mRNA vaccines as cancer therapies

Abstract

Cancer remains a major global health challenge, with conventional treatments like chemotherapy and radiotherapy often hindered by significant side effects, lack of specificity, and limited efficacy in advanced cases. Among emerging therapeutic strategies, mRNA vaccines have shown remarkable potential due to their adaptability, rapid production, and capability for personalized cancer treatment. This review provides an in-depth analysis of messenger RNA (mRNA) vaccines as a therapeutic approach for cancer immunotherapy, focusing on their molecular biology, classification, mechanisms, and clinical studies. Derived from reported literature and data on clinicaltrials.gov, it examines studies on mRNA vaccines encoding tumor-specific antigens (TSAs), tumor-associated antigens (TAAs), immunomodulators, and chimeric antigen receptors (CARs) across various cancer types. The review highlights the ability of mRNA vaccines to encode TSAs and TAAs, enabling personalized cancer treatments, and classifies these vaccines into non-replicating and self-amplifying types. It further explores their mechanisms of action, including antigen presentation and immune activation, while emphasizing findings from clinical studies that demonstrate the potential of mRNA vaccines in cancer therapy. Despite their promise, challenges remain in enhancing delivery systems, improving immunogenicity, and addressing tumor heterogeneity. Overcoming these obstacles will require further investigation to fully harness the potential of mRNA vaccines in personalized cancer treatment.

Introduction

Cancer continues to be a predominant global health challenge, leading to significant rates of illness and death worldwide, with recent statistics suggesting that accounts for nearly 10 million fatalities each year.^[1] Although traditional cancer treatments including chemotherapy and radiotherapy are pivotal, they are often associated with significant drawbacks such as severe side effects, a lack of specificity, and limited effectiveness in advanced cases.^[2,3] These findings highlight the need for effective treatment strategies and innovative therapeutic approaches. Among emerging approaches, messenger RNA (mRNA) vaccines have demonstrated significant promise in their adaptability, rapid production timelines, and potential for personalized cancer treatment, as evidenced by their success in enabling swift and potent immune responses during the coronavirus disease 2019 (COVID-19) pandemic.^[4,5]

Technically, mRNA vaccines use synthetic mRNAs that prompt cells to produce antigens that trigger a targeted immune response against cancer cells. Clinical trials have demonstrated that mRNA vaccines can safely stimulate antibody production in humans without entering the cell nucleus,^[6] thereby reducing the risk of genomic integration and mutation. To improve efficacy and reduce adverse effects, delivery systems with chemically modified mRNAs have been employed to overcome RNA instability and excessive immune activation.^[7] Through encoding tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs), mRNA vaccines have the advantage of offering personalized treatment strategies that target unique cancer profiles.^[8] Numerous clinical trials are presently underway to evaluate the effectiveness of novel medicines, with preclinical research serving as the basis for clinical evaluation.^[9,10] However, despite these promising attributes, mRNA cancer vaccines still face significant challenges that must be addressed to fully realize their potential. Tumor heterogeneity complicates target identification because of diverse genetic and phenotypic profiles. The tumor microenvironment (TME), which is enriched with immunosuppressive cells and cytokines, further limits vaccine-induced immune activation. Additionally, current vaccine administration routes, such as intramuscular delivery, may not effectively target tumor sites or ensure optimal antigen presentation. Monitoring the efficacy of mRNA vaccines also remains challenging, with limited tools for real-time assessment of therapeutic responses. Finally, practical barriers, including high production costs, storage requirements, and logistics, hinder widespread implementation. Addressing these challenges requires multifaceted strategies, including personalized vaccine design, advanced delivery systems, optimized administration routes, and scalable production methods. The particular drawbacks of mRNA cancer vaccines are listed in Table 1, along with solutions that will be covered in more detail in the sections that follow.

Table 1.

Current limitations of mRNA cancer vaccines in clinical application and potential strategies.

Challenge	Impact	Potential solution
Tumor heterogeneity	Diverse genetic and phenotypic profiles complicate the identification of universal antigens.	Develop personalized neoantigen vaccines using high-throughput sequencing combined with robust algorithm.
Immunosuppressive TME	Immunosuppressive cells and cytokines (e.g., TGF-β, IL-10) limit vaccine-induced immune activation.	Combine mRNA vaccines with immune checkpoint inhibitors (e.g., anti-PD-1/PD-L1) or other immunomodulatory agents.
Vaccine administration routes	Suboptimal delivery methods reduce biodistribution, immune response, and may increase side effects.	Optimize delivery routes (e.g., intratumoral or intravenous injection) and employ advanced monitoring technologies to track biodistribution.
Indications for monitoring vaccine efficacy	Lack of reliable biomarkers limits real-time assessment of immune response and therapeutic outcomes.	Integrate biomarkers such as miRNAs, neutralizing antibodies, and immunosuppressive markers (e.g., TIGIT) into efficacy evaluation.
Practical barriers to clinical application	High production costs and stringent cold chain requirements limit accessibility, especially in resource-limited settings.	Develop thermostable formulations and scalable manufacturing processes to reduce costs and enhance global accessibility.

ICIs: Immune checkpoint inhibitors; IL: Interleukin; mRNA: Messenger RNA; PD-1: Programmed death-1; PD-L1: Programmed death-ligand 1; TGF-β: Transforming growth factor-beta; TIGIT: T-cell immunoreceptor with Ig and ITIM domains; TME: Tumor microenvironment.

This review discusses the mechanisms, development, and future prospects of mRNA vaccines in cancer treatment, highlighting their potential to revolutionize cancer therapy.

Classification and Structure of mRNA Vaccines

Between transcription and translation, the mRNA is a byproduct that carries genetic information that is crucial for directing the synthesis of proteins. mRNA vaccines encode transcripts of one or more immunogens, which are subsequently translated into immunogenic proteins in the cytoplasm of host cells, thereby initiating both innate and adaptive immune responses.^[11] In addition to DNA vaccines, mRNA vaccines are classified as nucleic acid-based immunizations. These vaccinations can be divided into two groups: self-amplifying mRNA vaccines (saRNAs) and non-replicating mRNA vaccines.^[12] A typical non-replicating mRNA consists of a 5′cap, 5′-untranslated regions (UTRs), an open reading frame (ORF) that encodes the vaccine antigens, 3′-UTRs, and a poly(A) tail. Together, these components strengthen mRNA’s stability and lengthen its half-life in the body. Whereas the 3′-UTR and poly(A) tail affect the stability of the mRNA and translation efficiency, the cap structure and 5′-UTR are essential for translation initiation.^[13] The modification of structural elements has enabled conventional mRNA vaccines to be applied in clinical settings. Examples of non-replicating mRNA vaccines include the Pfizer-BioNTech and Moderna COVID-19 vaccines, which have demonstrated significant efficacy in preventing infection.^[14] However, these vaccines require high doses to elicit strong immune responses, which can be a limitation in terms of production and cost.^[11] The classification and structure of key mRNA vaccines are listed in Table 2. This review summarizes key mRNA cancer vaccines and concepts. Notably, the first two entries represent vaccines that have been clinically approved or utilized, whereas the remaining entries are still under investigation as candidates or theoretical approaches.

Table 2.

Classification and the structure of key mRNA vaccines.

