key: cord-0863194-zvvrctdn
authors: Wei, Jiao; Hui, Ai-Min
title: The Paradigm Shift in Treatment from Covid-19 to Oncology with mRNA Vaccines
date: 2022-05-05
journal: Cancer Treat Rev
DOI: 10.1016/j.ctrv.2022.102405
sha: 05f4315622062ef83dae8c5e563f5570d745b17c
doc_id: 863194
cord_uid: zvvrctdn

mRNA vaccines have gained popularity over the last decade as a versatile tool for developing novel therapeutics. The recent success of coronavirus disease (COVID-19) mRNA vaccine has unlocked the potential of mRNA technology as a powerful therapeutic platform. In this review, we apprise the literature on the various types of cancer vaccines, the novel platforms available for delivery of the vaccines, the recent progress in the RNA-based therapies and the evolving role of mRNA vaccines for various cancer indications, along with a future strategy to treat the patients. Literature reveals that despite multifaceted challenges in the development of mRNA vaccines, the promising and durable efficacy of the RNA in pre-clinical and clinical studies deserves consideration. The introduction of mRNA-transfected DC vaccine is an approach that has gained interest for cancer vaccine development due to its ability to circumvent the necessity of DC isolation, ex vivo cultivation and re-infusion. The selection of appropriate antigen of interest remains one of the major challenges for cancer vaccine development. The rapid development and large-scale production of mRNA platform has enabled for the development of both personalized vaccines (mRNA 4157, mRNA 4650 and RO7198457) and tetravalent vaccines (BNT111 and mRNA-5671). In addition, mRNA vaccines combined with checkpoint modulators and other novel medications that reverse immunosuppression show promise, however further research is needed to discover which combinations are most successful and the best dosing schedule for each component. Each delivery route (intradermal, subcutaneous, intra tumoral, intranodal, intranasal, intravenous) has its own set of challenges to overcome, and these challenges will decide the best delivery method. In other words, while developing a vaccine design, the underlying motivation should be a reasonable combination of delivery route and format. Exploring various administration routes and delivery route systems has boosted the development of mRNA vaccines.

Vaccinations play a vital role in reducing disease, disability, and mortality from a variety of infectious diseases [1] . The use of conventional vaccines such as live attenuated vaccines, inactivated pathogens, subunit vaccines or toxoid vaccines provides durable efficacy against various infectious diseases [2] . Nucleic acid vaccines mainly, plasmid DNA(pDNA) and messenger RNA (mRNA), came to existence in 1900s due to their innate ability to stimulate inoculation with live organism-based vaccines, notably for cell-mediated immune stimulation [3] .

For several decades later, pDNA-based approaches dominated the field, since mRNA-based approach was considered unstable due to inefficient in-vivo delivery and excessive stimulation of inflammatory responses [4, 5] . Eventually in late 2000s, a series of improvement in manufacture, modification and stabilization of mRNA led to its recognition as a resourceful platform for developing novel therapy [4, 5] . mRNA vaccines thus hold a lot of promise and confer several advantages over traditional vaccines.

The recent outbreak of SAR-CoV-2 and coronavirus disease has demonstrated an urgent need of rapid vaccine development. Two mRNA vaccines, BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna TX), have acquired authorization from FDA that are currently being used to prevent COVID-19 [6] . Both vaccines have good efficacy as demonstrated in the various phase III trials and real world studies [7] [8] [9] [10] [11] . Knowledge gained from these trials and versatile therapeutic potential of the mRNA can be applied for the development of vaccine for the infectious diseases and cancer. In this review, we focus on the therapeutic aspect of mRNA vaccines as a cancer therapy. In addition, we would apprise the literature on the various types of cancer vaccines, the novel platforms available for delivery of the vaccines, the recent progress in the RNA-based therapies and the evolving role of mRNA vaccines for various cancer indications, the available clinical and preclinical studies with the future chapter in treatment of patients.

Cancer vaccines are a promising new immunotherapeutic strategy for both prevention and treatment. Vaccines targeting tumor associated or tumor-specific antigens (TAAs or TSAs) can destroy malignant cells that overexpress the antigens due to immunologic memory, resulting in a durable therapeutic response. Compared to other immunotherapies, cancer vaccines provide a precise, safe, and acceptable treatment. Currently, 2 prophylactic vaccines have been approved by the U.S. Food and Drug Administration (FDA) for routine use in clinical practice. Gardasil-9 is approved for prevention of HPV infection that is the cause of most HPV cancers. The other one is hepatitis B (HBV) vaccine, for example HEPLISAV-B, to prevent HBV infection that is known to cause hepatocellular carcinoma [12, 13] .

