mRNA: A promising platform for cancer immunotherapy


 Messenger RNA (mRNA) is now in the limelight as a powerful tool for treating various human diseases, especially malignant tumors, thanks to the remarkable clinical outcomes of mRNA vaccines using lipid nanoparticle technology during the COVID-19 pandemic. Recent promising preclinical and clinical results that epitomize the advancement in mRNA and nanoformulation-based delivery technologies have highlighted the tremendous potential of mRNA in cancer immunotherapy. mRNAs can be harnessed for cancer immunotherapy in forms of various therapeutic modalities, including cancer vaccines, adoptive T-cell therapies, therapeutic antibodies, and immunomodulatory proteins. This review provides a comprehensive overview of the current state and prospects of mRNA-based therapeutics, including numerous delivery and therapeutic strategies.



Introduction
Over the past decade, mRNA has been increasingly recognized as a versatile tool for developing innovative therapeutics.In particular, the COVID-19 pandemic has kindled the interest in mRNA molecules as a means to transmit instructions into the cells to produce antigenic proteins for vaccination.Studies during the coronavirus pandemic have demonstrated that large-scale good manufacturing practice (GMP) production of mRNA is feasible and that mRNA-based vaccines are safe in humans [1,2].Behind the remarkable success of mRNA COVID-19 vaccines are painstaking efforts of researchers to develop mRNA therapeutics for different purposes, such as treating cancers.Scientists at Pfizer-BioNTech and Moderna developed COVID-19 vaccines building upon their experience in developing mRNA cancer vaccines.
Simultaneous advances in mRNA production and modification have enabled mRNA as a viable therapeutic product.The accepted method of mRNA-based vaccine production includes in vitro transcription (IVT) followed by 5 0 capping and polyadenylation (poly (A)) at the 3 0 end, which mimics the natural maturation process of mRNA in the cytoplasm of eukaryotic cells.Although the process of IVT is a relatively straightforward, production of high-quality therapeutic mRNAs that do not induce severe inflammation remains a key challenge.Recently, issues regarding inflammation and innate immunity have been mostly addressed by advancements in capping and tailing techniques, optimization of coding sequences, and incorporation of modified nucleosides such as pseudouridine (w), N6-methyladenine (m6A), 2-thiouracil (s2U), 5-methylcytosine (m5C), and 5-methyluracil (m5U) [3][4][5].These techniques may help improve the translation of mRNAs by reducing the immune-related signals of exogenous mRNAs.
Along with the promising results of mRNA vaccines against infectious diseases, research on mRNA vaccines for cancer treatment is gaining immense attention.Numerous clinical trials are currently underway to evaluate therapeutic mRNA vaccines for patients with different types of cancers (e.g.melanoma, glioblastoma, pancreatic, breast, prostate, colorectal, and lung cancer) [4,[6][7][8].Some vaccines are being examined in combination with other immunotherapies (e.g.pembrolizumab, cemiplimab, durvalumab, and atezolizumab) to synergistically enhance the anticancer immune response, yielding promising early results [9].Other applications of mRNA in cancer immunotherapy include engineering T-and natural killer (NK) cells to present antigen receptors.For example, chimeric antigen receptor T (CAR-T) cells are produced using IVT mRNA to possess controllable cytotoxicity, and their therapeutic potential and safety have been demonstrated in several preclinical and clinical studies [10][11][12][13].
This review mainly focuses on the recent progress and advances in research on mRNA therapeutics for cancer immunotherapy and discusses the hurdles to overcome.In addition, we address the potential of nanoparticle (NP)-based platforms to maximize the therapeutic effectiveness of mRNA-based drugs by improving their targeted delivery to specific tissues.This review provides the insights that can contribute to expanding the utilization of stateof-the-art mRNA-based cancer immunotherapy.

Messenger RNA (mRNA) cancer vaccines
Cancer vaccines are promising modalities for cancer immunotherapy.With prophylactic and therapeutic potential, can-cer vaccines stimulate and boost preexisting immune responses to tumor antigens.Cancer vaccines induce an immune response against tumor-associated or tumor-specific antigens (TAAs or TSAs) and consequently increase tumor cell recognition and clearance, leading to long-lasting therapeutic responses based on the established immunological memory.Selection of an appropriate target antigen is one of the most important factors in cancer vaccine design; tumor-specific neoantigens are favorable targets because they are recognized as foreign materials, thereby minimizing the induction of central and peripheral immune tolerance [14,15].Cancer vaccination can be implemented through multiple platforms such as tumor cells, peptides, viral vectors, dendritic cells (DCs), and nucleic acids (DNA or RNA) [16].
Recently, mRNA-based cancer vaccines have shown many advantages in cancer immunotherapy over or shared with other vaccine platforms because 1) nuclear localization is not required for antigen translation, unlike DNA-based vaccines; 2) multiple antigens can be delivered simultaneously with high potency; 3) mRNA-based cancer vaccines can induce simultaneous crosspresentation of multiple epitopes in antigen-presenting cells (APCs) with class I and II patient-specific human leukocyte antigen (HLA), thereby minimizing the restriction by the HLA type while enhancing the T cell response [17]; 4) mRNA is translated into functional antigens regardless of cell cycle status; and 5) rapid and scalable production are favorable for industrialization.Owing to these intrinsic advantages, mRNA cancer vaccines are now in the spotlight as a promising modality for cancer treatment.
Although progress has been made in the field of mRNA-based therapeutics, challenges to be addressed still exist on the development of mRNA-based cancer vaccines to expect FDA approval.The identification of tumor-specific mutated or non-conforming sequences and the prediction of corresponding neoepitopes for individual HLA alleles pose significant challenges, particularly in the context of personalized mRNA cancer vaccines [18,19].Moreover, lipid nanoparticles (LNPs), a prominent delivery system for mRNAs encoding tumor antigens, can induce a pronounced inflammatory response, as shown in the case of COVID-19 mRNA-LNP vaccines [20][21][22].There is also a need for further studies to validate the optimal route of administration, as both the distribution of mRNA and vaccine efficacy can be influenced by the chosen administration route [9].