Vaccine name	mRNA	Immunogen	Structure
Pfizer-BioNTech COVID-19 vaccine	Non-replicating mRNA	SARS-CoV-2 spike protein	5′cap, 5′UTR, ORF, 3′UTR, poly(A) tail
Moderna COVID-19 vaccine	Non-replicating mRNA	SARS-CoV-2 spike protein	5′cap, 5′UTR, ORF, 3′UTR, poly(A) tail
saRNA	Self-amplifying mRNA	Multiple (depending on the target antigen)	5′cap, 5′UTR, CDS, 3′UTR, poly(A) tail, viral replication machinery
Circular RNA vaccine	Circular mRNA	Multiple (depending on the target antigen)	Covalently closed loop mRNA, resistant to exonucleases
taRNA vaccine	taRNA	Multiple (depending on the target antigen)	Two-part system: non-replicating mRNA and TR RNA

CDS: Coding sequence; COVID-19: Coronavirus disease 2019; ORF: Open reading frame; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2; saRNA: Self-amplifying mRNA vaccine; taRNA: Trans-amplifying mRNA; TR: Trans-replicon; UTR: Untranslated region.

A 5′cap, a 5′UTR, a coding sequence (CDS), a 3′UTR, and a poly(A) tail are the five essential structural elements that make up saRNAs, another kind of mRNA vaccination, which are organized sequentially from the 5′ end to the 3′ end. Compared with non-replicating mRNAs, saRNAs address this issue by incorporating the viral replication machinery, allowing the mRNA to replicate within the host cell.^[15] The viral replication machinery can help encode an RNA-dependent RNA polymerase (RDRP) complex essential for self-amplification, which typically comes from alphaviruses like the Sindbis virus.^[16] The synthesis of the encoded immunogen is increased by this self-replicating mechanism, which also increases the construct’s magnitude and duration of expression. This leads to increased protein production and a more robust immune response, potentially reducing the required dose.^[13] For example, in non-human primates, administering low doses of a self-replicating mRNA vaccine resulted in increased production of immunogens, with levels peaking on the third day and remaining detectable for more than 14 days postimmunization.^[16] Additionally, by integrating multiple gene sequences into a single replicon, self-replicating mRNA constructs can simultaneously target multiple immunogens and immunomodulatory molecules to increase cancer vaccine potency.

While saRNA vaccines also provide high-level and prolonged protein expression owing to their self-amplification properties, they are typically more challenging to stabilize and require more complex delivery systems. The limitations of saRNAs include the development of circular RNA (circRNA) vaccines and trans-amplifying mRNA (taRNA) vaccines.^[17,18] In fact, unlike traditional linear mRNAs, circRNAs form a covalently closed loop, which makes them more resistant to exonucleases that degrade RNA. This circular structure lacks the free ends found in linear RNA, increasing the stability and prolonged expression of the encoded protein.^[19] Feng et al^[20] studied the use of in vitro-transcribed circRNAs to target EIF4G2(+)/PTBP1(+) pan-adenocarcinoma cells. They engineered a circRNA encoding a mutant gasdermin D (GSDMD) protein to specifically kill cancer cells overexpressing EIF4G2 and PTBP1. This work underscores the promise of circRNAs as stable and precise therapeutic tools, especially for cancers driven by particular genetic profiles.^[20] Next, taRNA vaccines contain two parts: a non-replicating mRNA encoding replicase enzymes, and a trans-replicon (TR) RNA carrying the genetic code of vaccine antigens. The replicase enzymes amplify the TR RNA, increasing antigen production without requiring the full viral genome, making it more efficient than saRNA vaccines. Furthermore, the efficacy of taRNA vaccines does not require a full viral genome, reducing the risk of viral recombination, and allowing for dose sparing and greater design flexibility, which can lower costs and simplify adaptation to various antigens.^[18,21]

Mechanisms of mRNA Vaccine-Mediated Cancer Therapy

The mechanisms underlying mRNA vaccine-mediated cancer therapy involve several critical processes that determine the efficacy and specificity of the vaccine, such as antigen presentation and both innate and adaptive immunity being activated [Figure 1].

Figure 1.

Mechanisms of mRNA cancer vaccines. The mechanism of action of mRNA cancer vaccines involves several critical processes, including antigen presentation, the enhancement of immunogenicity, and the activation of both innate and adaptive immunity. mRNA: Messenger RNA; MHC-I: Major histocompatibility complex I; TAAs: Tumor-associated antigens; TCR: T cell receptor; TLR: Toll-like receptor; TSAs: Tumor-specific antigens.

Antigen presentation

The delivery systems and the antigens encoded by mRNAs are two key factors influencing the efficiency of antigen presentation, which is central to the immunological reaction elicited by mRNA cancer vaccines. Formulated lipid nanoparticles (LNPs), the most common delivery system, not only bind to anionic mRNAs, but also fuse with membranes, promoting their cellular uptake and endosomal escape. During this process, antigen-presenting cells (APCs) mature and further enhance their antigen presentation capabilities, triggering a strong antitumor immune response.^[22] Mannosylated lipid nanoparticles (MLNPs) increase the accumulation of mRNA vaccines in APCs through mannose receptor-mediated endocytosis, significantly increasing the uptake capacity of APCs.^[23] Toll-like receptor 4 (TLR4)-mediated endocytosis subsequently internalizes LNPs, which then fuse with endosomes.^[24] Unlike LNPs, bacterial outer membrane vesicles (OMVs), engineered through surface modifications and conjugation with the RNA-binding protein L7Ae, enable the rapid adsorption and efficient delivery of mRNA antigens to dendritic cells (DCs).^[25] This plug-and-play feature makes OMVs highly suitable for the rapid preparation of various tumor antigens, thereby significantly reducing the time required for the clinical translation of mRNA cancer vaccines. Studies have reported that decorating mRNA vaccines with listeriolysin O can also effectively facilitate the cross-presentation of antigens, significantly inhibiting melanoma progression.^[25] The impact of various delivery systems on the efficacy of antigen presentation in mRNA cancer vaccines will be described in subsequent sections. Once released from endosomes, the mRNA enters the cytoplasm to produce various vaccine antigens, including TAAs or TSAs. Although TAA-based mRNA vaccines, such as melanoma-associated antigen A3 tyrosinase (MAGE-A3) and New York esophageal squamous cell carcinoma 1 (NY-ESO-1), have widespread expression across a variety of tumor types, they may encounter difficulties with immunological tolerance.^[26] TSA-based mRNA vaccines, exemplified by KRAS mutations, offer increased specificity and a reduced risk of off-target effects, making them ideal for targeting well-defined tumor subtypes.^[27] The translated antigen is subsequently processed into smaller fragments (antigenic peptides), which bind to major histocompatibility complex (MHC) molecules and are presented on the outer layer APCs. The KRAS mutations G12D, G12V, G13D, and G12C, for instance, are encoded by mRNA-5671. These mutations trigger immunological responses that are dependent on memory T cells and cytotoxic T lymphocytes (CTL) when they attach to MHC I molecules.^[28] A hybrid nanovaccine composed of mRNA encoding the model antigen ovalbumin (OVA) significantly induced peptide presentation by macrophages and DCs and upregulated costimulatory cluster of differentiation (CD86) expression in a mouse model, resulting in strong tumor inhibition.^[29]

In addition, the speed and intensity of antigen presentation are related to the rate of mRNA translation and the signal cascades activated by the adjuvant effect. The incorporation of chemically modified nucleosides such as N1-methylpseudouridine into mRNA, paired with optimization of codon usage in tRNA, significantly enhances translation efficiency and promotes antigen presentation.^[30] Adjuvant-related stimulator of interferon genes (STING) and TLR pathways can enhance the cross-presentation of tumor antigens through cyclic GMP-AMP synthase (cGAS), MYD88, and nuclear factor kappa B (NF-κB), subsequently inducing adaptive immune responses.^[31,32] In an example of in situ injection of engineered DCs, antigen-encoding mRNAs can be coinjected with TriMix to form a complex that better stimulates DC maturation and enhances cytotoxic T-cell-mediated tumor killing.^[33,34]