In 2010, PROVENGE (sipuleucel-T), an immune-cell based therapeutic cancer vaccine was granted approval for the treatment of individuals with asymptomatic or mild symptomatic metastatic castration-resistant prostate cancer (mCRPC) [14] . Besides, therapeutic vaccines are available for the treatment of early-stage bladder cancer (TheraCys ® and TICE ® Bacillus Calmette-Guerin (BCG)) [15] and melanoma [IMLYGIC ® (talimogene laherparepvec/T-VEC)] [16] . Despite significant attempts to produce cancer vaccines, clinical translation of cancer vaccines into effective therapeutics has remained difficult for decades due to the wide range of tumor antigens and low immune response [17] , originating the need to develop more potent vaccine approaches.

Furthermore, there is a growing demand for vaccine development, large-scale manufacture, and dissemination, particularly in the case of non-viral diseases such as cancer [18, 19] .

In general, cancer vaccine platforms are classified into tumor cell, peptide, viral vector, dendritic cell (DC), DNA and RNA types (Fig. 1) . Allogenic or autologous patient-derived tumor cells are used to make cellular vaccines [20] . This approach is beneficial in that target antigens does not have to be determined in advance [21] . The whole cell cancer vaccine approach using granulocyte-macrophage colony-stimulating factor (GM-CSF) has been studied in several types of cancer both in animal as well as human trials. The phase 1 and 2 studies with allogeneic GM-CSF-transduced vaccine post-radiation (derived from two pancreatic tumor lines) demonstrated durable efficacy and prolonged survival in patients with pancreatic cancer [22, 23] . The non-replicating mRNA encodes antigen of interest, while self-amplifying mRNA encodes antigen of interest and a replication machinery, a self-replicating single-stranded RNA virus) Peptide vaccines are made up of amino acid sequences that contain an epitope which can cause an immune response. Due to the difficulties of small peptides to attach directly to major histocompatibility complexes (MHC) I molecules, long peptides (containing of between [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] amino acids) are frequently favored over short peptides (consisting of approximately 10 amino acids). Short peptides also fail to activate CD4 helper T cells, which are required for full cytotoxic T lymphocyte activation (CTLs). These shortcomings can be overcome by using a long-peptide vaccine, that forces dendritic cells (DCs) to phagocytose the long-peptide before it is exposed on MHC I and attached to T cells. Long peptide vaccines also increase the HLA-related compatibility that exist with short-peptide vaccine. Furthermore, using a long peptide vaccine permits APCs to be presented via MHC II, which stimulates CD4+ lymphocytes, allowing for a more efficient immune response against tumor cells. However, because peptides are not self-immunogenic, administering an adjuvant at the same time is required for producing maximum efficiency [24] . So far, the peptide-based vaccines tested in laboratory has been able to elicit limited tumor-targeting immune responses, mostly because of intrinsic changes in cancer cells that reduce antigenicity and/or changes immunosuppressive alterations in the tumor microenvironment [25] . Therefore, other approaches are being developed including its combination with other immunotherapies, targeting antigenic epitopes arising from tumor cells and identifying target population [25] .

Genetically modified viruses are also used for mRNA delivery. Application of positive strand RNA viruses via translation with host ribosomal machinery. However, challenges with host genome integration and the likelihood of host rejection, as well as cytotoxicity and immunogenicity, remains the major challenges. The MHC allows cancer cells to create peptide antigens that are present on their membrane surface. T cell receptors (TCRs) on cytotoxic T lymphocytes (CTLs) identify these antigens, resulting in cancer cell lysis. The antiviral immune response neutralizes viral vectors, limiting the number of vaccines that can be given [21] .

Finally, to boost the adaptive immune system against tumor antigens, DNA cancer vaccines are created from bacterial plasmids (naked DNA) expressing one or more tumor antigens. The capacity of DNA vaccines lies in its ability to combine many genes expressing numerous tumorantigens to establish a precise and broader adaptive immune response at the same time. However, these vaccines are poorly immunogenic [24] . To improve the immunological response of DNA vaccines, researchers have looked into encoding xenogeneic versions of antigens, fusing antigens with compounds that activate T cells or trigger associative recognition, DNA vector priming followed by viral vector boosting, and immunomodulatory molecules [26] . In contrast, RNA cancer vaccines are superior to DNA vaccines. While RNA is more susceptible to RNase breakdown, this can be minimized through chemical changes and the insertion of modified nucleosides such as pseudo uridine. Furthermore, unlike DNA, which must reach the nucleus to overcome the second barrier, the nuclear membrane, RNA just needs to enter the cytoplasm [21] . The encoded proteins are converted into peptides that are present on MHC I and II to excite CD8+ and CD4+ T cells, respectively, after RNA translation. The fundamental pharmacology of mRNA vaccines is presented in fig. 2 . 1. In a cell-free system, mRNA is in vitro transcribed (IVT) from a DNA template. 2. IVT mRNA is then transfected into dendritic cells (DCs) by the process of (3) endocytosis. 4. Endosomal escape allows entrapped mRNA to be released into the cytoplasm. 5. The mRNA is translated into antigenic proteins using the ribosome translational mechanism. After post-translational modification, the translated antigenic protein is ready to act in the cell where it was produced. 6. The protein gets secreted by the host cell. 7. Antigen proteins are digested in the cytoplasm by the proteasome and transferred to the endoplasmic reticulum, where they are loaded onto MHC class I molecules (MHC I). 8. MHC I-peptide epitope complexes with loaded MHC I-peptide epitopes produced, resulting in induction. 9.