The mechanistic basis of mRNA cancer vaccines
After administration, mRNA-based cancer vaccines are taken up by nearby cells at the injection site, especially DCs, which are the most efficient APCs that act as surveillance agents of the immune system [23,24].Internalized mRNAs are then translated by cytoplasmic ribosomes, and the resulting antigenic proteins are subsequently subjected to post-translational modifications, including ubiquitination.Thereafter, antigen fragments are generated by degradation with the proteasome complex and then directed into two pathways for antigen presentation via the major histocompatibility complex (MHC) class Ⅰ.Some of the resultant peptides are transported to the rough endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP) protein and eventually mounted on the MHC class I to be presented on the cell surface [25].After recognizing the displayed antigens, activated CD8 + T cells secrete molecules such as perforin and granzyme to induce apoptosis in cancer cells [26,27].
Anti-cancer immune responses can be further boosted by the interaction between mRNA featuring pathogen-associated molecular patterns (PAMPs) and intracellular pattern recognition receptors (PRRs) [28].For instance, exogenously introduced mRNA is recognized by endosomal Toll-like receptors (TLRs) such as TLR7 and TLR8 during endocytosis.Subsequently, TLRs recruit Toll/IL-1 receptor domain-containing adaptor proteins, such as MyD88 and TRIF, to initiate signaling pathways that lead to the activation of NF-jB, IRFs, or MAP kinases.These pathways trigger the release of pro-inflammatory cytokines and type I interferon (IFN), leading to the activation of robust adaptive immune responses.This activation includes enhancing T cell immunity against tumors by promoting antigen presentation and releasing co-stimulatory molecules [29,30].However, excessive immune stimulation by type I IFN inhibits mRNA translation and degrades mRNA molecules.This can be further exacerbated by double-stranded RNA (dsRNA), a byproduct generated during the production of mRNA through IVT.Studies reported that multiple intracellular PRRs are activated by dsRNAs, including endosomal TLR3, cytoplasmic melanoma differentiation-associated-5 (MDA-5), retinoic acid inducible gene I (RIG-1), oligoadenylate synthetase (OAS), and RNAdependent protein kinase (PKR).TLR3 recognizes 40-45 bp-sized dsRNA, further activating type I IFN [31].MDA-5 and RIG-1 bind to dsRNA with a size of approximately 2 kb or more and 5 0triphosphate short dsRNA respectively, leading to induction of additional genes inhibiting mRNA translation [32][33][34][35].
Researchers have investigated various methods to alleviate the excessive immune response activated by mRNA, while maintaining its intracellular activity as a vaccine.In addition to incorporating modified nucleosides, researchers have also increased the purity of IVT mRNA to prevent immune stimulation by dsRNA [36].High-purity IVT mRNA can be obtained by removing the dsRNA using HPLC or cellulose purification techniques [37,38].
2.2.Non-replicating mRNA and self-amplifying mRNA mRNAs as cancer vaccines are generally classified into two types: nonreplicating mRNA (also called conventional mRNA) and self-amplifying mRNA (saRNAs).Both mRNAs are manufactured to meet a specific purpose through IVT and are mainly synthesized by T3, T7, or SP6 bacteriophage RNA polymerases using linearized DNA encoding the antigen as a template [39].The conventional mRNAs, structurally composed of 5 0 -m7G cap, 5 0 and 3 0 UTR, 3 0 Poly(A), and a coding region, are known to be approximately 1,000-5,000 nucleotides in length [40].Similarly, saRNAs also contain the basic elements mentioned above, but additionally have considerably larger (9-10 kb) sequences that encode engineered replicons derived from alphaviruses, such as the Sindbis or semliki-forest virus [41,42].While the protein expression of nonreplicating mRNA depends on the number of transcripts that are successfully delivered, saRNA produces a large amount of antigen even at low doses because of its self-replicating characteristics [43].
Although the clinical applications of saRNAs in preventing infectious diseases are promising, their utilization in cancer vaccines remains mostly limited to preclinical studies.Currently, only two clinical trials (NCT00529984 and NCT01890213) using virusbased particle (VRP)-delivered carcinoembryonic antigen (CEA) for colorectal cancers have been completed [44].In this clinical results, the five-year survival rates for patients with stage IV and stage III cancer were 17% and 75%, respectively.Additionally, all patients showed CEA-specific humoral immunity and increased CEA-specific, IFNc-producing CD8 + granzyme B + TCM cells.These results suggest that VRP-CEA induces antigen-specific effector T cells while decreasing Tregs, indicating a favorable immune modulation.
One representative consideration that limits saRNA application is its inherent dsRNA structure, which causes an excessive immune response and consequently restricts its function as a vaccine, and thus a small number of preclinical and clinical studies have been performed [39,41].

Clinical development of mRNA cancer vaccines
Numerous clinical trials on mRNA-based cancer vaccines have been conducted for multiple types of cancers.The primary types of mRNA-mediated cancer vaccinations evaluated in clinical trials include ex vivo mRNA-based DC vaccines and in vivo mRNA cancer vaccines.Recent clinical studies have utilized therapeutic antibodies (such as anti-PD1 or anti-PD-L1 antibodies), or chemotherapy (such as temozolomide), for combination treatment of cancer.This strategy in cancer immunotherapy has demonstrated superior antitumor effects and high response rates [45].It is well known that PD-1/PD-L1 blockade are responsible for rescuing T-cells from their exhausted state and reviving the immune response against cancer cells [46].Ongoing clinical trials of mRNA cancer initiated within the last five years are summarized in Table 1.

