mRNA Vaccines on The Frontline: Targeting Neoantigens in Cancer Immunotherapy

1.1. Overview of Cancer Immunotherapy

Immunotherapy is a new paradigm in cancer that harnesses the body's immune system to recognize, attack and destroy cancer cells. Immunotherapy works by improving the patient's natural immune defense against tumors, whereas conventional modalities like chemotherapy, surgery and radiotherapy work by killing cancer cells directly [1]. The immune system has the ability to recognize abnormal cells, but the immune system is often rendered ineffective against cancer cells by mechanisms of immune suppression, changes in antigen presentation and the formation of an immunosuppressive tumor microenvironment. Cancer immunotherapy targets these obstacles and re-engages beneficial anti-cancer immune response [2]. In recent years, a number of immunotherapeutic strategies have been shown to have a tremendous success. Immune checkpoint inhibitors like antibodies against programmed death receptor-1 (PD-1), programmed death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) have dramatically improved survival in melanoma, lung cancer, renal carcinoma and some types of haematological cancer [3]. Likewise, adoptive cell therapies like chimeric antigen receptor T-cell (CAR-T) therapy include the genetic alteration of patient-derived T cells to specifically target and attack tumor cells. In addition, monoclonal antibodies and cytokine-based therapies have added to the growing field of immuno-oncology. Another key therapeutic cancer vaccine study targets is cancer therapeutic vaccines [4-6]. The vaccines are designed to trigger an immune response against tumor cells that presents tumor antigens to immune cells. Although there are significant advances, immune resistance, tumor heterogeneity, lack of target antigens, and major immune related side effects are still major issues [7]. As a result, scientists are working on methods that are even more tailored to the individual, and that induce more selective, lasting immune responses [8]. The mRNA-based neoantigen vaccines have been singled out as a promising approach based on their versatility, swift production, and ability to generate strong cellular immune response in this context. Genomics, molecular biology and immunology have revolutionized cancer immunotherapy as a key component of precision oncology [9,10].

1.2. Emergence of mRNA Vaccine Technology

One of the most innovative biomedical innovations of the 21st century is the messenger RNA (mRNA) vaccine technology. mRNA vaccines are based on synthetic mRNA molecules that carry the antigen for the disease being targeted to the host cell for protein synthesis in the cell [11]. These new proteins are then targeted by the immune system, leading to humoral and cellular immune responses. The non-infectious and non-integrating nature of mRNA vaccines represents a more versatile and safer platform than current vaccines based on recombinant proteins and attenuated pathogens. The swift advancement and effective use of mRNA vaccines in the fight against COVID-19 proved their potential as therapeutics and speeded up their use in other areas, such as cancer immunology [12]. The stability, translational efficiency, and safety of mRNA therapeutics has improved significantly over the last decade with advances in nucleoside modification, codon optimization, and lipid nanoparticle (LNP) delivery. Fragile mRNA molecules are secured from enzymatic degradation by lipid nanoparticles, which also promote efficient endosomal escape and cell uptake. The main benefit of mRNA is its quick and flexible production process. Synthetic mRNA vaccines can be developed quickly once a sequence of a target antigen is identified and produced, making them very attractive for personalized medicine [13]. In oncology, mRNA vaccines can be designed to express tumor-associated antigens (TAAs) or patient-specific neoantigens, which in turn should elicit very specific immune responses against the tumor. Additionally, mRNA vaccines can also instruct CD4+ helper T lymphocytes and CD8+ cytotoxic T lymphocytes that are important for tumour clearance and immune memory [14]. The fact that they can display multiple antigens at once also increases the therapeutic potential and immune recognition. For this reason, mRNA vaccine technology is now thought of as a paradigm-breaking platform with the potential to treat individual cancer patients, prevent infectious diseases, and advance regenerative medicine [15].