Activation of innate and adaptive immunity

Like mRNA vaccines used for infectious diseases, cancer vaccines activate the immune system through the same pathways, as illustrated in Figure 1. The initiation of innate immunity involves engaging pattern recognition receptors (PRRs) and stimulating DCs to trigger signals that result in the creation of proinflammatory factors. During the innate immune system, single-stranded RNA (ssRNA) serves as a ligand for TLR7 and TLR8, which are recognized by APCs.^[35] The body’s immunity is enhanced by this recognition, triggering the production of interferon-α (IFN-α) and other inflammatory factors, including interleukin-12 (IL-12), interferon-gamma (IFN-γ), and tumor necrosis factor-α (TNF-α). The critical function of lymph node (LN) macrophages in promoting innate immune responses has been brought to light by recent research. By activating the stimulator of interferon genes (STING) pathway, these macrophages play a crucial role in inducing the production of IFN-I, which greatly improves DC maturation and downstream adaptive immunity. Notably, a novel LNP formulation, L17-F05, has self-adjuvant properties that target LN macrophages, thereby increasing the antitumor efficacy of mRNA vaccines.^[36] Another example is an mRNA-based vaccine with dual activity that leverages TLR7 activation to induce balanced adaptive immune responses. This vaccine’s two-component formulation combines free mRNA and protamine-complexed mRNA to simultaneously increase immune activation and antigen expression.^[37] The TLR7-dependent pathway promotes the activation of cytotoxic CD8⁺ T cells and antigen-specific CD4⁺ T helper cells. It causes a powerful Th1 immunological reaction.

A significant anticancer effect has been shown by this pathway in preclinical models, underscoring the function of TLR signaling in innate immune activation and its future application in cancer immunotherapy.

Similarly, cytoplasmic non-self mRNAs are recognized by other PRRs, such as retinoic acid-inducible gene-I (RIG-I) receptors,^[38–40] which ultimately contribute to the production of IFNs. This activation enhances the function of DCs, making them more effective at maturing and activating T cells. A clinical trial (NCT01302496) used an mRNA electroporation DC vaccine encoding gp100 and tyrosinase. The occurrence of IFNγ-producing TAA-specific CD8⁺ T cells was significantly associated with differences in the median overall survival of patients alongside stage IV melanoma. In the activation of adaptive immunity, the mRNAs that escape from the endosome further express TAAs or neoantigens. On the one hand, these antigens are degraded by the proteasome, processed further, and shown on major histocompatibility complex I (MHC-I) molecules, encouraging CD8⁺ T cell maturation and differentiation, and leading to CTL-mediated direct killing of tumor cells.^[41] On the other hand, they can be degraded by lysosomes and eventually bind to MHC-II molecules, supporting CD4⁺ T cell as well as B cell maturation and differentiation, which results in the production of matching antibodies and the destruction of tumor cells.^[41] A significant hurdle in cancer therapy is immune evasion, whereby tumors develop strategies to escape immune detection. mRNA vaccines have shown considerable promise in counteracting these challenges by bolstering immune surveillance. This mechanism involves the induction of a wide and diverse immunological activation that targets multiple tumor antigens, thereby minimizing the risk of tumor cells evading immune recognition. mRNA vaccines can also encode other immune-regulating molecules, such as chemokine receptors and costimulatory molecules. By participating in immune cell enrollment and activation, these substances strengthen the anticancer effects of immune cells. Moreover, the incorporation of adjuvants in mRNA formulations has been proved to further amplify the immune response, supporting T cell penetration into the surrounding TME.^[42] In summary, clarifying the processes by which the two types of immunity interact is essential to the therapeutic effectiveness of mRNA cancer vaccines. This includes modulating adjuvant effects to maximize vaccine activation while minimizing adverse effects.

Increased immune memory

Tumor vaccines based on mRNA are a revolutionary approach to cancer immunotherapy, primarily because of their capacity to induce effective memory T cell responses. The molecular mechanism by which mRNA-based tumor vaccines activate memory T cells and memory B cells lies in their ability to encode and express TAAs or TSAs. As an example, mRNA vaccines that express a chimeric protein from human papillomavirus (HPV)-16 E7 and herpes simplex virus type 1 glycoprotein D (gDE7) can stimulate E7-specific CD8⁺ T cells and generate memory T-cell responses to prevent tumor recurrence.^[43] In individuals suffering from overtime lymphocytic leukemia who received BNT162b, vaccine-specific CD8⁺ T cell activation and memory T cell persistence were maintained six months postvaccination and significantly increased following the second booster dose.^[44] Notably, mRNA vaccines not only elicit immediate immune responses but also foster the development of long-lived memory T cells, which, when exposed to the same antigen again, can cause the presenter to develop a quick reaction.^[45] Specific transcription factors such as T-bet and eomesodermin are essential for memory T cell differentiation, ensuring their survival and functionality. Furthermore, an optimal production of memory CD8⁺ T cells depends on the participation of CD4⁺ helper T cells, emphasizing the complex interplay within the T-cell response that mRNA vaccines harness to enhance antitumor immunity.^[11] Besides T-cell-mediated immunity, mRNA vaccines significantly stimulate robust B-cell responses, which are integral to humoral immunity. The process begins when DCs present TAAs to B cells, triggering their activation and subsequent differentiation into antibody-secreting plasma cells. The unique ability of mRNAs to encode multiple antigens enables a diverse antibody repertoire, increasing the likelihood of neutralizing tumor cells effectively. Recent studies have underscored the role of mRNA vaccines in promoting affinity maturation, a crucial process that increases the binding affinity of antibodies to their respective antigens through mechanisms such as somatic hypermutation and clonal selection inside the LN’s germinal centers.^[46] Additionally, memory B cells and long-lived plasma cells are essential for both sustained antibody production and quick reactions to antigens. The capacity of mRNA vaccines to induce class switching, particularly to immunoglobulin G (IgG) isotypes, is also of paramount importance, as it facilitates effective opsonization and complement activation against tumor cells, boosting the immunological response even further.^[47] Moreover, it has been shown that adding adjuvants to mRNA formulations enhances immune reaction and encourages T cell penetration into the TME.^[42] This multifaceted approach not only enhances the initial immune assault on tumors but also establishes a memory response capable of swiftly addressing potential tumor recurrences.^[42,48]

Strategies to Improve the Efficacy of mRNA Vaccines

Balancing the immunogenicity of mRNAs

Several tactics have been created to increase the effectiveness of mRNA cancer vaccines [Figure 2]. Due to its necessity for systemic immune activation, the immunogenicity of mRNAs is a significant factor in triggering an immunological response. However, excessive immune activation can lead to adverse effects and diminished tolerability of vaccines, as evidenced by the local pain, headache, and fatigue observed with BNT162b2.^[6,49,50]

Figure 2.

Potential strategies to improve the effectiveness of mRNA cancer vaccines. The potential strategies to improve the effectiveness of mRNA cancer vaccines include the refinement of mRNA sequences, the innovation of delivery systems, and the integration of immunological adjuvants. APC: Antigen-presenting cell; CTLA4: Cytotoxic T lymphocyte-associated antigen 4; mRNA: Messenger RNA; IFN-γ: Interferon-gamma; LNP: Lipid nanoparticle; PD-1: Programmed death-1; PD-L1: Programmed death-ligand 1; STING: Stimulator of interferon genes; TLR: Toll-like receptor; TNF-α: Tumor necrosis factor-α.

