNA molecules and cell membrane, relatively large size of mRNA molecules and degradability by ribonucleases existing in skin and blood.¹⁴³ To overcome this, a number of mRNA delivery methods and mRNA delivery carriers have been explored and used currently, including naked mRNA delivery strategies and conjugation with delivery vehicles, such as lipid-based materials, polymers or peptides.^{144, 145}

The traditional and self-amplifying forms of naked mRNA, injection including intramuscular/subcutaneous/intradermal/intravenous/intranasal/intramodular/intratumoural injection, can provoke the immune-therapy response, which effectively stimulates antigen presentation and initiates immune responses¹⁴⁶ (Figure 4). Moreover, a study has demonstrated that subcutaneous injection of naked mRNA in mice triggered immune responses more than mRNA nanoparticle carriers, while mRNA nanoparticle carriers perform better when administrated intranasally and intravenously.¹⁴⁷ Intratumoural injection of tumour-associated antigen mRNA is believed to be a promising vaccination method because of the induction of an appropriate immune response¹⁴⁸ (Figure 4). Besides, common physical approaches like electroporation, gene gun and microneedles partially assist in improving mRNA antigen presentation.¹⁴⁹ However, neither the injection of a naked mRNA delivery system nor physical ways to deliver mRNA can be applied in human patients because they are primitive and dangerous and may impact cell activities, or even cause abnormal cell death.¹⁵⁰

Liposome complexes or liposome nanoparticles (LNPs) are one of the most promising mRNA delivery tools because they can transport hydrophobic or hydrophilic molecules (e.g., small molecules, proteins and nucleic acids). Cationic liposomes which encapsulate mRNA were the first liposome delivery materials, it prevents mRNA from being degraded by RNase.¹⁵¹ pH-responsive cationic lipids are developed as mRNA delivery vehicles to improve delivery efficacy,¹⁵² since other negatively charged molecules also interact with positively charged cationic lipids which can be captured by immune cells as well,¹⁴⁴ LNPs, originally explored for siRNA delivery, are currently the most advanced delivery system for mRNA vaccines.¹⁵³ The basic structure of LNPs includes an aqueous core encompassed by a lipid bilayer shell of cationic lipids, auxiliary lipids, cholesterol and polyethylene glycol, which stabilizes the particles.¹⁵⁴ LNP-mediated delivery of mRNA vaccines can induce durable, protective immune responses against multiple infectious pathogens, such as Zika¹⁵⁵ and influenza,¹⁵⁶ and encouraging results have been found in combatting cancer as well¹⁵⁷ (Figure 6).

There are several polymer-based vectors, such as poly(l-lysine) (PLL), poly(amido-amine) (PAA), poly(beta amino-esters) (PBAEs) and poly(ethylenimine) (PEI), whereas, only PEI has been widely used for mRNA vaccine delivery.^{158, 159} Although PEI offers high gene transfection efficiency, its severe cytotoxicity limits its application, so it is often modified by fatty chains.^{160, 161} A novel lipid-containing polymer called charge-altering releasable transporters (CARTs) has been explored to effectively deliver mRNA molecules. mRNA molecules and the synthetic Toll-like receptor-9 agonist CpG can be encapsulated into a nanoparticle complex by CARTs, then the antigen-coding mRNA can be safely delivered to antigen-presenting cells (APCs). After delivery, mRNA is validly translated, processed and presented by MHCs. Also, the codelivery of mRNA and TLR by CARTs simultaneously transfect and activate target cells to motivate an immune response that can clear established tumours in mice.¹⁶² A study has also found that the mixed-lipid CARTs are more effective in transfecting lymphocytes, CD4 T cells and CD8 T cells than single-lipid CART.¹⁶³ Furthermore, polymers are capable of building scaffolding for mRNA vaccination as well. Studies have proved that scaffold-based mRNA delivery stimulates antigen-specific antibody in mice^{164, 165} (Figure 7).

Although less explored, a peptide-based delivery method can also be used. The tight combination of mRNA with protamine provides strong protection of mRNA from being degraded by RNases, as well as induces intense immune responses of various immune cells like DCs, monocytes and B cells.^166-168 The clinical trials of the protamine-formulated mRNA delivery system have proved its great therapeutic effects in a variety of diseases, such as rabies and non-small cell lung cancer^{169, 170} (Figure 6). Cell-penetrating peptides (CPPs) served as a new delivery approach that promotes the immune reaction of T cells in vivo,¹⁷¹ regulates innate immune response and strengthens protein expression in DCs and human cancer cells by facilitating mRNA release from the endosomes and thereby allow expression of mRNA inside the DCs cytosol.¹⁷² Anionic peptides can increase cell uptake without causing cytotoxicity in DCs through activating both endosome and cytosolic pattern recognition receptors (PRRs) and inducing markers of adaptive responses in primary human DCs in vitro, with prevalent Th1 signature.¹⁷³ Another study has developed a ferritin nanoparticle vaccine to deliver PreS1 to specific bone marrow cells, which induces a durable anti-PreS1 response and eradicates HBV in mice.¹⁷⁴