1.3. Importance of Neoantigens in Personalized Medicine          

Neoantigens are peptides with novel sequences that are only found in cancer cells as a result of somatic mutations, gene fusions, insertions, deletions or abnormal protein processing events in cancer cells. Neoantigens are not found in normal tissues and so are regarded as foreign by the immune system and are therefore very good targets for cancer immunotherapy. Neoantigens are more specific than tumor-associated antigens, which can also be found at low levels in normal tissues, and are much less likely to lead to autoimmune toxicity [16]. Neoantigens are now central to the field of personalized cancer medicine, and their identification and use is now critical. The latest breakthroughs in next-generation sequencing (NGS), bioinformatic algorithms and computational immunology have made it possible to rapidly interrogate tumour mutational profiles and identify immunogenic neoepitopes that bind major histocompatibility complex (MHC) molecules [17]. These advances have led to the creation of personalized vaccines that are specially designed for a person's own tumor with its peculiar molecular profile. Compared to traditional therapeutic strategies, Neoantigen-based mRNA vaccines offer a number of benefits. These vaccines are able to generate strong activation of CTLs that specifically kill the tumor cells while leaving normal tissues unharmed, as they carry multiple patient-specific neoantigens [18]. Furthermore, neoantigen vaccines could elicit long-lasting memory that could inhibit the growth of tumor recurrence and metastasis. Immune responses and the clinical outcomes of patients after neoantigen-targeted immunotherapy have been promisingly shown in clinical studies in melanoma, pancreatic, glioblastoma and colorectal cancer [19]. Combining neoantigen identification with mRNA vaccine technology is a significant step forward in precision oncology. Personalized neoantigen vaccines can be quickly synthesized and modified based on the evolution of each tumor, which can overcome the problems of tumour heterogeneity and immune escape. As a result, neoantigens are emerging as a key therapeutic target for the development of next-generation cancer vaccines and personalized immunotherapeutic approaches [20].

1.4. Historical Development of mRNA Therapeutics

The concept of mRNA therapeutics has emerged from the identification of mRNA as a short-lived intermediate molecule, which carries the genetic information from DNA to the ribosome involved in protein synthesis, in the early 1960s. The use of mRNA for therapeutic applications looked promising but initial studies were marred by the inherent instability of mRNA molecules, their tendency to be rapidly degraded by enzymes, and their ability to trigger excessive innate immune response [21]. However, in the 1990s, scientists showed that synthetic mRNA could be delivered to living cells to cause the synthesis of proteins encoded by the mRNA, laying the groundwork for mRNA therapeutics. But clinical applications were limited due to limited delivery efficacy and the occurrence of high inflammatory reactions. A major advance in the early 2000s was the use of modified nucleosides in synthetic mRNA sequences, which greatly reduced the unwanted immunogenicity, but increased the translational efficiency and stability. The findings paved the way to the use of mRNA as a therapeutic platform. Another important milestone was the emergence of lipid nanoparticle delivery systems for the evolution of mRNA therapeutics. Lipid nanoparticles protected the mRNA from ribonuclease-mediated degradation and facilitated its entry into the cell where it produced the desired effects, addressing one of the major challenges of mRNA-based drug delivery [22]. The protein expression and therapeutic effectiveness were further optimized by optimizing untranslated regions (UTRs), cap structures, and poly(A) tails of the mRNA. The world-wide success of mRNA COVID-19 vaccines was a historic achievement in biotechnology and pharmaceutical science. Both Pfizer/BioNTech and Moderna's vaccines proved to be highly effective, quickly scalable and safe, and mRNA technology has become widely accepted. This accomplishment continued the advancement of mRNA therapeutics in the fields of cancer, infectious diseases, rare genetic diseases, and regenerative medicine. In the modern era, mRNA therapeutics are considered as one of the most promising innovations of modern medicine. Given the continuous innovations in the delivery technologies, the development of antigen predictions using artificial intelligence and personalized vaccine design, there is hope that the clinical use of mRNA-based therapeutics will continue to grow, especially in cancer immunotherapy [23].