In recent years, studies have focused on reducing the immunogenicity of mRNAs through chemical modifications, purification techniques, and codon optimization. The incorporation of N1-methyl-pseudouridine (1mΨ) reduces mRNA immunogenicity by minimizing innate immune activation and enhances translation efficiency across cell types and in vivo models.^[30] High-performance liquid chromatography (HPLC) and other sophisticated purification methods can minimize double-stranded RNA impurities, which lowers TLR-mediated immune activation. HPLC-purified nucleoside-modified mRNA avoids triggering type I interferons or proinflammatory cytokines.^[51] Moreover, by altering the codon usage to favor GC-rich sequences, degradation and immunological reaction are less likely to occur in mRNA constructions.^[52]

In the context of mRNA-based cancer vaccines, adjuvants are crucial because they stimulate various immunological pathways, increase the absorption of antigens by APCs, and encourage the production of cytokines. Mechanistically, certain delivery systems that also function as adjuvants are called vaccine adjuvant delivery systems (VADSs), alongside immunostimulants, representing advancements in mRNA cancer vaccine development [Figure 3].

Figure 3.

Mechanisms of adjuvants in the context of mRNA cancer vaccines. (A) Comparison of immune responses with and without adjuvants. Vaccines without adjuvants induce APC maturation and cytokine production, but present low-affinity antigens, leading to limited immune responses. Adjuvants enhance antigen uptake, cytokine profiles, and high-affinity antigen presentation, promoting stronger CD8⁺ T cell responses and long-term immune memory. (B) Intracellular pathways activated by adjuvants. Adjuvants stimulate pattern recognition receptors (e.g., TLR4, TLR3), activating STING-cGAS, TBK1, and NF-κB signaling, which leads to type I interferons (IFN-α/β) and cytokine production, amplifying adaptive immunity. APCs: Antigen-presenting cells; cGAS: Cyclic GMP-AMP synthase; IFN-α: Interferon-α; IFN-γ: Interferon-gamma; IL: Interleukin; IRF-7: Interferon regulatory factor 7; LNP: Lipid nanoparticle; mRNA: Messenger RNA; MHC-I: Major histocompatibility complex I; NF-κB: Nuclear factor kappa B; STING: Stimulator of interferon genes; Th1: T-Helper 1; TNF-α: Tumor necrosis factor-α; TLR4: Toll-like receptor 4.

LNPs can enhance adjuvant effects through structural modifications. mRNA-LNP_ssPalmE, an ionizable liposome that uses vitamin E as a scaffold, produces cytotoxic T-cell responses specific to OVA and has anticancer effects in the E.G7-OVA tumor model.^[53] Vitamin E enhances CTL responses by stimulating antigen expression in conventional dendritic cells (cDC2s), demonstrating its adjuvant effect.^[53] Additionally, adjuvants can be embedded or encapsulated within LNPs. For example, codelivery of all-trans-retinoic acid with mRNA significantly increased antigen-specific T cell infiltration in colorectal tumors, markedly extending the survival time of treated animals.^[54] The integration of C16-R848, a TLR7/8 agonist, into LNPs containing ovalbumin-encoding mRNAs significantly improved mRNA transfection and MHC-I antigen presentation in APCs.^[32] This highlights the potential for further development of LNPs with adjuvant effects for future applications in mRNA cancer vaccines. Another self-adjuvating nanovaccine is polyguanidine (PolyGu), which is made from polyethylenimine (PEI) and modified with different guanidine groups to transform its cytotoxicity into innate immune activation. Poly(biphenyl)guanidine (Poly-PBG)-based PolyGu nanovaccines efficiently activate DCs, accelerating their maturation via the NLRP3 and TLR4 pathways. It dramatically reduced the growth of tumors and prolonged survival in animal experiments.^[55] Different pathogen-associated molecular patterns (PAMPs) found in OMVs have the ability to efficiently stimulate both innate and adaptive immunity. Engineered OMVs can quickly bind mRNA antigens tagged with box C/D sequences through the RNA-binding protein L7Ae and enable cross-presentation via endosomal escape mediated by listeriolysin O. This OMV-LL-mRNA not only inhibits melanoma progression but also induces long-term immune memory.^[25] Porous silica nanoparticles (PSNs) can act as self-adjuvants by mimicking natural infection processes to enhance immune responses. When coated with PEI, hollow mesoporous silica nanoparticles (H-XL-MSNs) change their surface charge in addition to acting as adjuvants, which makes it easier to load and release model antigens gradually. This surface modification enhances the activation of DCs, thereby improving vaccine efficacy.^[56]

Although the clinical applications of adjuvants in mRNA cancer vaccines are limited, their role parallels that of traditional cancer vaccines. Liu et al^[57] used 2′,3′-cGAMP as an adjuvant, which was coloaded with tumor antigen-encoding mRNA into pardaxin-modified liposomes. These LNPs enhance the transfection efficiency of DCs and lysosomal degradation of the cargo. In vitro and in vivo, 2′,3′-cGAMP stimulates immunological responses specific to tumor antigens by activating the STING pathway, which in turn induces downstream activation of NF-κB and interferon regulatory factor 3 (IRF3).^[57] Adjuvants that target TLRs and the cGAS-STING pathway still hold promise for further research.^{[58, 59]}

Optimizing mRNA vaccine stability through sequence refinement

Increasing the stability of an mRNA vaccine is a strategic approach to improve its efficacy. Various methods of sequence optimization can be applied to different segments of the mRNA sequence. Specifically, optimization of codon usage leads to a better match of tRNA abundance in the host cell,^[52,60] and alteration of the GC content in the mRNA sequence,^[52,61] all of which effectively increase the translation efficiency and protein expression levels. For different diseases and cell types, the effectiveness of 5′UTRs and 3′UTRs in maintaining mRNA stability varies.^[60,62] One approach is to use the UTR combinatorial library to identify alternative UTR sequences that can be used to achieve stability and protein levels.^[63] It has been demonstrated that the addition of 2′-O-methylation to the mRNA 5′ cap affects protein synthesis levels in a cell-specific way and aids in RNA immune evasion.^[60,64] Additionally, the poly(A) tail stabilizes mRNA, and its length strongly affects mRNA stability and processing.^[65,66] And the mRNA degradation has been revealed to be specifically influenced by the poly(A)-binding protein Pab1’s C-terminal region.^[67] Replacing codons with modified or synonymous nucleotides can also improve mRNA stability and may affect its secondary structure and posttranslational modifications, without altering the amino acid sequence.^[68] mRNAs with high GC content exhibit improved translational stability, suggesting that a moderate increase in GC content may enhance the immunogenic efficacy of vaccines in combating tumors.^[61,69] Notably, adjustments and modifications of mRNA sequences, which are often accompanied by changes in mRNA structure, translation accuracy, and protein folding mechanisms, influence the ultimate efficacy of the vaccine. As noted earlier regarding taRNAs, Perkovic et al^[18] streamlined that the TR structure replaced Sindbis virus (SINV) with Semliki Forest virus (SFV), resulting in increased taRNA replication and immune responses in vivo. In summary, future mRNA cancer vaccine developers need to comprehensively consider the impacts of mRNA sequence optimization, each of which contributes to achieving the optimal balance in efficacy and stability.