Ex vivo mRNA-based DC vaccines
The mRNA-loaded DC vaccine is the first clinically proven type in which mRNA can function as a cancer vaccine.Despite the limited T-cell responses and suboptimal clinical effectiveness observed in mRNA-based DC vaccines [47], several studies have indicated their potential in mitigating or postponing disease recurrence, thereby potentially extending overall survival [48,49].This strategy ensures direct delivery of mRNA vaccine into harvested DCs but requires complex and expensive manufacturing processes, as follows: 1) DC extraction from patients' peripheral blood [50].2) Differentiation and maturation of DCs through treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), and delivery of antigen-coding mRNAs into DCs using electroporation [51].3) Reinfusion of an mRNA-based DC cancer vaccine into the patient via subcutaneous, intranodal, or intravenous injection.
In phase I and I/II clinicals, an mRNA-based DC vaccine derived from autologous tumors demonstrated safety in various cancer patients, including those with pediatric and adult brain cancers, melanoma, pediatric neuroblastoma, androgen-resistant prostate cancers, and renal cell carcinoma [52].Moreover, many complete or ongoing human studies have shown positive clinical effects.For example, a clinical study (NCT02808364) of DCs treated with personalized TAAs mRNAs, in combination with an anti-PD-1 antibody (nivolumab), in glioblastoma multiforme (GBM) or advanced lung cancer demonstrated TAA-specific T cell response and favorable overall survival among the treated patients without serious adverse effects [53].A phase II clinical study (NCT00965224) treated acute myeloid leukemia patients in remission with Wilms' tumor 1 (WT1) mRNA-based DC vaccines, and 43% of the vaccinated patients exhibited relapse prevention or delay, as well as favorable five-year overall survival rates [41,54].In a phase III clinical trial, patients diagnosed with metastatic renal cell carcinoma received DCs loaded with amplified tumor RNA and CD40Lencoded mRNA, in conjunction with sunitinib, a tyrosine kinase inhibitor.The immunologic rationale for the use of sunitinib is that it induces tumor regression through inhibition of VEGF activity and exerts a non-specific immunomodulatory effect consisting of the well-known reduction of Treg cells [55].However, the administration of this combination therapy did not result in a significant enhancement in patient survival [56].In a phase II study, researchers found that the administration of Trimix DC vaccines with TAA mRNAs in combination with ipilmumab, the anti-CTLA-4 antibody, resulted in higher numbers of peripheral blood CD62L high Tregs and exhibited 38% of partial response or a complete response [48].Three ongoing clinical trials are evaluating DC vaccines loaded with mRNAs encoding various antigens in glioblastoma patients as combination therapy with chemotherapy or anti-CD27 antibodies (NCT03548571, NCT04911621, NCT04911621).

In vivo mRNA cancer vaccines
In vivo mRNA cancer vaccines are another mRNA vaccine type actively being tested in clinical trials due to a few advantages over the mRNA-based DC vaccine, including its relative lower cost, convenient production process with less inconsistencies between batches, and easier scalability [57].This approach involves direct administration of mRNA vaccines, with or without being packaged in a delivery platform, into patients with various cancers, such as melanoma (NCT03394937), prostate cancer (NCT01817738), nonsmall-cell lung cancer (NCT00923312), and gastrointestinal cancer (NCT03480152).An ongoing clinical trial is evaluating the effectiveness of liposomal RNA (RNA-LPX) vaccine, FixVac (BNT111), which targets four non-mutated TAAs that are prevalent in melanoma.In this study, patients with advanced melanoma received intravenous administration of RNA-LPX either as a monotherapy or in combination with an anti-PD-1 antibody.Encouragingly, durable objective responses have been observed (NCT02410733) [58].Moreover, mRNA-2752, a mixture of three mRNA species encoding OX40L, IL-23 (an inflammatory regulator), and IL-36c (an inflammatory cytokine that acts as an alarmin), is currently being tested in a dose-escalation study in patients with solid tumors and lymphomas (NCT03739931), based on preclinical results in which intratumoral infusion of the formulated vaccine led to regression of existing tumors in three syngeneic tumor models.

Personalized mRNA cancer vaccines
Personalized mRNA cancer vaccines based on TSAs provide enhanced vaccination efficiency compared to TAA-based vaccines, as they are tailored to each cancer patient.Neoantigens, or TSAs, are generated from random somatic mutations that occur during the carcinogenesis of malignant cells and thus exhibit protein sequences that are not observed in normal cells [59].To produce this ''patient-tailored immunotherapy," an excisional biopsy is taken from the patient's tumor to acquire the genomic information of the tumor through next-generation sequencing.Thereafter, patient-specific somatic mutations are confirmed by comparison with matched healthy tissues and neoantigens are obtained using a prediction algorithm for MHC class I epitopes [60].Subsequently, through additional screening using an in vitro binding assay with neoantigen candidates, a final personalized vaccine is prepared and administered to the patients.Considering that there are several dozen neoantigens in certain cancer types, cancer vaccination with mRNAs encoding multiple neoantigens is an ideal strategy for cancer treatment as it can also present multiple epitopes without limitation of HLA type.For example, Kreiter et al. revealed that computationally engineered mRNA cancer vaccines for several MHC class II neoepitopes completely eradicated tumors in preclinical studies [61].Moderna and Merck also developed LNP-formulated mRNA-4650 and mRNA-5671 KRAS personalized cancer vaccine products for patients with pancreatic carcinoma for its evaluation in combination with pembrolizumab [62].In addition, BioNTech has conducted several clinical trials using the LNP-formulated personalized cancer vaccine candidates, including BNT121 and BNT122.BNT121 was administered repeatedly in the inguinal lymph nodes of 13 patients who had metastatic melanoma (NCT02035956) [63].The findings of the study were promising, as there were strong immunological responses observed and indications of clinical effectiveness.In preliminary results, BNT122 (RO7198457), which includes up to 20 personalized patient neoepitopes, was intravenously administered alone or in combination with the anti-PD-L1 antibody atezolizumab and showed a satisfactory safety profile.The reported adverse events are mostly temporary and include infusion-related reactions and/ or cytokine-release syndrome, which may manifest as fever and chills.Currently, BNT122 is undergoing evaluation in clinical studies of colorectal (NCT04486378) and pancreatic cancer (NCT04161755) [64,65].