2. Cancer Immunotherapy: Current Landscape

2.1. Conventional Cancer Therapies

For many types of cancer, the traditional cancer treatments have been surgery, chemotherapy, and radiotherapy. For localized tumors, surgery is one of the most effective methods, and the goal of surgery is to destroy cancerous tissues physically before metastasis [24]. Whilst surgery can offer a cure in early cancers, it is less likely to cure advanced metastatic disease and can cause complications from the damage to tissues and recovery from surgery. Chemotherapy uses cytotoxic drugs to disrupt the DNA synthesis, mitosis or cell metabolism of rapidly growing cells. Commonly used chemotherapeutics are alkylating agents, antimetabolites, anthracyclines and taxanes. Chemotherapy has increased survival in some malignancies but, due to its lack of specificity, it can result in significant systemic toxicity such as myelosuppression, gastrointestinal issues, alopecia and organ damage [25]. Furthermore, resistance to multiple drugs is a significant clinical problem. Radiotherapy involves exposure to ionizing radiation that creates damage and cell death in cancer cells. Depending on the location of the tumour and the stage of the disease, it can be used as external beam radiation therapy or internal brachytherapy. Radiotherapy is very effective for treating localized tumors and may be used in combination with chemotherapy or surgery to improve the response to treatment. Radiation can also cause damage to healthy tissues surrounding the area, which can cause complications like fibrosis, inflammation and secondary cancers. Current methods for cancer diagnosis and treatment have certain drawbacks such as lack of selectivity, resistance to therapy, tumor recurrence and high side effects. All these challenges have spurred the development of treatment targets and individualization, and led to the development of cancer immunotherapy as a promising alternative approach in current oncology [26].

2.2. Immune checkpoint inhibitors

Immune checkpoint inhibitors (ICIs) are a significant advancement in cancer immunology, and have revolutionized the treatment of various types of cancer. In healthy physiology, immune checkpoints serve as regulatory mechanisms to keep the immune system in check and prevent overactive immune responses. Tumor cells, however, take advantage of these pathways to escape the immune system and block anti-tumor T-cell responses. The immune checkpoints most studied are programmed death receptor-1 (PD-1), programmed death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Immune checkpoint inhibitors are monoclonal antibodies that bind to these inhibitory receptors and remove the inhibition, allowing T cells to be activated and to destroy tumors via an immune response [27]. Agents against PD-1 and PD-L1 have demonstrated outstanding clinical results in melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer and Hodgkin lymphoma. Likewise, CTLA-4 inhibitors promote T-cell priming and proliferation in the early phases of immune activation. The use of immune checkpoint inhibitors has greatly enhanced prognosis for several advanced cancers that were once considered to be low prognosis [28]. Moreover, combination immunotherapy with several checkpoint inhibitors was shown to have synergistic therapeutic effects or combination with chemotherapy and radiotherapy showed synergistic therapeutic effects. But this approach for immune checkpoint blockade is not effective in all patients, and only a small percentage of patients respond in a durable manner. Another important problem with checkpoint inhibition therapy is immune-related adverse events (irAEs) [29]. Excessive activation of the immune system can lead to autoimmunity of various organs including the skin, liver, lungs, endocrine glands and gastrointestinal tract. Furthermore, the tumor heterogeneity, low neoantigen load and immunosuppressive tumor microenvironment can also play a role in therapeutic resistance. Current research is focused on finding predictive biomarkers and improving the combination strategies for efficacy and safety of immune checkpoint inhibitors in precision oncology [30].

Fig.1: Immune activation and tumor cell killing

2.3. CAR-T Cell Therapy

Chimeric antigen receptors T-cell (CAR-T) therapy is a sophisticated type of adoptive cell immunotherapies in which tumor-targeting T cells generated by genetic engineering and derived from patients are used to kill tumor cells [31]. The strategy involves extracting the patient's T cells, genetically engineering the receptor to express an artificial chimeric antigen receptor (CAR) ex vivo, multiplying the cells in culture and then re-engrafting them back into the patient. These designed receptors allow a T cell to specifically recognize tumor-associated antigens without major histocompatibility complex (MHC) presentation. CAR-T therapy has shown spectacular results in hematological malignancies including B-cell acute lymphoblastic leukaemia (B-ALL), diffuse large B-cell lymphoma and multiple myeloma [32]. The majority of currently approved CAR-T therapies target the CD19 antigen found on malignant B cells. After infusion, CAR-T cells divide and directly kill tumor cells by releasing cytokines and by activating immune effector mechanisms [33]. CAR-T therapy has several significant benefits, most notably that it can deliver lasting and potent tumor-killing effects, including in patients with recurrent or treatment-resistant (refractory) cancers. But there are still a number of hurdles that prevent its broader use. Two potentially life-threatening adverse effects of excessive immune activation are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Also, engineered cells can be limited in persistence, antigen escape, and T-cell exhaustion can all play a role in disease relapse [34]. The use of CAR-T therapy in solid tumors is still challenging because of the low tumor infiltration, the presence of antigen heterogeneity and the immunosuppressive tumor microenvironment. The next generation of CAR-T cells under development will try to address these issues with increased persistence, targeting multiple antigens, and better safety features. While there are still hurdles to overcome, CAR-T therapy is a major breakthrough in personalized cancer immunotherapies and an exciting and evolving platform on which to develop therapeutic solutions [35].