Carriers for mRNA delivery

The development of efficient and reliable delivery systems to ensure mRNA integrity is crucial for achieving effective immune activation. These systems must protect mRNA from nucleases in physiological fluids, evade the mononuclear phagocyte system, facilitate cellular uptake via endocytosis, and enable endosomal escape into the cytoplasm.^[70] Choosing the right carrier is crucial for vaccine success and influences the immune response’s strength and precision of the immune response. Advances in nanotechnology have enabled diverse carrier systems to improve mRNA delivery and expression in target cells. Table 3 presents the main delivery carriers focused on in mRNA vaccine development, including lipid-based, polymer-based, and virus-like particle (VLP)-based systems.

Table 3.

Delivery strategies of mRNA vaccines.

Delivery strategies	Structure	Study findings	References
Lipid-based
3-Comp	Composed of nAcx-Cm lipids, permanent cationic lipids, and PEG lipids	Better delivery efficiency and lung targeting than four- or five-component systems containing cholesterol and phospholipids, while still maintaining nanoparticle stability	[77]
4N4T LNP (4-Comp)	Composed of multicharged lipids with 4N4T, cholesterol, phospholipids, and PEG	Providing enhanced mRNA delivery, robust immune responses, and improved safety compared to traditional LNP systems	[78]
Inhalable LNP (5-Comp)	Composed of ionizable lipids, helper lipids, phospholipids, and PEG	The iLNP formulation is more suitable for inhalation and respiratory disease treatments because it can form effective and intact aerosolized particles.	[79]
Polymer-based
F-PEI-based nanoparticle	Composed of a F-PEI shell and an mRNA core	Effective delivery of mRNA vaccines to exert anti-tumor effects without the need for adjuvants.	[22]
PEG-Plys-based nanoparticles	Composed of polyethylene glycol-polylysine (PEG-PLys) modified with triphenylphosphonium	Significantly extended blood circulation time and increased mRNA distribution within solid tumors	[83]
Anionic polymers nanoparticles	Composed of a PLGA shell and an mRNA core	Achieved a delivery efficiency of up to 80%, rapidly inducing protein translation	[84]
VLP-based
Chimeric VLP	Composed of antigen-encoding mRNA, unmethylated CpG oligonucleotides, and positively charged proteins within a phospholipid bilayer.	Exerting adjuvant effects through structural modifications to elicit potent humoral and cellular immunity in prophylactic vaccines against HPV, cervical cancer, and breast cancer	[86–89]

CpG: Cytosine-phosphorothioate-guanine; F-PEI: Fluoroalkane-modified polyethylenimine; iLNP: Inhalable LNP; LNP: Lipid nanoparticle; mRNA: Messenger RNA; PEG: Polyethylene glycol; PEG-PLys: PEG-poly(l-lysine); PLGA: Poly(lactic-co-glycolic acid); VLP: Virus-like particle; 3-Comp: Three-component LNP; 4N4T: Four tertiary amine nitrogen atoms.

The LNP-based delivery platform is the most widely employed and technologically mature delivery system for mRNA vaccines.^[70] LNPs consist of four key lipid components, each serving a distinct role: cationic or ionizable lipids protect mRNAs from RNase degradation, polyethylene glycol (PEG) lipids reduce clearance by the reticuloendothelial system (RES), and phospholipids along with cholesterol stabilize the overall LNP structure.^[71,72] pH variations will improve mRNA packaging and make endocytosis easier.^[73] Notably, owing to the tendency of cationic lipids and cholesterol to drive hepatic accumulation, hepatotoxicity is the most likely toxic side effect of lipid-based mRNA vaccines, which requires further development of novel lipids to expand their clinical applications.^[74,75] Specifically, Liu et al^[76] optimized the composition of LNPs and reported that cholesterol and phospholipids were not necessary for effective delivery. They thus developed a three-component LNP (3-Comp) strategy, and showed that mRNA delivery systems made of nAcx-Cm lipids, permanent cationic lipids, and PEG lipids had better delivery efficiency and lung targeting than four- or five-component systems did.^[76] Song et al^[77] innovatively designed a series of novel multicharged lipids with four tertiary amine nitrogen atoms (4N4T) for mRNA vaccine delivery and demonstrated a strong and sustained humoral response in mice. Zhang et al^[78] optimized LNP nanomedicines via the “LOOP” process, identifying an optimal formulation capable of withstanding shear stress during nebulization. The inhaled LNP (iLNP) formulation containing scFV significantly alleviated BLM-induced pulmonary fibrosis, which enhances the potential application of LNP-based mRNA vaccines in treating lung cancer.^[78]

Although not as clinically advanced as LNPs are, polymeric nanomaterials are gaining increasing attention because of their distinctive features in vaccine development. For the delivery of mRNA vaccines, PEI has been used extensively among several polymer-based vectors.^[4,74,79] Fatty chain modification is a commonly employed method in current research to mitigate this limitation.^[80,81] Peng et al^[22] demonstrated that the fluoroalkane-modified polyethylenimine (F-PEI)-based personalized nanovaccine, when formulated with mRNA encoding neoantigens from MC38 tumors and combined with immune checkpoint inhibitors (ICIs), was able to effectively eliminate established tumors.^[2] In addition, Norimatsu et al^[82] substituted the amine group in PEG-poly(l-lysine) (PEG-PLys) with triphenylphosphine (TPP) to formulate a single-component polymer micelle loaded with mRNA for biological characterization. In a mouse model, this approach significantly extended the blood circulation time and increased the mRNA distribution within solid tumors. Meanwhile, Yasar et al^[83] showed that Poly(lactic-co-glycolic acid) (PLGA)-incorporating nanoparticles coated with LNPs achieved a delivery efficiency of up to 80%, rapidly inducing protein translation that peaked within a short time frame and dissipated after 48h.

VLPs can encapsulate saRNAs that encode antigens and deliver them into the cytoplasm, which is analogous to the viral infection approach. This allows the mRNA to be translated without escaping from the endosome.^[84] Chimeric VLPs are capable of delivering epitopes of various cancer-associated viruses on their own right and can exert adjuvant effects through structural modifications to elicit potent humoral and cellular immunity in prophylactic vaccines against HPV, cervical cancer, and breast cancer.^[85–87] One study showed effectiveness both in vitro and in vivo by encapsulating positively charged proteins, unmethylated cytosine-phosphorothioate-guanine (CpG) oligonucleotides, and mRNA in a phospholipid bilayer to simulate the viral structure.^[88] Other delivery carriers, including extracellular vesicles (EVs), engineered DCs, and peptides, have been created to effectively and securely convey mRNAs. In particular, engineered DCs can also be employed to deliver antigen-encoding mRNAs for cancer biotherapy via both ex vivo and in situ loading strategies, such as electroporation and lipofection.^[89,90] Through electrostatic interactions, mRNAs can be bound to peptides with positive potentials, thus preventing RNAase degradation.

Clinical Trials of mRNA Cancer Vaccines

Since the initial invention and validation of mRNA cancer vaccines, numerous preliminary and clinical studies have proved the potential of mRNA vaccines as a feasible method for cancer treatment [Table 4]. These trials aim to assess their pharmacological properties, dosage responses, and effects on tumor recurrence and survival rates. However, most of these studies are still in the early stages, and further validation along with long-term follow-up is essential. In this review, on the basis of different mechanisms of mRNA-mediated immunotherapy, the clinical trials discussed here are categorized into four types: mRNAs encoding TAAs, mRNAs encoding TSAs, mRNAs encoding cytokines, and mRNAs encoding tumor suppressor genes.

Table 4.

mRNA cancer vaccines registered on clinicaltrials.gov.