mRNA-based therapeutic protein production
Protein replacement therapy is used to substitute or replenish deficiencies in a specific protein that is absent or dysfunctional owing to genetic mutations in the affected patient.Alternatively, gene therapy has been actively explored to mediate the production of functional proteins in patients, leading to the recent FDA approval of hemophilia gene therapy (Hemgenix) [66].In cancer treatment, mRNA can be used to introduce immunotherapeutic or immunomodulatory molecules, such as anti-cancer antibodies and immunostimulatory cytokines.When using mRNA to produce immunomodulators in vivo, high protein production rates are required to reach effective therapeutic levels, suggesting the possibility of repeated dosing [67].Furthermore, the systemic administration of mRNA-LNP complexes, the widely adopted mRNA delivery strategy, results in their significant unintended accumulation in the liver [68].Therefore, achieving optimal therapeutic efficacy necessitates the development of an efficient delivery approach targeting specific tissues.Of note, extensive investigations are underway to determine the ideal lipid ratios and composition alterations within LNPs [69,70].Although mRNA-based protein production approaches pose several challenges including the delivery to target sites and high production efficiency, they are being actively investigated in several clinical trials for various cancers such as melanoma, lymphoma, breast cancer, and ovarian cancer.Considering the heterogeneous and evolving nature of various cancers, this generally applicable strategy regardless of specific cancer type has been of great interest to further promote an anti-cancer immune response through monotherapy or in combination with other immunotherapies.

mRNA-encoded antigen receptors
Immunotherapy using TSA receptors is a promising strategy for the treatment of malignancies.This approach involves genetically engineering T cells harvested from cancer patients to display either T cell receptors (TCRs) or CARs on their surfaces, allowing them to recognize and kill tumor cells [71].TCRs recognize epitopes from tumor antigens present on the MHC, whereas CARs recognize tumor surface antigens in an MHC-independent manner.This personalized cell therapy type can be effectively manufactured by the electroporation of mRNAs encoding antigen receptors, which is a practical way to achieve significantly high transfection efficiency [72][73][74].
To produce mRNA CAR-T cells, T cells are extracted from patients and genetically engineered to express CARs via mRNA electroporation.Engineered CAR-T cells are then expanded in large quantities and stored cryogenically for future treatment.Patients receive a lymphodepleting chemotherapy regimen to remove CAR-lacking T-cells, followed by adoptive cell transfer (ACT) of the engineered cell products.Several factors, such as the molecular structure of CAR (which includes costimulatory domains), influence the efficacy of mRNA-based CAR-T cells, affecting their expression and duration [75,76].Higher CAR expression correlates with increased potency in anti-cancer effects, and in vitro experiments have demonstrated that high levels of CAR expression can induce activation-induced cell death (AICD) [77].Additionally, optimization of structural elements, such as the 5 0 cap, UTRs, and poly(A) tail, along with dsRNA removal and modified nucleosides, can improve CAR expression levels and duration [78,79].
Several studies have explored the potential of mRNAtransfected CAR-T cells in cancer therapy.Rabinovich et al. reprogramed lymphocytes with CD19-targeting CAR mRNA transfection to target and kill both hematological and solid tumor cells.One study showed that mRNA-modified human T cells efficiently inhibit lymphoma growth in NOD/LtSz-Prkdc SCID mice [13].Another study demonstrated that mRNA-transfected CAR-T cells specific for the cell surface molecule c-Met was well-tolerated and resulted in extensive tumor necrosis in patients with metastatic breast cancer [80].Additionally, Descartes-08 is an engineered T-cell product engineered by transfecting CD8 + T cells with anti-BCMA CAR mRNA and has been confirmed for effective killing of primary myeloma and MM cell lines.Preliminary clinical studies of Descartes-11 suggested its possibility of achieving an efficient, durable response without significant CAR-T cell-related toxicity, and thus a phase II study of was initiated for frontline myeloma (NCT03448978) [81].
A revolutionary T-cell therapeutic modality of current interest and exploration is a mRNA-based in vivo T-cell reprogramming strategy which involves delivery of mRNA encoding antigen receptors to T cells in vivo [82,83].This approach eliminates the need for ex vivo manipulation of T cells, thus enabling the co-delivery of additional RNAs for the simultaneous manipulation of lymphocytes.