2.4. Therapeutic Cancer Vaccines

Therapeutic cancer vaccines aim to trigger the immune system to target and eliminate existing tumor cells by using tumor-specific or tumor-associated antigens (TAAs). Therapeutic cancer vaccines are used to boost tumour immunity and stop the spread or recurrence of disease after the diagnosis of cancer, as opposed to prophylactic vaccines which are given to prevent infectious diseases. Peptide-based vaccines, dendritic cell vaccines, DNA vaccines, viral vector vaccines and mRNA vaccines are all vaccine platforms being investigated for immunotherapy of cancer [36]. The mechanism of action of these vaccines is that the tumor antigens are transported to APCs, which activates the CTLs and generates tumor-specific immune responses. One of the first approved therapeutic cancer vaccines, dendritic cell-based vaccines is a prostate cancer vaccine (sipuleucel-T). The discovery of highly immunogenic tumor-specific peptides known as neoantigens has been made easier by recent developments in genomics and bioinformatics. Targeting neoantigen has received a lot of interest due to its greater specificity and the decreased risk of autoimmune toxicity [37]. mRNA vaccines have emerged as highly promising vaccine platforms, as they are quickly made, scalable, and safe, and are capable of delivering multiple neoantigens. Cancer therapeutic vaccines could also be used alongside immune checkpoint inhibitors, chemotherapy or radiotherapy to improve the effectiveness of treatment. Despite the promising immune responses seen in several clinical trials, issues associated with limited immunogenicity, tumor immune evasion, and variability in patient responses remain to limit the broader clinical success. However, continual progress in personalized vaccine development and delivery systems are anticipated to enhance the efficacy of therapeutic cancer vaccines in precision oncology [38].

3. mRNA Vaccine Technology

3.1. Structure and Mechanism of mRNA Vaccines

Advanced therapeutic platform, messenger RNA (mRNA) vaccines are engineered to introduce synthetic genetic information into host cells that can be used to manufacture specific antigenic proteins that can trigger immune responses against. mRNA vaccines do not use weakened pathogens or recombinant proteins, but rather make use of the cell's machinery for in-vivo synthesis of the antigen [39]. This not only offers greater flexibility but also quick production and better safety profiles. The basic architecture of an mRNA vaccine is similar to naturally occurring eukaryotic messenger RNA with the following essential features: 5′ cap structure, 5′ untranslated region (UTR), open reading frame (ORF), 3′ untranslated region and polyadenylated (poly(A)) tail. The coding sequence of the desired antigen (e.g. tumor neoantigens and viral proteins) is contained within the ORF [40]. These structural features all play a role in the stability, efficiency of translation, and persistence of mRNA inside the cell. The mRNA vaccine is taken up into host cells primarily through the process of endocytosis by the lipid nanoparticles to which the vaccine is delivered. After entering into the cell, the mRNA is extruded into the cytoplasm where the message is translated into antigenic proteins by the ribosomes [41]. The proteins are then broken down and displayed to T lymphocytes by major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells. The activation of antigen presentation leads to the activation of CD4+ helper T cells and CD8+ Cytotoxic T lymphocytes and subsequent humoral and cellular immune response. The mRNA vaccines are one of the most promising features as they are not able to integrate into the host genome, reducing the risk of insertional mutagenesis. Moreover, mRNA vaccines are easily adaptable to target a combination of multiple antigens or patient-specific neoantigens, making them highly promising in personalized medicine and cancer immunity. The ability to provoke strong immune responses with good safety properties has made mRNA vaccines a leader in next-generation therapeutic technologies [42].