Target antigen	Cancer type	Phase	NCT number	Results
mRNA cancer vaccines encoding TAAs
NY-ESO-1, tyrosinase, MAGE-A3, TPTE	Advanced melanoma	Phase I	NCT02410733	Lipo-MERIT is safe and well-tolerated in patients with advanced melanoma
BNT113 (HPV16 E6 and E7 proteins)	Unresectable recurrent, or metastatic head and neck squamous cell carcinoma	Phase II	NCT04534205	Recruiting
BNT113 (HPV16 E6 and E7 proteins)	Human papillomavirus-related carcinomaHead and neck neoplasmCervical neoplasmPenile neoplasms malignantUnknown primary tumors	Phase I/II	NCT03418480	BNT1113 showed acceptable safety and tolerability in patients with head and neck neoplasm
BNT114 plus BNT122 (personalized set of premanufactured non-mutated shared TAA plus a personalized cancer vaccine)	TNBC	Phase I	NCT02316457	BNT114 showed acceptable safety and tolerability, along with a temporary increase in cytokine levels
BNT112 (PAP, PSA, PSMA, NKX3.1, HOXB13)	Prostate cancer	Phase I/II	NCT04382898	BNT112 alone or with cemiplimab has shown good safety in prostate cancer patients
BNT111(NY-ESO-1, tyrosinase, MAGE-A3, and TPTE)	Melanoma stage IIIMelanoma stage IVUnresectable melanoma	Phase I	NCT04526899	BNT111 possesses improvement in ORR in the combination therapy with cemiplimab, showing good safety whether used alone or in combination
Ovarian cancer TAA	Ovarian cancer	Phase I	NCT04163094	W_ova1 vaccine showed good immune response and tolerability in ovarian cancer patients, but then it was terminated
mRNA-4359 (IDO1, PD-L1)	Advanced solid tumors	Phase I/II	NCT05533697	mRNA-4359 holds promise in controlling tumor progression in patients with advanced solid tumors
mRNA cancer vaccines encoding TSAs
BNT122 (personalized cancer vaccine encoding TSA)	MelanomaNon-small cell lung cancerBladder cancerColorectal cancerTNBCRenal cancerHead and neck cancerOther solid cancers	Phase I	NCT03289962	BNT122 is safe and well-tolerated in advanced solid tumors
BNT122 (personalized cancer vaccine encoding TSA)	Colorectal cancer stage II/III	Phase II	NCT04486378	BNT122 in combination with atezolizumab demonstrates good immunogenicity and safety
mRNA-4157 (personalized cancer vaccine encoding TSA)	Solid tumors	Phase II	NCT03313778	mRNA-4157 has an acceptable safety profile along with observed clinical responses in combination with pembrolizumab
mRNA-4157 (personalized cancer vaccine encoding TSA)	Melanoma	Phase II	NCT03897881	mRNA-4157 combined with pembrolizumab can prolong RFS in high-risk melanoma patients postresection
mRNA cancer vaccines encoding immunomodulators
CD40L, CD70, caTLR4; melanoma-TAA, gp100, MAGE-A3, MAGE-C2, and PRAME	Melanoma	Phase I	NCT03394937	ECI-006 is safe and well-tolerated in advanced melanoma
mRNA-2752 (OX40L, IL-23, IL-36γ)	Relapsed/refractory solid tumor malignancies or lymphoma	Phase I	NCT03739931	mRNA-2752 is well tolerated in patients with solid tumors and exhibits sustained immunomodulatory effects
mRNA-2752 (OX40L, IL-23, IL-36γ)	Carcinoma, intraductal, non-infiltrating	Early phase I	NCT02872025	Recruiting
mRNA cancer vaccines encoding CARs or TCR
MET CARs with tandem TCRζ and 4-1BB	Malignant melanomaBreast cancer	Early phase I	NCT03060356	RNA CART-cMET is safe and feasible in patients with metastatic melanoma
HBV-specific T-cell receptor (TCR	Recurrent hepatocellular carcinoma	Phase I	NCT02719782	Unpublished results
MESO CAR T-cell	Refractory malignant solid neoplasm	Phase I	NCT04981691	Unpublished results
Allogeneic NKG2DL-targeting CAR-grafted γδ T cells	CancerMalignancyRefractory cancerRelapsed cancer	Phase I	NCT05302037	Unknown status

CAR: Chimeric antigen receptor; HOXB13: Homeobox B13; IDO: Indoleamine 2,3-dioxygenase; IL-23: Interleukin-23; IL-36γ: Interleukin-36 gamma; MAGE-A3: Melanoma-associated antigen A3 tyrosinase; MAGE-C2: Melanoma antigen gene-C2; MESO: Anti-mesothelin; mRNA: Messenger RNA; NKG2DL: Natural killer group 2 member D ligand; NKX31: NK3 homeobox 1; NY-ESO-1: New York esophageal squamous cell carcinoma 1; ORR: Overall response rate; PAP: Prostatic acid phosphatase; PD-L1: Programmed death-ligand 1; PSA: Prostate-specific antigen; PSMA: Prostate-specific membrane antigen; RFS: Recurrence-free survival; TAAs: Tumor-associated antigens; TCR: T-cell receptor; TLR: Toll-like receptor; TNBC: Triple-negative breast cancer; TPTE: Transmembrane phosphatase with tensin homology; TSA: Tumor-specific antigen.

mRNA cancer vaccines encoding TAAs

TAAs are a group of proteins or molecules that are either lacking or expressed at lesser amounts in healthy cells but are present in tumor cells. The mechanisms of mRNA-based cancer vaccines encoding TAAs include the stimulation of targeted immune responses against antigens that are predominantly overexpressed on cancer cells rather than normal cells. Therefore, despite the high heterogeneity of tumors, TAA-based vaccines still hold potential as anticancer therapy. Nevertheless, TAAs often exhibit low T-cell receptor (TCR) affinity and face limitations in overcoming immune tolerance caused by the TME. Therefore, to increase the effectiveness of vaccines in clinical applications, vaccines encoding a mixture of multiple TAAs are commonly used. An mRNA cancer vaccine (Lipo-MERIT) was tested for safety and tolerability in patients with advanced melanoma in a clinical trial (NCT02410733) conducted by BioNTech. The subsequent trial of BNT111 showed promising results in a phase I clinical study. BNT111 monotherapy induced T-cell responses against at least one TAA in 64% of the evaluable patients who had evidence of disease (ED) and 68% of the patients with no evidence of disease (NED). The clinical efficacy was particularly encouraging in NED patients, with a median disease-free survival of 34.8 months.^[91] In a phase II trial in combination with a programmed death-1 (PD-1) inhibitor (cemiplimab),^[91,92] BNT111 significantly improved the overall response rate (ORR) in patients with unresectable advanced melanoma. These results indicated that combination therapy with BNT111 and cemiplimab could significantly increase the ORR in these patients. In subsequent clinical trials of several mRNA vaccines encoding TAAs, such as those for the treatment of head and neck squamous cell carcinoma (NCT04534205), ovarian cancer (NCT04163094), prostate cancer (NCT04382898), and adult glioblastoma (NCT04526899), the Lipo-MERIT vaccine platform was also utilized. BNT112 (NCT04382898) encodes five prostate cancer-associated antigens (prostate-specific antigen [PSA], prostatic acid phosphatase [PAP], prostate-specific membrane antigen [PSMA], NK3 Homeobox 1 [NKX3.1], and Sperm Acrosome Associated 3 [SPACA3] ). Among the seven patients who received treatment, two individuals receiving BNT112 monotherapy showed a decrease in prostate-specific antigen (PSA) levels, and there were no significant side effects associated with BNT112, which may suggest that the drug is safe and effective against tumors. BNT114 (NCT04163094), an mRNA vaccine encoding a mixture of three TAAs for triple-negative breast cancer (TNBC), is being utilized in conjunction with mRNAs encoding p53 and other LNP-formulated TAAs. According to the results of phase I clinical trials, the BNT114 vaccine is viable in terms of patient burden, logistics, and scheduling.