Exogenous proteins are taken up DCs. 10. They are degraded in endosomes and delivered via the MHC II pathway.

Furthermore, to obtain cognate T-cell help in antigen-presenting cells, the protein should be routed through the MHC II pathway. 11. The generated antigenic peptide epitopes are subsequently loaded onto MHC II molecules.

Given the importance of DCs in initiating adaptive immunity in vitro and in vivo through generating CTLs, cmRNA-transfected DC vaccine is an approach gaining interest for cancer vaccine development [24] . DC-based mRNA cancer vaccines have shown promising effects in various phases of clinical trials. Boczkowski and colleagues in 1996 first demonstrated that electroporation of DCs with mRNA could elicit potent immune responses against tumor in mice [27] . Since then, several human trials with electroporation of DCs have been conducted [28, 29] . Bulk mRNA isolated from autologous tumors is another method for pulsing DCs with tumor antigen-loaded mRNA [30, 31] . Direct injection of mRNA can be used instead of DC vaccines since it eliminates the need for DC isolation, ex vivo cultivation, and re-infusion [32] .

Directly injecting the mRNA into secondary lymphoid tissue aids in delivering antigen to APCs at the T cell activation site, circumventing the need for DC movement [33] .

The cancer vaccines have the ability to elicit immune response to tumor antigens. The selection of a suitable target antigen is pivotal in the development of a vaccine design. Currently, the majority of vaccinations are TAAs, which are self-proteins that are improperly expressed by cancerous cells [21] . Developing vaccines against TAAs is challenging, as B-and T-cells might be subjected to removal by central and peripheral tolerance [34] . Besides, along with overexpression on tumor cells, TAAs might also be expressed in normal healthy cells leading to collateral damage [21] . In contrast, TSAs, which consists of neoantigens and viral oncoproteins are expressed only in cancerous cells. The prophylactic viral oncoproteins work by inducing the production of powerful neutralizing antibodies that block viral entrance into host cells and neoplasia caused by viruses [21] . However, these vaccines were ineffective in curing cancer as humoral immunity cannot effectively eliminate larger number of virus-infected cancer cells [21] . Neoantigens, like viral oncoproteins, are specific to tumor cells and are recognized by the immune system as foreign substances, hence they are not destroyed by tolerance mechanisms. Lately, neoantigens are being considered as a potential target in the progress of anti-cancer vaccine development. Numerous preclinical trials and early phase clinical trials have shown the ability of neoantigen based vaccines to minimize the potential induction of central and peripheral tolerance as well as the risk of autoimmunity [35, 36] .

TAAs with shared expression across cancer types, such as melanoma-associated antigen (MAGE1) and NY-ESO-1.37, has encouraged studies to target TAAs that are habitually overexpressed in a certain type of cancer, along with the prospect of generating a common vaccine per tumor type [37] . Empirical clinical experience has also suggested that vaccines targeting specific tumor antigens are ineffective in tackling tumor heterogeneity, as well as in dealing with the challenges of clonal evolution and immune evasion by the tumor [38] . As a result, with the increasing importance of therapeutic cancer vaccines, the rapid development and large-scale production using mRNA platform introduces the potential for the development of both personalized vaccines and off-shelf cocktail vaccines.

The neoantigens remain unique for each individual, with their numbers varying on the type of cancer. This necessitates for a tailored approach in which the tumor genome is sequenced and mutations are detected, neoantigens are predicted using computerized algorithms and a vaccine is then created and delivered to the patient. Mice vaccinated with a computationally engineered synthetic mRNA comprising numerous MHC class II neoepitopes showed 100% tumor rejection in preclinical studies, demonstrating antigen distribution [39] . The safety and efficacy of this approach was established in a first-in-human clinical study involving 13 patients with metastatic melanoma. Each patient was given a vaccine that contained 10 neoepitopes specific to their tumor.