mRNA-encoded antibodies
Monoclonal antibodies (mAbs) are promising anti-cancer agents that target and eliminate cancer cells by enhancing the host immune system against tumors [84].The successful engineering of IgG mAbs has facilitated the development of various antibody variants, such as antibody fragments (e.g., Fab and single-chain variable fragments (scFv)), bispecific antibodies, and antibody derivatives [85].To date, more than 100 mAbs have been approved by the FDA for various diseases, including cancers, and are accepted as a primary therapeutic class in the field of cancer immunotherapy [86].However, antibody manufacturing is hampered by high production costs, purification issues, the need for post-translational modifications, and aggregation during longterm storage.In addition, antibody fragments have a short plasma half-life and require repeated administration, resulting in high treatment costs [87,88].Alternatively, researchers have found that fully bioactive mAbs can be produced in vivo by delivering mRNA.Unlike proteins that require complicated optimization of the manufacturing process and storage buffers depending on their amino acid sequences, mRNAs are composed of simple sugar-phosphate backbones and are relatively easy to manage [89,90].Additionally, the expression of mRNA encoding mAbs in vivo can be observed within 2 h after the administration and expression can last for hours or days [91][92][93].
A representative example of an mRNA-encoded mAb is rituximab, a clinically approved IgG 1 that targets CD20 for treating B-cell lymphomas.Thran et al. constructed plasmids encoding the heavy (H) and light (L) chain sequences of rituximab to generate respective unmodified mRNA and subsequently determined the optimal H-to-L chain mRNA molar ratio to be 1.5:1 for the full antibody production.Repeated intravenous administration of mRNAencoded rituximab formulated in LNP to a non-Hodgkin's lymphoma xenograft mouse model resulted in significant slowing or even inhibition of tumor cell growth [94].
Another promising approach is mRNA-based expression of a bispecific T-cell engager (BiTE) that forms a bridge between tumor cells and T cells and induces target-dependent T cell activation.The BiTEs are encoded in 1-methylpseudouridine-containing mRNA (called RiboMABs), which simultaneously target CD3 and one of the three TAAs: claudin 6 (CLDN6), claudin 18.2 (CLDN18.2),or epithelial cell-attached molecules (EpCAM).RiboMAB was shown to induce effective T-cell activation and target cancer cell lysis at low concentrations to a similar extent as its corresponding recombinant protein.In a xenograft mouse model of human ovarian tumors (300 mm 3 in size at the initiation of the treatment), injection of 3 lg (6 pmol) of CD3 Â CLDN6 RiboMAB mRNA once a week for a total of 3 weeks resulted in complete tumor elimination [95].
Lastly but not least, mAbs that regulate immune checkpoints have been encoded in mRNA to enhance anti-tumor T cell activity.Pruitt et al. transfected DCs with mRNAs encoding cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoidinduced TNFR-related protein (GITR) targeting mAbs.The administration of these transfected DCs in a murine melanoma model stimulated anti-tumor immunity and improved survival without signs of autoimmunity These results were further evaluated in a phase I clinical trial among patients with metastatic melanoma (NCT01216436) [96].Recently, the co-delivery of mRNAs that encode agonists for T cells (CD137 or OX40) has been shown to improve anti-tumor immunity compared to anti-OX40 antibody alone in various tumor models [97].In conclusion, mRNAencoded antibodies are emerging as an alternative therapeutic option for recombinant proteins, and will advance further as effective mRNA modifications and administration routes are optimized.

mRNA-encoded immunomodulatory proteins
Multiple immunomodulatory proteins such as cytokines, TLRs, chemokines, and co-stimulatory ligands reprogram the tumor immune microenvironment and allow effective immune responses against tumors [98].However, the systemic delivery of immunomodulators has some limitations in clinical practice because of their short half-lives and dose-related toxicities.mRNAs within delivery carriers are ideal treatments for overcoming these obstacles; various cytokines and co-stimulatory ligands have been combined with mRNAs, and their efficacy has been evaluated through clinical trials.

Cytokines
IL-12 mediates T H 1 immunity and promotes effective anticancer activity in mouse tumor models; however, it is lethally toxic when administered systemically to humans [99].Therefore, MEDI1191, an IL-12 mRNA agent, was administered intratumorally together with durvalumab (an anti-PD-L1 antibody) to patients with advanced solid tumors (NCT03946800).Data from this clinical study showed that administration of MEDI1191 with durvalumab was well tolerated and resulted in a transient increase of IL-12 in serum levels, as well as demonstrated tumor shrinkage [100].In another study, Hotz et al. investigated the anti-tumor effect of intratumoral administration of mRNAs encoding four cytokines for tumor regression (IL-12 single chain, IFN-a, GM-CSF, and IL-15).Delivery of a saline solution of the mRNA mixture resulted in intratumoral IFN-c induction, antigen-specific T cell expansion and infiltration, and development of immunological memory in multiple tumor models.Consequently, a clinical trial of the mRNA mixture encoding cytokines in patients with advanced solid tumors was initiated (NCT03871348) [101].

Co-stimulatory ligands
OX40L, which binds to OX40 to promote CD4 + and CD8 + T cell expansion and memory T cell development, and inhibits Treg cell function, was developed as a therapeutic mRNA (mRNA-2416) and entered its first clinical study for patients with solid tumors (NCT03323398).In this clinical trial, intratumoral administration of mRNA-2416 alone or in combination with durvalumab increased OX40L protein expression and elevated pro-inflammatory activity [102].Similarly, mRNA-2752 encoding OX40L, IL-23, and IL-36c pro-inflammatory cytokines was investigated in a first-in-human study of 23 solid tumor patients as monotherapy or in combination with durvalumab (NCT03739931).The administration of mRNA-2752 alone and in combination with durvalumab was well tolerated at all dose levels tested, and treatment was associated with tumor shrinkage [103].TriMix is a cocktail of mRNAs encoding antigens or immunostimulatory proteins such as CD40L, CD70, and TLR4.Injection of TriMix mRNA into tumor-bearing mice led to the maturation of tumor-associated CD8a + DCs and activation of T cells, resulting in delayed tumor growth [104].Clinical trials are currently underway to validate the safety and immunomodulatory effects of intratumoral TriMix injections in patients with early stage breast cancer (NCT03788083).

Delivery systems forthe mRNA cancer therapeutics
Despite the advantages of using mRNA in cancer therapeutics, challenging biological barriers encountered following systemic administration remain major obstacles to its clinical application.mRNA is chemically unstable because of the 2'hydroxyl group of the ribose sugar and is prone to hydrolysis by enzymatic degradation in the human body [105].mRNA that has managed to reach target cells cannot readily cross the anionic cellular membranes due to its high-density negative charge and large molecular weight [106].Moreover, endocytosed mRNA-based drugs are incapable of escaping from the endosome into the cytosol [107].Collectively, naked mRNA, regardless of stability-enhancing chemical modifications, can only induce weak transient protein expression at best in vivo [24].Recently, various vehicles, such as LNPs, polymerbased NPs, peptide-based NPs, have been developed to address these issues, thereby improving mRNA stability, delivery, and expression [107].In particular, mRNA delivery via NPs is widely applied in cancer immunotherapy, including the production of monoclonal antibodies, vaccines, and CAR T-cell therapeutics, and some of them are being evaluated in clinical trials [108].In this section, we discuss the latest research in the development of mRNA delivery platforms for cancer treatment (Fig. 1).Representative delivery systems and their components, mRNA payloads, and therapeutic effects on specific diseases are overviewed (Table 2).