3.2. Types of mRNA Vaccines

Non-Replicating mRNA

The most widely used mRNA vaccine platform is a non-replicating mRNA vaccine which contains synthetic mRNA that encodes an antigen without any replication machinery. These vaccines aim to directly enter host cells and take advantage of the host's own ribosome system to make proteins transiently [43]. After its translation, the antigen will elicit adaptive immune responses by engaging antigen presenting cells and T lymphocytes. Non-replicating mRNA vaccines have several benefits such as being simple to design, relatively easy to manufacture, and less likely to uncontrollably replicate inside cells. High scalability and flexibility of the manufacturing process that enables rapid modification depending on the evolution of the therapeutic needs or the patient-specific neoantigen profile. In addition, modifications of the nucleosides and optimized UTRs greatly enhance translational efficiency and dampen innate immune activation. Non-replicating mRNA vaccines, however, have a number of disadvantages, including the need for higher doses for adequate antigen expression due to the relatively short duration of persistence of the delivered mRNA in the cells and its inability to self-replicate. However, their favorable safety profile and clinical efficacy, especially in the context of COVID-19, has proven their therapeutic promise in infectious diseases and oncology [44].

Self-Amplifying mRNA

S. self-amplifying mRNA (saRNA) vaccines are a more advanced mRNA platform based on alphavirus replicon systems. SaRNAs not only encode the target antigen, but also genes for viral replication machinery that can increase the amount of RNA inside the cell [45]. This self-replication mechanism leads to greater production of antigen in the host cells, which boosts the immunogenicity of vaccines at lower doses. The increased expression of the antigen from saRNA vaccines allows higher and more prolonged immune responses than the non-replicating mRNA vaccines. Thus, saRNA technology has attracted significant research interest in the field of cancer immunotherapy, prevention of infectious diseases, and development of personalized vaccines. Reducing the dose needs could also decrease production expenses and make the drug more available worldwide. Self-amplifying mRNA vaccines, however, are larger and more complex, and can pose stability, formulation and intracellular delivery challenges. The optimization of excessive innate immune activation and potential inflammatory response are critical. Regardless of these constraints, continued development of saRNA engineering and delivery systems continues to enhance the therapeutic potential of this novel RNA vaccine platform [46].

3.3. Components of mRNA Vaccine Platforms

Cap Structures

 An important modification at the 5′ end of the mRNA molecule is the cap structure, which is critical for mRNA stability, ribosome recognition and translational initiation. Synthetic cap analogs can be used for protecting mRNA from degradation by exonuclease and increase the efficiency of protein synthesis. Optimized caps successfully also minimize the inherent immune recognition and hence performance of the vaccine [47].

 Untranslated Regions (UTRs)

In the mRNA molecule, there are non-coding sequences at the 5′-untranslated and 3′-untranslated regions, known as the 5′ and 3′ untranslated regions (UTRs), respectively. These regions control mRNA stability, localization within cells and translational efficiency. Optimized UTR sequences improve antigen expression and promote cell longevity of mRNA. The modification of UTRs is also an important technology to enhance the therapeutic efficacy of mRNA vaccines [48].

Poly(A) Tail

The polyadenylated [poly(A)] tail is a string of adenine nucleotides at the 3′ end of the mRNA molecule. It prevents mRNA from being degraded by enzymes and helps to recruit a ribosome and mRNA to round the circle around for efficient translation. Protein expression and vaccine stability can be greatly affected by the length and composition of the poly(A) [49].

 Lipid Nanoparticles (LNPs)

The most popular delivery systems for mRNA vaccines are lipid nanoparticles. Fragile mRNA molecules are wrapped in LNPs to prevent degradation by ribonuclease and enhance cellular uptake. The nanoparticles are usually made of ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids. Following endocytosis, LNPs promote endosomal escape and release of mRNA into the cytoplasm. The clinical success of today's mRNA vaccines has been greatly aided by the use of lipid nanoparticle technology, which promotes enhanced vaccine stability, delivery, and immunogenicity [50, 51].