In certain patients, antitumor responses were discovered in metastases removed after immunization, where T-cell infiltration and neoepitope-specific apoptosis of autologous tumor cells were discovered after vaccination. All patients exhibited CD4+ and CD8+ T-cell responses [40] . Since then, therapeutic cancer treatment with tailored mRNA vaccines has received a lot of interest, and several clinical trials are presently underway, according to the US National Library of Medicine. A recent study with mRNA-4650, a KRAS personalized vaccine, developed by Moderna and Merck, in combination with or without pembrolizumab was conducted to treat patients with pancreatic carcinoma. The lipid nanoparticles (LNPs) approach for delivery of mRNA-4650 showed well-tolerated anti-tumoral immune response [41] . Another personalized vaccine, mRNA-4157, targeting 20 TAAs and useful in treating various types of tumors, in single or in combination with pembrolizumab demonstrated acceptable safety profile with cytotoxic Tlymphocyte (CTL)-and memory T-cell-dependent immune responses [42] . Based on the ability of mRNA-4157 to elicit clinical response, a phase II trial is currently undergoing to evaluate the efficacy of the postoperative adjuvant therapy with mRNA-4157 and pembrolizumab in comparison with pembrolizumab monotherapy in high-risk recurrent individuals with complete resection of tumor (NCT03897881). A first-in-human phase Ib study of RO7198457, a combination of systemically administered RNA-Lipoplex iNeST with the PD-L1 antibody atezolizumab is presently conducted in patients with locally advanced or metastatic solid tumors.

The preliminary results of this study suggest significant level of neoantigen immune-tumor response. A randomized phase 2 study of RO7198457 in first-line for patients with melanoma in combination with pembrolizumab is currently ongoing, and 2 randomized clinical trials are planned for the adjuvant treatment of individuals with non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) [43] .

A tetravalent RNA-lipoplex cancer vaccine, BNT111, contains 4 types of naked RNA such as RBL001.1, RBL002.2, RBL003.1, and RBL004.1 encoding 4 melanoma-associated antigens (MAAs), the cancer-testis antigen NY-ESO-1, the human MAGE-A3, tyrosinase, and putative tyrosine-protein phosphatase (TPTE), encapsulated in liposomes. The vaccine upon intravenous administration is taken up by the APCs, and after being translocated to the cytoplasm, is translated into the 4 tumor-associated proteins. As a result, CD8+ and CD4+ T-cell responses against 4 selected antigens are produced [44] . A phase 1 trial showed that this vaccine alone and in combination with immune checkpoint inhibitors (ICIs) induced durable objective responses and exhibited a favorable safety profile among patients with advanced melanoma [45] . A phase 2 trial is ongoing to evaluate the vaccine candidate in combination with the anti-PD-1 antibody cemiplimab for patients with unresectable stage III or stage IV melanoma who are refractory to or relapsed after anti-PD-1 therapy [46] . mRNA-5671, another tetravalent vaccine, is an LNP-formulated mRNA-based vaccine that targets 4 of the most frequent KRAS mutations (G12D, G13D, G12C and G12V). APCs take up and translate mRNA-5671 after immunization. Following translation, the MHCs displays the epitopes on the surface of APCs, resulting in the development of both cytotoxic T-lymphocyteand memory T-cell-dependent immune responses directed at tumor cells with KRAS mutations [47] . CD8 T cell responses to KRAS antigens were considerably improved in preclinical investigations after immunization with mRNA encoding KRAS mutations [48] . Patients with advanced or metastatic NSCLC, colorectal cancer, or pancreatic adenocarcinoma and KRAS mutations are being enrolled in a phase 1 research using mRNA-5671 with or without pembrolizumab (NCT03948763).

Due to heterogenous and ever evolving nature of cancer mechanisms, the clinical benefit of monotherapy regimen in patients with advanced cancer is not adequate. Tumor-specific T lymphocytes produced by vaccines do not operate efficiently against the tumor due to their lack of motility and/or gradual depletion. As a result, combining procedure that prevent immune escape pathways is critical [49] . For instance, a phase II clinical trial in chemotherapy treated patients with metastatic castration-resistant prostate cancer (mCRPC) showed similar and durable tumor immune responses on addition of DC vaccines [50] . Monoclonal antibodies (mAbs) targeting CTLA-4 and the PD-1/PD-L1 expression have revolutionized the treatment paradigm for several types of cancers, including renal cancer, melanoma, bladder cancer, lung cancer and Hodgkin's lymphoma [51] . CureVac GmbH systemic mRNA immunotherapy and local irradiation therapy can eradicate established macroscopic E.G7-OVA and LLC cancers in a synergistic manner.