Virus-like particles (VLPs) use viruses as the carrier to express or present antigens, including poxvirus, adenovirus and herpesvirus, however, merely replication-defective viruses or attenuated viruses are adopted due to safety reasons.^{175, 176} Replication-defective viruses perform a virus-infecting manner by encapsulating antigen-encoding saRNA for delivering into the cytosol, and attenuated viruses sustain the ability of self-replication.¹⁷⁷ Compared with the abovementioned delivery systems, VLPs have been successfully applied to promote cancer immunotherapy benefiting from safety and a simple preparation approach. VLPs can be delivered through intradermal inoculation which precludes vaccine particles from leaking into non-lymphatic organs such as the liver. Also, VLPs reduce the burden of combination therapy with a checkpoint antibody.^178-180 A study designed an mRNA virus-mimicking vaccine platform using a phospholipid bilayer encapsulated with a protein–nucleotide core consisting of antigen-encoding mRNA molecules, unmethylated CpG oligonucleotides and positively charged proteins, which offers a potent platform for therapeutic mRNA vaccines confirmed both in vivo and in vitro¹⁸¹ (Figure 6).

Other delivery systems include extracellular particles and microneedles. Extracellular particles can be used as an efficient delivery platform based on their physiochemical characteristics, high bioavailability and low non-targeted cytotoxicity. Exosomes are most used in cancer vaccine development, which exerts dendritic cell-released major histocompatibility complex (MHC) class I/peptide complexes for efficient CD8⁺ T cell priming to suppress tumour growth.^{182, 183} Microneedles, both solid microneedle patches and hollow microneedles for intradermal injection, not only simplify vaccine distribution and improve patient compliance but stimulates the immune responses of the skin. Moreover, microneedles coated with VLPs can elicit stronger immune responses and enable dose sparing compared to intramuscular injection in mice.^{184, 185}

4.2.1 Biosafety of physiological disposition

The innate immunity is supposed to be properly activated in order to initiate the adaptive immune response, at the same time, averting the toxic overactivations, which inhibit antigen protein expression as well as immune response. Innate immune response is usually activated by host immune system through pathogen-associated molecular patterns (PAMPs) from PRRs, the detecting exogenous motifs.^{190, 47} However, innate immune sensing of RNAs may dampen the immune response, because of the association with inhibition of antigen expression. Specifically, phage RNA polymerases produce dsRNA that is not desired, which may activate innate immunity through PKR, causing the phosphorylation of eIF-2, which can block mRNA translation.¹⁹⁰ In addition, the dsRNA activates RNase L upon binding to OAS,¹⁹¹ leading to degradation of the exogenous RNAs. Moreover, dsRNA bound with MDA-5 and TLR-3 can activate type I IFN. Therefore, several other genes are elicited and inhibit the translation of mRNA.¹⁹² Nevertheless, when mRNA structure is improperly designed, PRRs may also be activated, thus abolishing antigen expression (Figure 7).

4.2.2 Biosafety in various crowds

To accelerate the vaccine development, the population enrolled in trials may be limited than expected. It becomes a concern when the vaccine is designed for people throughout the world, for unknown side effects may emerge in the larger population. Certain groups have specific conditions. In older individuals, a different vaccine formulation or a booster dose is supposed to improve immune responses.¹⁹³ In children, as they usually show increased reactogenicity compared to adults, low-dose vaccines might be required, particularly for mRNA-based vaccines. In pregnant women, the data on the mRNA vaccine is limited, which makes it difficult to expect whether an equivalent immunological response occurs.¹⁹⁴ Furthermore, some evidence suggests that babies suffering enduring adverse consequences may be related to variant CD4⁺ T cell responses in their mothers.¹⁹⁵ In patients on immunosuppressive therapy, immune response to vaccinations is attenuated, which deserves special consideration.¹⁹⁶

4.3.1 Limitations in type

Although some kinds of bacteria and parasites may cause cancer, researches on mRNA vaccines against bacteria and parasites are rare. It can hardly find antigens in the reproduction cycle of bacteria and parasite which can be made into vaccines. Moreover, some bacteria and parasites have the ability to escape from immunity, at the same time, effective but cheap anti-bacterial and anti-parasitic drugs have existed. Therefore, vaccines are intended to fail when assessed by cost/benefit ratio.^{197, 198}

4.3.2 Limitations in effectiveness

The vaccination program's effectiveness depends on convincing efficacy and safety data accompanied by popular public acceptance and inoculation.¹⁹⁹ Vaccine immunogenicity and efficacy depend on the points including packaging, storage, preparation and administration. The risks of improperly performing supply chains are detrimental to the safety and effectiveness of the vaccines, with latent consequences of adverse events.²⁰⁰ Moreover, vaccine hesitancy remains a noteworthy challenge, which has been described as a ‘lack of confidence in vaccination and/or complacency about vaccination’, which may result in deferment or failure to vaccinate.²⁰¹ There are also worries that the political pressure may hasten the development and approval processes, resulting in an ineffective vaccine being released to the public.²⁰²

Taken together, the safety of mRNA cancer vaccines is associated with their ability to encode multiple antigens simultaneously as well as being non-integrating, highly degradable and having no insertional mutagenic potential. In order to develop a safe and effective mRNA vaccine, pre-clinical trials must be done with caution to avoid severe adverse events. Even after approval, long-term safety and efficacy data are required. Moreover, cooperation between international organizations provides solid funding insurance for mRNA vaccines.