Fig.2: Nanoparticlebased delivery of mRNA vaccine

3.4. Delivery systems and formulation strategies

The clinical use of mRNA vaccines will depend on efficient delivery systems as naked mRNA is very unstable and rapidly degraded by enzymes. A number of different formulation strategies have been developed to improve mRNA stability, cellular uptake, and expression of intracellular antigen, reduce toxicity and immune-related adverse effects. The current gold standard method of mRNA vaccine delivery is through lipid nanoparticles. These nanoparticles are able to inhibit the degradation of the mRNA and to facilitate the endocytosis of the mRNA into antigen presenting cells. Biodistribution, pharmacokinetics, and endosomal escape efficiency are enhanced by optimization of lipid composition, particle size, surface charge and PEGylation [52]. Apart from lipid nanoparticles, other types of delivery platforms have been studied such as polymeric nanoparticles, liposomes, dendrimers, nanoemulsions, and peptide-based carriers. Electrostatic binding to mRNA is supported by cationic polymers, like polyethyleneimine (PEI), and membrane fusion and release of the mRNA into the cell is supported by liposomal systems. Combination nanocarriers, with different delivery methods, are also being investigated for enhanced therapeutic activity. Additional formulation approaches involve nucleoside modification, codon optimization and lyophilization to improve vaccine stability and minimize storage restrictions. Immune adjuvants and targeting ligands might enhance antigen presentation and target antigens to dendritic cells or tumor sites [53]. Nanotechnology and biomaterials have seen some great developments in recent years, greatly enhancing the delivery of mRNA vaccines for cancer treatment. Future developments will be directed towards tissue-specific targeting, improved intracellular trafficking, decreased toxicity, and scale up production processes. The innovations will have a significant impact on the effectiveness and availability of personalized mRNA based cancer vaccines and next generation therapeutic platforms [54].

4. Neoantigens: The Core Targets

4.1. Definition and Characteristics of Neoantigens

Neoantigens are ‘newly' formed peptide antigens that arise from genetic changes in tumors (somatic mutations, insertions or deletions, gene rearrangements, or abnormal RNA processing events). The antigens are expressed exclusively on cancer cells and not on normal healthy tissues and are therefore highly specific targets for cancer immunotherapy [55]. Neoantigens are intracellularly processed and presented on the surface of tumour cells by major histocompatibility complex (MHC) molecules that can be recognized by cytotoxic T lymphocytes. High immunogenicity is one of the distinguishing features of neoantigens. Neoantigens are not used in the development of the immune system and therefore do not gain central immune tolerance and are seen as foreign by immune cells. This offers the potential to trigger potent anti-tumour immune responses with relatively low risk of damaging normal tissues [56]. Neoantigens offer several advantages over the traditional tumour-associated antigens, which are generally not immunogenic because they are either not recognized by the immune system or are poorly recognized. Another unique characteristic of neoantigens is that they are individualized. Patients have a different mutational profile, and so do their tumors, leading to a unique repertoire of neoantigens in each patient [57]. Highly mutational tumors like melanoma, lung cancer and bladder cancer tend to generate more neoantigens and may be more sensitive to immunotherapeutic treatments. Tumor heterogeneity and continuous genetic changes can however affect the expression of neoantigens and help in immune escape. This combination of neoantigen identification and advanced vaccine technologies, such as mRNA vaccines, has ushered in a new era in precision oncology, offering the potential to tailor cancer immunotherapies to a patient's unique cancer profile, thereby creating highly specific and long-lasting immune responses against the cancer cells [58].

4.2. Sources of Tumor Neoantigens

Tumor neoantigens are generated by different genetic and molecular changes during the process of cancer progression. It is these changes that give rise to abnormal proteins or peptides, which are recognized as foreign by the immune system. Identifying neoantigens is a crucial step in creating effective personalized cancer vaccines and immunotherapies [59].

Somatic Mutations

Tumor neoantigens are the most frequently found somatic mutations. They are mutations that happen in somatic cells throughout a person's lifetime, as the result of errors in DNA replication, exposure to carcinogens, oxidative stress and environmental insults. Nonsynonymous mutations change the sequences of amino acids within proteins, which would then generate the mutated peptides that could be processed and presented by MHC molecules to T cells. Highly mutational cancers, such as melanoma and smoking related lung cancer, produce numerous neoantigens, which makes them likely to be recognized by the immune system. These neoantigens, derived from mutations, have been highly correlated with good response to immunotherapy with immune checkpoint inhibitors or personalized vaccines. Neoantigens resulting from somatic mutations are still important targets for ongoing cancer immunotherapy research [60].