Moreover, this combination boosted CD4 + , NKT and CD8 + cell infiltration in tumor infected mouse [52] . CV9202, vaccine encoding 6 NSCLC-associated antigens (NY-ESO-1, MUC-1, MAGE-C2, MAGE-C1, 5T4 and survivin) have been proven to induce targeted immune responses.

The combination of this vaccine with radiotherapy in a phase Ib clinical trial in 26 stage IV NSCLC patients revealed elevated CV9202 antigen-specific immune responses in 84% of patients, with 80% increased antigen-specific antibody levels, 40% patients with functional T cells and about 52% of patients had multiple antigen specificities [53] . In another study, researchers used an mRNA vaccine expressing the TAA MUC1 in combination with an anti-CTLA-4 monoclonal antibody to boost the vaccine's immune response against triple-negative breast cancer (TNBC) by improving T cell activity [54] .

Preclinical and clinical evidence have shown that using mRNA for prophylaxis and therapy can help prevent infectious disease and treat cancers, and that mRNA vaccines are safe and well tolerated in both animal models and humans. Further enhancements might also boost antigenspecific immune responses as well as B and T cells immune responses [55] . As of 21 st December 2021, 23 RNA vaccines are currently under phase I/II/III clinical trials, while 24 vaccines are at pre-clinical stage.

Breast cancer remains a cause of mortality for women globally [56] . More often, 81% women suffer from invasive breast cancer, which comprises of at least 21 distinct histological subtypes and 4 molecular subgroups (luminal A, luminal B, triple-negative and HER2-enriched) that differ in risk factors, presentation, response to treatment, and outcomes [57] . Invasive breast cancer can spread to adjacent lymph nodes or other organs over time. It is because of widespread metastasis that a woman dies from breast cancer[58]. Using modern methodologies for mRNA sequencing, such as The Cancer Genome Atlas (TCGA) data, it has been established that increased expression of T-and B-cell predicts higher overall survival (OS) in a variety of tumor types, including breast cancer [59] . The current treatment approach for breast cancer includes radiation therapy, surgery, chemotherapy, as well as hormonal and targeted therapies. Lately, the development of medications that can prevent breast cancer from developing in the first place, as well as their recurrence, has gathered attention. The overexpression of high-affinity transmembrane receptors such as HER3, HER2, c-MET, EGFR, and the transmembrane protein epithelial mucin-1 (MUC-1) are the key oncogenic drivers for breast cancer [60] . Treatment of breast cancer, especially, TNBC is gaining importance, since lack of therapeutic targets makes such type of cancer unresponsive to typical endocrine therapies and HER2-targeted therapy. In such a case, cancer vaccines which aid in activation and amplification of TAA-specific immunity combined with a sustained memory T cell immune response may be an effective therapy for preventing breast cancer recurrence in patients [61] . Previous vaccination strategies in adjuvant settings, against HER2+ self-antigens have shown substantial efficacy in patients with breast cancer [62] [63] [64] . However, such an approach is usually weak as immune response as T-lymphocytes have affinity to HER2+ and thus are subject to central tolerance [65] . An ongoing phase I/II trial is being conducted in patients with TNBC and who completed standard of care chemotherapy, where patients are allocated to receive either 8 vaccination cycles of mRNA WAREHOUSE vaccine (containing pre-formulated, shared tumor antigens, non-mutated) or mRNA MUTANOME vaccine (containing individual mutations). The preliminary data of this trial showed mRNA WAREHOUSE is feasible approach for treatment of TNBC [66] . Another phase I trial from the [67] . Theoretically, this treatment regimen will lead to a transition from an individualized therapy targeting a single biomarker (e.g., HER2) to a fully specialized treatment targeting specific mutations in each patient.

The ongoing trials related to mRNA vaccine in breast cancer is listed in table 1. The standard treatment for prostate cancer includes androgen deprivation and chemotherapy. However, patients become resistant after prolonged treatment with these agents.

Relapse or progression of disease occur even after complete androgen blockage and when plasma concentrations of testosterone are reduced to <50 ng/dL by castration or gonadotropin-releasing hormone analogs, and the effects of the remaining androgens are suppressed by androgen receptor antagonists [69] . With the advent of Sipuleucel-T, a dendritic-cell based vaccine, for treatment of advanced stages of prostate cancer, immunotherapy for prostate cancer has come into limelight. of responders had antigen-specific T-cells. One patient displayed >85% drop in his PSA-level [71] .