mRNA-4359 targets two TAAs: indoleamine 2,3-dioxygenase (IDO) and programmed death-ligand 1 (PD-L1).^[93] The results from its phase I clinical trial (NCT05533697) revealed that, among 16 evaluable patients, 8 experienced no tumor growth or new lesions, resulting in a 50% disease control rate. mRNA-4359 showed good safety and tolerability both when used alone and in conjunction with the ICI pembrolizumab. Other strategies for applying TAA mRNA cancer vaccines include a CLDN6 mRNA-LPX vaccine that effectively induces APC-mediated antigen presentation,^[94] facilitating the controlled growth of low-dose chimeric antigen receptor (CAR)-T cells by enabling the number of CLDN6-CAR-T cells to peak three to four days after immunization.

mRNA cancer vaccines encoding TSAs

Unlike TAAs, TSAs (also known as neoantigens) are expressed exclusively in tumor tissues and are generated by mutations in tumor cells. Mutated neoantigens dominate the antitumor immune response in naturally occurring T cells, leading to neoantigen-based immunotherapy. An overview of registered clinical trials with TSA-encoding mRNA cancer vaccines is given in Table 4. In general, the distinct mutational profile of tumor cells in cancer patients can be utilized to design personalized vaccines via the next-generation sequencing (NGS). Owing to leukocyte antigen (HLA) restrictions, not all mutations lead to the immune system recognizing new epitopes. Therefore, patient HLA typing and computational methods are needed to predict MHC-I binding epitopes and identify peptides likely to induce CD8⁺ T cell responses.

In patients with resected pancreatic ductal adenocarcinoma, RO7198457 (also known as BNT122 or autogene cevumeran) is a customized mRNA cancer vaccine intended to boost immunity against neoantigens.^[95] In a phase Ib clinical trial, it was used in combination with the PD-L1 inhibitor atezolizumab, demonstrating certain efficacy and safety. Among the 108 evaluable patients with solid tumors, 9 responded to combination therapy, including 1 with a complete response, while 53 patients had stable disease. Additionally, in peripheral blood, 77% of patients showed neoantigen-specific T-cell responses produced by RO7198457 (NCT03289962). A phase II clinical trial has also been completed (NCT04486378), which was conducted to compare the efficacy of RO7198457 with that of active surveillance in patients with resected stage II (high-risk) and stage III colorectal cancer. The injectable cancer vaccine BNT133 (HPV16 E7 mRNA) has demonstrated promising results for improving tumor regression and avoiding recurrence in two animal tumor models (TC-1 and C3).^[96] A synergistic effect was also demonstrated when PD-L1 inhibitors were used. As a result, BNT133 has been used in two clinical trials involving HPV patients to evaluate its safety and efficacy: NCT03418480 and NCT04534205.

At present, multiple institutions, including BioNTech and Moderna, are collaborating in an effort to develop personalized mRNA cancer vaccines, with several clinical trials actively in progress. BioNTech SE has created the iNeST platform for personalized cancer antigen therapy, which includes treatments such as BNT121 and BNT122. Moderna created mRNA-4157, a customized mRNA cancer vaccine that contains 20 highly immunogenic neoepitopes based on the patient’s immunological profile and particular mutations. The mRNA is encapsulated in LNPs and administered via intramuscular injection. The safety and clinical effectiveness of this medication are presently being assessed in patients with melanoma (NCT03897881) and solid tumors (NCT03313778). With a hazard ratio of 0.561 (95% CI: 0.309–1.017), the combination of mRNA-4157 with pembrolizumab in the KEYNOTE-942 study reduced the risk of mortality or recurrence by 44% when compared to pembrolizumab alone. Moreover, in a 3-year updated study, combination therapy demonstrated a 2.5-year recurrence-free survival (RFS) rate of 74.8% vs. 55.6% with pembrolizumab alone, along with a significant improvement in the distant metastasis-free survival (DMFS) rate (HR = 0.384; 95% CI: 0.172–0.858).^[97,98]

mRNA cancer vaccines encoding immunomodulators

Cytokines and immune costimulatory molecules can act as immunomodulators by activating antitumor effector cells, altering the tumor immune microenvironment, reducing tumor immune evasion, and enhancing the magnitude of the immune response against malignant cells.^[99,100] In clinical practice, injecting cytokines into cancer patients has emerged as a treatment approach. With some patients experiencing complete response (CR), the Food and Drug Administration (FDA) has approved high-dose recombinant interleukin-2 (IL-2) for the treatment of melanoma and metastatic renal cell carcinoma (RCC).^[101] In addition to IL-2, other cytokines such as IL-7 and IL-15 have also shown potential in cancer treatment. IL-7 can induce an inflammatory TME, and more than 170 clinical trials related to IL-15 have been initiated.^[99] BNT131 (SAR441000) from BioNTech, which encodes IL-12, IL-15sushi, and IFN-α for intratumoral delivery, is under assessment both as a standalone therapy and in combination with cemiplimab for patients with advanced solid tumors, aiming to modify the TME.^[102] No dose-limiting toxicities were observed, and no grade 3, 4, or 5 adverse events related to the treatment were reported. Adverse events related to treatment that occurred in a minimum of two patients in each group were restricted to grade 1 or 2 non-serious occurrences. Additionally, some patients presented increased plasma levels of IP10 and IFN-γ, along with increased CD8⁺ T-cell infiltration in tumor biopsies.^[103] In 2024, Beck et al^[104] revealed the role of IL-2-activated CD8⁺ T cells and macrophages in the treatment of MHC class I-deficient tumors, and how they promote an inflammatory tumor environment with antitumor activity through the cross-presentation of tumor neoantigens. These findings suggest that the use of mRNA technology to restore immune responses in the TME may help increase the success rate of cancer treatments. Other mRNA therapies have also demonstrated potential, including ECI-006, which combines TriMix with melanoma-specific TAAs for intranodular administration and is currently being evaluated in a phase I trial for resected melanoma (NCT03394937). Additionally, MEDI1191, an immunomodulatory fusion protein consisting of the IL-12a and IL-12b subunits, was developed for intratumoral injection.^[105] An observational research combining mRNA-2752 with the anti-PD-1 antibody pembrolizumab in ductal carcinoma and a dose-escalation trial of mRNA-2752 in several advanced malignancies are presently in progress (NCT03739931, NCT02872025). Table 4 provides a list of clinical trials for other mRNA cancer vaccines encoding immunomodulators, as retrieved from clinicaltrials.gov. We anticipate further research to explore the clinical potential of cytokines and immune costimulatory molecules in reshaping the functionality of the TME. It is important to determine the optimal delivery methods and maximize the efficacy of vaccines.

mRNA cancer vaccines encoding CARs or TCRs

Using transient mRNA transfection technology, specific CARs or TCRs can be constructed through in vitro transcription and then introduced into T cells, increasing their ability to recognize and kill tumor cells.^[106] This approach decreases the time needed to produce the target-engineered T cells. The duration for which CARs are expressed on the surface of T cells is directly related to the duration for which CAR-encoding mRNAs remain active within the cells. Once the transfected CAR mRNA becomes inactive, the CARs are no longer displayed on the T cell surface. The transfection of mRNAs also enables the introduction of additional mRNAs to encode for other immunologically active substances, leading to synergistic therapeutic effects. In a recent study, Tong Aiping et al^[107] confirmed the ideal functional characteristics of mRNA-CD5-CAR-γδT^CD5− cells, which show promise as effective treatments for T-ALL. Additionally, in preclinical solid tumor models, chimeric antigen receptor-engineered macrophages (CAR-Ms) demonstrate extraordinary efficacy in lowering the tumor burden and increasing overall survival due to their strong phagocytic activity against target cells and ease of penetration into solid tumors. Tan Weihong et al^[108] explored 36 CAR combinations containing different macrophage intracellular domains (ICDs) and developed an mRNA-LNP delivery system that targets macrophages to achieve efficient in vivo CAR-M construction. Intraperitoneal programming of CAR-M cells and anti-PD-1/L1 immune checkpoint blockade (ICB) therapy together produced a strong activation of the adaptive immune response in immunosuppressed solid tumor mouse models.