Lipid nanoparticles (LNPs)
Clinical validation of the SARS-CoV-2 mRNA-LNP vaccines from Pfizer/BioNTech spurred clinical investigation of LNPs as an mRNA drug delivery platform for treating various diseases, including cancer.A typical LNP is composed of four components: 1) ionizable or cationic lipids, 2) helper phospholipids, 3) cholesterol, and 4) polyethylene glycol (PEG)-lipids.Cationic or ionizable lipids electrostatically interact with and compact negatively charged mRNAs to form and stabilize NPs to protect the payloads from the site of administration to the intracellular compartments [109].While per-manently cationic lipids exhibit less efficient transfection and more toxic pro-inflammatory responses [110], the currently used ionizable lipids have low toxicity and immunogenicity because they are protonated at acidic pH and neutralized at physiological pH [106].Thus, ionizable lipids exhibit positive charges at acidic endosomal pH after endocytic uptake to mediate fusion with the anionic endosomal membrane, thereby facilitating endosomal escape and cytosolic release of mRNA [111].Phospholipids and cholesterol maintain the structural integrity, while PEG-lipids provide particle colloidal stability and prolong blood circulation time by minimizing serum protein adsorption and opsonization [112,113].Owing to these characteristics, LNPs are widely explored for mRNA cancer therapeutics, and many LNP studies have been conducted in combination with mRNA-based cancer therapeutics.As a representative case, the efficacy of the LNP-mRNA personalized cancer vaccine mRNA-4157 was recently evaluated in a phase I clinical study (NCT03897881).This vaccine encodes up to 34 neoantigens that induce antigen-specific T cell and anti-tumor responses.It was administered alone or in combination with a checkpoint inhibitor (pembrolizumab) to patients with resected solid tumors or advanced solid tumors, respectively.From 16 patients who received monotherapy, 14 remained disease-free, and the response rate of the combination therapy was 50% median progression-free survival (mPFS) of 9.8 months in 10 CPI-naïve HPV-neg HNSCC patients [114].
Similar to other synthetic NPs, the practical aspect of LNPs is the ability to co-deliver mRNAs and other types of nucleic acids (e.g., small interfering RNA (siRNA), microRNA (miRNA), and single guide RNA (sgRNA)) or a mixture of mRNAs encoding multiple proteins.For example, researchers have shown that the combination of both si-PD-L1 and OX40L mRNA in a single LNP formulation can efficiently knockdown and express immune checkpoint targets.To this end, intratumoral treatment with LNPs resulted in reduced tumor growth, enhanced intratumoral infiltration of CD4 + and CD8 + T cells, and immune activation in the tumordraining lymph nodes [115].In another study, delivery of mRNAs encoding multiple tumor antigens and adjuvants (STING V155M ) via LNPs showed antigen-specific CD8 + T-cell responses by activating NF-jB, IRF3, and IRF7.Optimization of the antigen/STING V155M mass ratio (5:1) maximized CD8 + T cell responses and increased the efficacy of cancer vaccines in mouse subcutaneous tumor models and lung metastasis models [116].
Fig. 1.Delivery systems for mRNA cancer therapeutics.(A) There are three main categories of NPs that are employed for mRNA delivery in cancer therapy: LNPs, polymeric NPs, and peptide-based NPs.LNPs typically contain cationic ionizable lipid, cholesterol, helper phospholipid, and PEG-lipid, and they are capable of co-delivering mRNAs with other forms of nucleic acids.Polymeric NPs and peptide-based NPs (CPPs), which consist of cationic amine groups or amino acids, respectively, can bind with anionic mRNA through electrostatic interaction to form nanosized particles.(B) Passive targeting and active targeting are two approaches for drug delivery to specific cells.Passive targeting uses natural transport systems, whereas active targeting involves the addition of targeting moieties to achieve on-target delivery.The composition, size, charge, and hydrophobicity of delivery vehicles can impact the binding of proteins to nanoparticles in the bloodstream, which affects their trafficking and uptake in passive targeting.Active targeting uses ligands such as antibodies and aptamers to selectively target specific cells, which can provide precise control of drug distribution in organs or cancer.
Recently, attempts have been made to increase LNP delivery efficiency by tuning the structure of ionizable lipids given their critical roles on governing NP formation, cellular uptake, and endosome escape [117].Novel ionizable lipids can be designed through the number and structure of tails, the presence of biodegradable and unsaturated bonds, and their hybrid delivery with polymers or dendrimers [109].Liu et al. synthesized new ionizable lipids containing diamino groups with various head groups.This diamino lipid derivative (DAL4) was used with cholesterol, phospholipids, and PEG-lipids to encapsulate mRNAs and to form LNPs. DAL4-LNP showed superior mRNA delivery efficiency compared to conventional ionizable lipid MC3-LNP in vitro and also induced efficient protein expression in tumors and B cells upon intratumoral injection.In addition, in vivo delivery of DAL4-LNP-mediated IL-12 and IL-27 mRNA induced robust anti-tumor immune responses and sustained tumor suppression even after the discontinuation of the treatment [118].Of note, despite numerous benefits of LNPs as a mRNA delivery platform, targeted delivery to specific tissues or cells in vivo remains challenging.To address this limitation, studies on LNP composition and appropriate administration routes are being actively conducted, and the contents are discussed in Section 4.4.