Though the initial responses in these trials were encouraging, the subsequent trial with CV9104

for prostate cancer were terminated due to no significant effect on OS [72] . These findings indicate that selection of antigen is crucial for activating APCs and immune response. Several studies have highlighted the efficacy of mRNA vaccine in other therapeutic areas [73, 74] 

Lymphomas are a biologically and clinically heterogeneous group of carcinomas that develop in secondary lymphoid organs from mature B-or T-lymphocytes [78] . Global statistics reveal Hodgkin lymphoma occurs in 0.4% of total cancer population, while, non-Hodgkin lymphoma is more frequent and accounts for 2.8% of all types of cancer [56] . With the increase in the incidence of lymphoma and no known effective treatment, there is an urgent need to develop novel therapies [79] . Patients with lymphoma have been benefitted from monoclonal antibodies such as rituximab, however, majority of patients remain incurable or die of the disease [79] . The identification of B-cell receptor variable regions as B-NHL unique antigens aided the development of tailored made vaccines to protect patients against their own tumors. Despite promising early results, this technique has yet to demonstrate enough clinical value to gain regulatory approval [80] .

Use of personalized and standardized approach have been tested earlier, but have experienced drawbacks, with slim chance of success in clinical trials. Further, tumor-induced immunosuppressive factors and immune regulatory mechanism might limit the ability of immune system to generate antitumor immune response. Currently, the mRNA vaccine approaches are mostly in the nascent stage with preclinical studies demonstrating promising efficacy in reducing tumor growth. As mentioned earlier in the paragraph for prostate cancer, the co-delivery of C16-R848 adjuvant-pulsed mRNA vaccination with OVA RNA significantly reduced tumor growth in syngeneic allograft mouse models of lymphoma [77] . Similarly, in another study, 6 female C57BL mice were subcutaneously injected with E.G7 OVA expressing lymphoma cells to test the therapeutic efficacy of mRNA Galsomes over NPs containing unmodified mRNA. After intravenous administration, mRNA Galsomes transmits nucleoside-modified antigen-encoding mRNA as well as the glycolipid antigen and immunopotentiator α-galactosyl ceramide to APCs.

Both the treatments showed significant tumor reduction in 40% of animals and prolonged OS.

Further combination of mRNA Galsomes with PD-L1 checkpoint inhibitor indicated a synergistic behavior in tumor reduction [81] . The clinical trials related to mRNA vaccine in lymphoma is listed in table 4. 

Pancreatic cancer is a fatal malignancy with survival rate of 10.8% over 5 years [82] . TPX2 and TMOD3 for mRNA vaccine development [89] . Patients with immune subtypes 4 and 5 considered as immunological "cold" phenotypes were found to be suitable for vaccination [89] . A list of clinical trials for the development of pancreatic cancer vaccine is reported in table 5. [91] . Table   7 . 

The development of cancer vaccine depends on type of cancer, vaccine design and its modification and route of delivery (Fig. 3) . The mRNA vaccine is presented under 2 categories: 

Translation efficiency is known to be influenced by codon composition. The rate of protein production and the time spent in the ribosome repository can be affected by mRNA sequence codon optimization [96] . It was discovered that replacing a nucleotide with N1-methylpseudouridine (1mΨ) improves base pair stability, resulting in a complex secondary structure and better mRNA translation [96] . Substituting rare codons with regular identical codons that contain plenty of similar tRNA in the cytosol is a common practice to alleviate mRNA production [97] .

Although a high GC sequence may cause problems with mRNA secondary structure, it translates 100-fold higher than a low GC sequence [98] .

The 5ʹ and 3ʹ UTR elements bordering the coding sequence have significant impact on the stability and translation of mRNA, both of which are crucial considerations in the optimal vaccine design. These optimization increases the efficiency and half-life of mRNA [99, 100] . For effective mRNA protein synthesis, a 5ʹ cap structure is essential [101] . This can be achieved by applying 5ʹ cap in multiple versions during or after the transcription process by using a vaccinia virus capping enzyme [102] or by incorporating synthetic cap or anti-reverse cap analogues [103] . An appropriate length of poly(A) tail also plays a critical role in regulation of mRNA translation and stability, thus it must be inserted directly from the encoding DNA template or with poly(A) polymerase [104] . A recent study suggested that mRNAs with phosphorothioate groups within the poly(A) tail were less sensitive to 3'-deadenylase degradation than unmodified mRNA and were more efficiently produced in cultured cells, paving the way for future progress of mRNA-based therapeutics [105] .