NCT03060356 is a phase I clinical trial aimed at evaluating the safety and feasibility of intravenously injecting RNA-electroporated cMET-targeting CAR T cells in patients with melanoma and TNBC.^[109] After six CAR T cell infusions, four out of seven patients maintained stable disease, and none of the patients experienced grade 3 or higher toxicity, neurotoxicity, or treatment interruption. Unfortunately, the sample size of this clinical trial was too small, necessitating larger-scale follow-up studies. Driven by the promising therapeutic results of CAR T cells in the treatment of cancer, additional CAR T products that target distinct tumor antigens have been created and evaluated in phase I and II clinical trials for a variety of solid tumors (NCT02719782, NCT04981691, and NCT05302037). Importantly, compared with mRNA vaccines, CAR-T-cell therapy requires higher doses of mRNA and repeated administration to achieve lasting therapeutic effects. Additionally, engineered T cells are personalized cell therapy products that are expensive and time-consuming. Studies have reported that LNPs outperform electroporation in mRNA transfection.^[107] Therefore, developing ex vivo CAR-T cell engineering methods based on mRNA-LNP delivery to replace traditional electroporation will be crucial in future research.

Overall, the vaccine candidates discussed here exhibit distinct mechanistic advantages. mRNA vaccines encoding TAAs address tumor heterogeneity by targeting shared tumor antigens. In contrast, TSA-encoding vaccines offer greater precision, avoiding normal tissue interference by targeting tumor-specific mutations. Immunomodulator-encoded vaccines excel in reshaping the TME, thereby boosting the immune response. Finally, CAR/TCR mRNA vaccines leverage transient gene expression to swiftly generate tailored immune responses, offering a dynamic approach for advanced cancer immunotherapy. These mechanistic innovations collectively highlight the potential of mRNA vaccines as versatile tools in cancer treatment.

Challenges and Perspectives

In light of the successful clinical safety and efficacy of mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and their continued clinical application in inducing cross-neutralizing antibodies against variants, the importance of mRNA cancer vaccines in advancing tumor immunotherapy research is promising.^[10,49] While mRNA-based cancer vaccines have shown encouraging outcomes in both preclinical studies and early phase clinical trials, their successful application in clinical practice remains challenging, as shown in Table 4.

First, one major challenge is tumor heterogeneity, where the diverse genetic and phenotypic profiles of tumors complicate the identification of universal antigens. More studies are warranted to uncover novel tumor antigens. Optimizing the processes for screening and identifying tumor antigens, particularly neoantigens, is essential for the development of systematic approaches that enable the cost-effective and rapid production of new antigen-based mRNA vaccines. This will necessitate the further advancement of high-throughput deep gene sequencing technologies, combined with robust algorithm development and sophisticated data analysis tools.^[110]

In addition, the TME also poses a significant barrier to the efficacy of mRNA-based cancer vaccines. Within the TME, regulatory T cells, myeloid-derived suppressor cells, and suppressive cytokines such as transforming growth factor-beta (TGF-β) and IL-10 actively inhibit the immune response.^[111] Addressing this requires innovative strategies such as combining mRNA vaccines with immune checkpoint inhibitors or other immunomodulatory agents to counteract TME-mediated suppression and boost vaccine-induced immune activation.^[112]

Another key limitation lies in vaccine administration routes. The delivery route of mRNA vaccines has a significant effect on their targeting, safety, and efficacy. Currently, although the approved SARS-CoV-2 mRNA vaccines use intramuscular injection, no consensus has been reached on the optimal delivery route for mRNA vaccines. Different delivery methods affect the efficiency of mRNA vaccine delivery, the strength of the immune response, and the manifestation of side effects. mRNA vaccines are administered primarily via intramuscular injection, subcutaneous injection, intravenous injection, or local injection, each requiring specific considerations in the design and adjustment of vaccine components. Each route of administration of mRNA cancer vaccines has its own unique advantages and limitations.^[113] A non-invasive technique using dual-radioisotope near-infrared probes has been used recently by researchers to monitor the spatiotemporal transit of vaccinations after intramuscular administration. This technique offers crucial guidance for precisely determining vaccine dose, injection locations, and biodistribution.^[114] To determine the most effective vaccination routes and optimize vaccine efficacy, new approaches capable of accurately monitoring and analyzing the spatiotemporal dynamics of vaccines are urgently needed.

The lack of reliable biomarkers to monitor vaccine efficacy further complicates the clinical application of mRNA cancer vaccines. Identifying robust indicators of immune activation and therapeutic response is essential for evaluating the success of these therapies. Advanced imaging techniques and real-time biomarker tracking are promising solutions to address this challenge, enabling more precise and timely assessment of vaccine performance. As a compelling example, miRNAs such as miR-21, miR-375, and miR-141 have shown high specificity as cancer biomarkers due to their significant overexpression in cancer samples compared with healthy controls, with a 1.8-fold increase and a highly significant P-value of 1.6 × 10⁻²².^[115] Similarly, neutralizing antibody responses, such as those observed with the mRNA-1273 vaccine in cancer patients, demonstrated a significant GMT increase 28 days postvaccination and maintained elevated levels at 6 months, highlighting their utility in evaluating vaccine protection.^[116] Moreover, immunosuppressive markers such as T-cell immunoreceptor with Ig and ITIM domains (TIGIT), where lower expression levels correlate with stronger vaccine immunogenicity, present potential as predictive biomarkers for assessing immune responses.^[117] These examples underscore the value of integrating biomarker tracking into vaccine development for improved efficacy monitoring and predictive assessment.

Finally, the practical barriers of high production costs and complex logistics limit the accessibility of mRNA cancer vaccines. The manufacturing process, which involves in vitro transcription and stringent purification, significantly increases costs, while ultracold chain storage requirements pose additional logistical challenges. The development of thermostable formulations and streamlined production processes are critical steps toward enhancing the scalability and affordability of these vaccines.^[118]

Thorough investigation and resolution of these challenges, achievements with SARS-CoV-2 mRNA vaccines, together with increased knowledge of tumor biology, are anticipated to lead to significant breakthroughs in mRNA-based cancer vaccines, revealing new possibilities for effective cancer treatment.

Acknowledgment

All the figures in this review were created with BioRender.com.

Funding

This work was supported by grants from the 1.3.5 project for disciplines of excellence from West China Hospital of Sichuan University (No. ZYGD23038), the National Science Foundation for Excellent Young Scholars (No. 32122052), the project fund for the development of a clinical evaluation technology platform for novel corona-virus vaccines and the research and development of a new preventive nasal spray coronavirus vaccine, the Young Scientists Fund of the National Natural Science Foundation of China (No. 32401268), the Postdoctoral Fellowship Program of CPSF (No. GZB20240500), and Sichuan Science and Technology Program (No. 2024NSFSC1200).

Conflicts of interest

None.