Polymeric nanoparticles (Polymeric NPs)
Leveraging on their endless synthetic tunability, versatile structure, and stability, polymeric NPs have attracted significant interest as a nucleic acid delivery platform [119].Polymers are made of simple structural repeating units adjoined via covalent bonds.The formation of nucleic acid-loaded NPs is influenced by the number of charged groups, degree of branching, and other formulation factors, such as polymer and nucleic acid concentrations and molar ratio [120].Commonly used polymers are positively charged to electrostatically condense negatively charged mRNA and the resulting cationic NPs promote association with anionic cell membranes and subsequent transfection in vitro [121].
One of the polymeric NPs used in mRNA cancer immunotherapy is an injectable GO-LPEI hydrogel (GLP Gel) made of graphene oxide (GO) and cationic low molecular weight polyethylenimine (LPEI).A transformable GLP hydrogel was able to encapsulate TLR7/8 agonist resiquimod (R848) and ovalbumin mRNA (mOVA) via p-p stacking and electrostatic interaction (GLP-RO Gel).Once injected into the subcutaneous layers, GLP-RO NPs were released from the gel and delivered to the lymph nodes.Consequently, APCs that take up GLP-RO NPs induce immune activation and prevent metastasis by expressing OVA antigens [122].A second example is charge-altering releasable transporters (CARTs) based on biodegradable poly(-carbonate)-b-(a-amino ester)s, which comprise amphiphilic molecules and lipid-like head and tail structures.In this work, the cationic amines of a CART were shown to rearrange into neutral amides after entering the cell, thereby promoting mRNA release and stable protein expression.Local administration of CARTs carrying mRNA encoding immunemodulators induced a systemic anti-cancer immune response to treat both local and distal tumors [123].Recently, polymers have been hybridized with lipids to enhance the performance as a mRNA delivery paltform.Shi et al. developed an mRNA delivery NP based on a methoxy poly(ethylene glycol)-poly(lactic-co-glycolic acid) (mPEG-PLGA) copolymer and cationic lipid-like material G0-C14 where mPEG-PLGA polymers are self-assembled to form NPs while G0-C14 lipids condense mRNA.This hybrid platform was used to form NPs carrying PTEN mRNA and was shown to induce antitumor immune responses by triggering autophagy and DAMP release and improved therapeutic efficacy when combined with anti-PD-1 antibody in Pten-mutated tumors [124].

Peptide-based nanoparticles (Peptide-based NPs)
Peptides have long been explored as a nucleic acid delivery platform due to the relatively simple synthesis process as well as their proteolytic nature that often imparts efficient transfection and favorable biocompatibility [125].In particular, cell-penetrating peptides (CPPs) composed of 4-40 amino acids have emerged as promising tools for mRNA delivery.With their cationic sequences, CPPs can electrostatically interact with anionic nucleic acids to form nanosized particles and promote the cellular uptake of the encapsulated cargos [126].Moreover, various functions such as endosomal escape can be introduced through countless combinations of amino acid compositions and sequences [127].In this regard, CPPs have recently been explored for mRNA-based cancer therapy.
A representative example is RNActive (CureVac AG), a twocomponent mRNA vaccine platform which involves naked mRNA to express the antigen and mRNA complexed with polycationic protamine as an adjuvant to activate TLR7 and TLR8 signaling [128][129][130].This type of cancer vaccine has demonstrated to induce sufficient immune responses in several preclinical animal studies [131].PepFect14 (PF14) is 21 amino-acid cationic amphipathic CPP with 5 amino acids being positively charged and stearylated at the N-terminus.When PF14/mRNA NPs were intraperitoneally injected into a xenograft model of primary ovarian cancer, reporter protein expression was detected in tumor cells, immune cells, and fibroblasts in tumor tissues, outperforming a commercial lipidbased transfection agent [132].A recent study compared the cancer cell transfection efficiency of three cationic and five amphipathic CPPs that formed NPs via electrostatic interactions with mRNA.All CPPs formed NPs with the mRNAs and protected them from endonuclease-mediated degradation.However, only amphipathic CPPs smaller than 200 nm induced cellular uptake and protein expression in CT26.CL25 cancer cells, suggesting that the conformational state and physicochemical properties of amphipathic CPPs play important roles in complex formation with mRNA [133].These studies provide insights into rational approaches for the development of peptide-based mRNA delivery systems.