Modifications of untranslated regions of mRNA also represents one of the approaches to enhance both mRNA efficiency and stability. Warren et al. used a synthetic 5′ UTR with a strong Kozak translation signal and the alpha globin 3′UTR to increase protein synthesis during fibroblast conversion to induced pluripotent stem cells [106] . Elsewhere, the globin 3′UTR has been used to increase mRNA stability since globin mRNAs generate large amount of protein with longer halflife [99] . Recently, in a review article by Miao et al. suggested 3 steps for modification of UTR as:

"avoid the presence of start codon (AUG), and non-canonical start codons (CUG) in the 5′ UTR, second, avoid the presence of highly stable secondary structures, which can prevent ribosome recruitment and codon recognition. Thirdly, shorter 5'UTR may be introduced as previous studies have shown that this type of 5'UTR is more conducive to mRNA translation process" [17] . A screening method using a diverse set of 5′UTR and 3′UTR combinations for better expression of the Arginase 1 protein highlighted 5′ UTR as an essential driver in protein expression for exogenously delivered mRNA [107] .

Elimination of pathogen-associated molecular patterns in mRNA via incorporation of modified nucleosides, such as pseudouridine [108] and 1-methylpseudouridine (m1Ψ) [109] , and fast protein liquid chromatography purification to remove double-stranded RNA contaminants [110] is another approach to improve mRNA therapeutic efficiency. An advantage of such optimization is that the vaccine is able to bypass the transcription process directly starting the translation phase to produce the immunogenic protein inside the human cells [33] .

Self-amplifying mRNA (SAM) vaccines are derived from an α-virus genome, which enables the intact RNA replication but the structural protein genes substituted with the antigen of interest. Due to intracellular replication of the antigen-encoding RNA, the SAM can produce a significant amount of antigen from a very little vaccination dose [33] . SAM vaccines are capable of creating their own complements of dsRNA structures, replicate intermediates and other features which could contribute to their high effectiveness. However, due to the inherent nature of these RNAs, modulating the inflammatory profile or reactogenicity of SAM vaccines may be difficult [33] . The applications of SAM in cancer vaccine development are however limited to animal models, and 2 clinical trials against colorectal cancers (NCT01890213 and NCT00529984).

Due to negatively charged structure of naked RNA and large molecular size, mRNA is prone to nuclease degradation and cannot cross the cell membrane. Thus to overcome this obstacle, several mRNA vaccine delivery strategies have been employed such as, naked mRNA delivery, mRNA delivery through viral vectors, polymer-based vectors, lipid-based vectors, lipid-polymer hybrid nanoparticles, and peptide-based vectors [4, 111] , Subcutaneous administration has been found very efficient for translation of encoded protein for mRNA, with the ability to mRNA to induce both cellular and immune response through this route. However, the outermost layer of skin represents a tough barrier for drugs absorption and hence various approaches have been adopted to overcome this barrier, including microneedles, microporation, and jet injection, electroporation, iontophoresis, sonophoresis, formulation as NPs and liposomes [112] .

The LNPs are derived from cationic lipids containing tertiary or quaternary amines to encapsulate polyanionic mRNA. A study reported antigen-specific CTL activity and suppressed the OVA-suppressing tumors in mice injected with OVA-encoding mRNA in 1,2-dioleoyl-3trimethylammonium-propane (DOTAP) and/or DOPE liposomes [113] . Coadministration of the mRNA for GM-CSF increased OVA-specific cytolytic responses in the same research. Another study found that subcutaneous distribution of LNP-formulated mRNA expressing two melanomaassociated antigens inhibited tumor growth in mice, and that co-delivery of lipopolysaccharide (LPS) in LNPs boosted CTL and antitumor activity [114] . A study by Kranz et al. [115] reported that mRNA-lipoplexes encoded with DOTMA/DOPE lipids were able to protect antigen-encoding mRNA against extracellular ribonucleases, which accumulated in the spleen and successfully delivered the mRNA into DCs following systemic treatment, leading in the development of an antigen-specific immune response. A preclinical study in mice injected with antibody-encoding mRNA delivery showed promising response against cancer [116] . Similarly, another study on mice inoculated with luciferase expressing Raji lymphoma cells and treated with mRNA-LNP encoding rituximab revealed diminished tumor growth, underscoring the importance of m RNA coated antibodies as a viable therapeutic option for treatment of cancer [117] . In general, mRNA cancer vaccines have shown to be immunogenic in people, but further improvement of vaccination methods based on fundamental immunological studies will almost certainly be required to gain higher clinical effects.

Polymeric materials though are less clinically investigated than ionizable lipids, they coat mRNA without the hassles of self-degradation and also promote protein expression. The drawback of polymeric materials however are polydispersity and the clearance of large molecules [91] . To improve the stability of the polymeric platforms, structural modifications such as lipid chains, expansion of branch structures and construction of biodegradation-promoting domains is considered [118] . A polyethyleneimine-polyplex nanoparticle carrying mRNA expressing the influenza virus hemagglutinin and nucleocapsid was employed in a research of mRNA vaccinations. mRNA was successfully transported to dendritic cells, transferred to the cytosol, and translated into proteins in this study, resulting in both humoral and cellular immunological responses [119] . However, because extremely positively charged polyethylene-based formulations attach to negatively charged serum proteins, they are more hazardous; as a result, new cationic polymers, such as poly(2-dimethylaminoethyl methacrylate) have been created [120] . Polymerbased delivery system research is still in the early stages of development. [4] . In short, the route of administration is significant in determining the efficacy of mRNA vaccine [111] .

Both naked and lipid-formulated mRNA administered subcutaneously cause cell transfection, with naked mRNA surpassing lipid-formulated mRNA in terms of translational efficiency. Both formats have demonstrated to induce antigen-specific T cells, but neither has been shown to transfect nodal cells [121, 122] . In contrast, a study using lipid nano formulations (approx.70-100 nm) found high and long-lasting translation at the injection site, as well as in CD11c+ cells in draining lymph nodes, leading to delayed tumor growth [114] . Kreiter et al. found that intranodal delivery of adjusted naked antigen-encoding mRNA elicited effective antitumor immunity and mRNA was internalized and translated via micropinocytosis by lymph node resident conventional and cross-presenting CD8a+ DCs [121] . Another study by Thielemans et al. validated the potency of intranodal delivery and format in additional tumor models [123] . Thielemans et al.

pioneered intertumoral administration to DCs. When in vivo transfected with TriMix, their findings show that naked mRNA is mostly picked up by cross-presenting CD8a+ DCs, and that these cells can reawaken T cells at the tumor site as well as move to the draining lymph. They also that mRNA-encoded secreted proteins might relieve part of the load on immune cells by lowering MDSC repression, boosting DCs, and activating T cell lysis, which improved tumor growth delay when paired with PD-1 inhibition [123] .

With the development and global approval of mRNA vaccines against SARS-CoV-2 virus in the last year have outscored the potential of mRNA technology. Most patients with cancer are non-responsive to current immunotherapies, frequently patients experience relapse and subsequently toxicities to therapies. In this context, therapeutic cancer vaccines is an appealing option to immunotherapy setting because of their potential for safety, specificity, and long-term response due to immunological memory stimulation [21] . The favorable features of potency, fast and relative low-cost production of mRNA vaccine provide an attractive platform for cancer therapy. The mRNA cancer vaccine can be a preferred combination agent with currently available therapies for long-term cancer treatment considering the favorable safety profile observed to date.

Apart from recent progress in the lipid-based delivery system for mRNA vaccines, chimeric antigen receptor (CAR) -T cell immunotherapy is emerging as an encouraging treatment approach for treating malignancies. CAR-T therapy is a personalized form of cell therapy where patient-T cells are genetically engineered to express receptors allowing them to recognize tumor antigens. The adoptive transfer of genetically modified T cells for expressing a CAR have shown encouraging response against hematological tumors. Given this approach, mRNA electroporation has been utilized to generate T cells with CAR expression in preclinical trials [124, 125] and subsequently in human trials [126, 127] . The preclinical data showed that Descartes-08, an autologous CD8+ CAR T therapy inhibits development of BCMA CAR-specific myeloma and substantially prolongs host survival. Furthermore, an ongoing clinical trial of Descartes-08 reported a favorable therapeutic index with durable responses upon preliminary analysis in patients with relapsed/refractory myeloma (NCT03448978), thus providing a framework for another study using Descartes-11 which is an optimized or humanized version of Descartes-08 in patients with newly diagnosed myeloma patients and having residual disease after induction therapy [128] . 

All authors contributed to manuscript conception, preparation, and approved the final version of the manuscript for submission.

 The recent success of COVID-19 mRNA vaccine has unlocked the potential of mRNA technology  Combination of delivery route and format should be considered for vaccine design  The rapid production of therapeutic mRNA cancer vaccine has led to development of both personalized and tetravalent vaccines  mRNA vaccines combined with checkpoint modulators and immunosuppressants show promise

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The authors acknowledge Anwesha Mandal and Dr. Amit Bhat of Indegene Pvt Ltd. For their medical writing and editorial support

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Medical writing and editorial support were funded by Shanghai Fosun Pharmaceutical Industrial Development, Co., Ltd.

All authors contributed to manuscript conception, preparation, and approved the final version of the manuscript for submission.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.