Progress and challenges in targeted delivery
Although several mRNA therapeutics have been commercialized beyond clinical trials, achieving targeted mRNA delivery to specific organs remains elusive.Most delivery systems predominantly home to the liver upon intravenous administration due to the low hepatic blood flow rate as well as the highly vascular and leaky nature of liver endothelium [134].Moreover, serum apolipoprotein E (ApoE) binds to the surface of circulating LNPs and induces receptor-mediated uptake by hepatocytes [135], which can cause immune-mediated hepatitis or liver toxicity due to undesired protein expression [136].Two major strategies have been developed to achieve extrahepatic organ-targeted delivery.
Passive targeting is based on modifying the physicochemical properties, such as the structure, molar composition, and charge of the components used in NPs.One of the best-known examples, named selective organ targeting (SORT), is characterized by the addition of a fifth SORT lipid to a conventional LNP to alter the internal charge and promote tissue-specific targeting [69].Chen et al. synthesized 113-O12B, a lymph node-targeting lipid for cancer treatment, [136], and its relatively short tails ( 12 carbons),an ester bond linker, and methyl-free head amine increased mRNA expression in the lymph nodes.Compared to ALC-0315, a synthetic lipid in FDA-approved vaccines, 113-O12B showed higher expression in the lymph nodes and reduced mRNA expression in the liver after subcutaneous injection.Moreover, 113-O12B LNPs carrying full-length proteins or short peptide-encoding mRNAs exhibited a CD8 + T-cell response and excellent tumor inhibition in vivo [136].In another study, LNP uptake switched from the liver to the lungs when the ester bond of the lipid tail linker was replaced with an amide bond [137].They found that the tail structure altered the serum proteins adsorbed onto the particle surface, thereby affecting organ-selective delivery.With this lung-targeted LNP, they not only achieved genome editing in the lungs by co-delivering Cas9 mRNA and sgRNA but also inhibited tumor cell growth in a mouse lymphangioleiomyomatosis model by delivering Tsc2 mRNA [137].LNPs targeting tumor-specific CD8 + T cells can also be optimized by tuning the lipid ratio and PEG-lipid chemistry, such as the acyl chain length and PEG molecular weight [138].An optimal LNP composition, low percentages of PEG lipids and DOPE, and high percentages of SS-EC ionizable lipids, enhanced mRNA uptake by splenic immune cells and elicited a robust CD8 + T cell response and anti-tumor efficacy in a mouse TC-1 tumor model upon intravenous administration [138].
Active targeting involves decorating the particle surface with targeting moieties, such as antibodies and peptides.By specifically interacting with surface receptors overexpressed on the target cells, the targeting moieties enable cell type-specific delivery.Kheirolomoom et al. developed an in situ-T cell targeted LNP by coating CD3 F(ab') 2 on the surface [139].They first synthesized LNPs containing 0.5 mol % DSPE-PEG5K-Maleimide and then conjugated to thiol-aCD3-targeting F(ab') 2 .These aCD3-LNPs encapsulating the reporter mRNA transfected approximately 97% of Jurkat T cells in vitro and accumulated in the spleen 30 min after systemic injection.In addition, aCD3-LNP injection resulted in the accumulation of transfected T cells within tumors and tumor-draining lymph nodes [139].Rosenblum et al. designed a novel amino-ionizable lipid, L8, and developed LNPs that efficiently encapsulated both Cas9 mRNA and sgRNA targeting PLK1, a kinase required for mitosis [140].Specifically, the surface of LNPs were engineered with the anchored secondary scFv-enabling targeting (ASSET) strategy where the lipid portion of the membraneanchored lipoproteins were incorporated into LNPs while the other side was bound to cell-targeting antibodies.The strategy was used to coat LNPs with epidermal growth factor receptor (EGFR) antibodies to target EGFR-expressing ovarian cancer cells.These EGFR-targeted LNPs achieved specific targeting and accumulation in tumors when intraperitoneally inoculated [140].In addition to LNPs, biodegradable poly(ß-amino ester) (PBAE) polymer has emerged as a polymer-based delivery platform for targeted mRNA-based cancer therapy.Specifically, PBAE was utilized to package mRNA and then the particles were coupled with a T celltargeting CD8 antibody-polyglutamic acid (PGA) by electrostatic absorption [141].In various orthotopic tumor xenograft mouse models, PBAE/PGA-anti-CD8 NPs formulated to carry CAR-or TCR mRNA reprogrammed circulating T cells and induced anti-tumor responses [82].

Conclusions and outlook
mRNA-based cancer immunotherapy has emerged as a promising approach for cancer treatment thanks to the advancement and convergence of mRNA and nanoparticle technologies.Recent clinical trials have demonstrated safety and efficacy of mRNA-based cancer immunotherapy, with some patients achieving therapeutically meaningful tumor remission.The strength of mRNA lies in its broad application window in various cancer immunotherapy modalities.mRNA can be used to encode virtually all players of the anti-cancer immune response, including cancer antigens, antigen receptors, therapeutic antibodies, and immunomodulatory cytokines, thereby enabling the development of highly sophisticated or personalized therapies for individual cancers.It is important to note that the effect of mRNA, due to its transient nature, is short-lived and easily controllable and thus can minimize the risk of off-target effects and long-term toxicity.Innovations of chemical modification endowed mRNA with improved stability and translation efficiency to enhance the production efficacy of encoded proteins.Unlike viral vectors, LNP lacks immunogenicity and thus can used for mRNA-based cancer immunotherapy in a repeated manner if needed for sustained therapeutic efficacy.The future direction of mRNA cancer immunotherapy is expected to be towards combination therapy of mRNA-based personalized therapeutic modalities, such as personalized vaccines, with immune checkpoint inhibitors.In light of the unsatisfactory clinical results associated with TAAs lacking tumor specificity, the imperative lies the employment of specific tumor antigens and the individualization of TSA-based mRNAs.Indeed, the recently updated study results from ongoing clinical trial by Moderna and Merck (NCT03897881) indicate that a personalized mRNA cancer vaccine, named mRNA-4157 (V940) containing up to 34 different neoantigens combined with the checkpoint inhibitor Keytruda (pembrolizumab), effectively diminished the risk of metastasis in individuals diagnosed with high-risk advanced melanoma.these results are along with the expectation of a larger phase III randomized clinical trial [142].
Collectively, preclinical and clinical studies on mRNA-based cancer immunotherapy have opened a new avenue for enhanced cancer patient care.However, there remain pending tasks to fully exploit this advanced therapeutic modality, which include optimizing the neoantigen-specific cancer vaccines, understanding the biology of immune escape in cancer and orchestrating and balancing the anti-cancer immune response.Advancements in the development of LNPs and other mRNA delivery platforms will only broaden and enhance the applicability of mRNA-based cancer immunotherapy.
Note that while mRNA-based cancer immunotherapy holds immense potential, it is still a relatively new and rapidly evolving field.Continued researches, clinical trials, and collaboration between scientists, clinicians, and industry partners are essential for advancing this innovative approach to cancer treatment.

Table 1
Summary of ongoing clinical trials for mRNA cancer vaccines.

Table 2
Overview of mRNA delivery systems for cancer treatment.