Gene Fusions

Fusion genes are genes that are fused together by a chromosomal rearrangement, resulting in proteins that were not previously present in the cell. The abnormal proteins have peptide junctions that are not present in normal tissues and hence are highly immunogenic neoantigens. Neoantigens, in the form of peptides derived from fusion genes, are frequently seen in hematological malignancies and some solid tumours. Examples include chronic myeloid leukemia (BCR-ABL fusion proteins) and lung cancer (EML4-ALK rearrangements). Because fusion neoantigens are highly tumor specific, they are a good choice for therapeutic vaccines, T-cell receptor therapies, and adoptive cellular immunotherapy [61].

Alternative Splicing

Alternative splicing is a physiological process whereby one gene can produce several different protein products. Abnormal splicing machinery can lead to the generation of abnormal transcripts and aberrant proteins with novel peptide sequences in cancer cells. Such modified proteins can produce neoantigens from splicing which can trigger immune reactions. New developments in RNA sequencing and transcriptomic analysis have led to a better understanding of the role of abnormal splicing events in the tumor immunopeptidome. Splicing-derived neoantigens expand tumour antigen diversity and can serve as novel targets for personalised immunotherapy. Their discovery has broadened our knowledge of neoantigen biology and has enabled novel vaccine design approaches [62].

4.3. Tumor-Specific vs Tumor-Associated Antigens

Tumor antigens can be divided into two main groups, according to their expression pattern and biological origin, namely tumor-specific antigens and tumor-associated antigens. Neoantigens, also known as tumor-specific antigens, are unique antigens that are only expressed on the surface of malignant cells caused by the genetic changes that occur in the tumor. These antigens are not present in normal tissues and therefore have a very high degree of specificity and a low risk for autoimmune toxicity [63]. The Neoantigen is highly immunogenic as it is regarded as a foreign molecule. This enables efficient activation of the cytotoxic T lymphocytes and better tumour immunogenic responses. Targeting neoantigens with personalized cancer vaccines is therefore a very promising approach in precision oncology. Tumors that develop in the body contain proteins that are either over-produced or mis-produced in cancer cells, but are not completely absent from normal tissue [64]. Some examples are HER2, MUC1, prostate-specific antigen and carcinoembryonic antigen. TAAs can be targets for immunotherapies, but they can also be present in healthy tissues, giving rise to immune tolerance and limiting the effectiveness of therapy and risking off-target side effects. Neoantigens are more specific, more immunogenic and have better safety profiles than tumor associated antigens. Thus, the development of personalized therapeutic vaccines and adoptive T-cell therapy has become a new focus in current cancer immunotherapy research by targeting neoantigens [65].

4.4. Identification and prediction of Neoantigens

Identification and prediction of neoantigens are essential steps in personalized cancer immunotherapies. New next generation sequencing technologies have led to a comprehensive characterization of tumor genomes and transcriptomes to find somatic mutations that can produce immunogenic neoepitopes. Typically, the neoantigen discovery process starts with RNA sequencing or whole-exome sequencing of tumor tissue and matched normal tissue samples. These altered protein sequences are then predicted using bioinformatics pipelines that are able to detect mutations which distinguish tumor cells from cells of the normal tissue [66]. Neoantigens are then tested for binding to patient-specific MHC molecules so as to be able to promote effective antigen presentation and T-cell activation. Peptide-MHC binding affinity, antigen processing efficiency and immunogenic potential can be estimated by several computational algorithms.

Further validation methods such as immunopeptidomics using mass spectrometry and functional T-cell assays are used to validate neoantigen presentation and biological relevance. Despite the numerous advances in technology, neoantigen prediction is still a difficult challenge because of tumor heterogeneity, evolutionary dynamics of mutations, variations in antigen processing pathways and differences in the immune response of patients. Sophisticated computational models and integrative molecular analysis is still needed to accurately identify clinically relevant neoantigens. However, progress in sequencing and immune informatics has significantly speeded up the development of personalized cancer vaccines [67].

4.5. Bioinformatics and AI-Based Neoantigen Screening

The era of neoantigen research and personalized vaccination has entered the Bioinformatics and Artificial Intelligence era. Computational methods are crucial to the identification of clinically relevant neoantigens capable of inducing strong immune responses in the face of the large number of mutations in tumor genomes. Bioinformatics pipelines merge information from genomic sequencing, mutation analysis, transcriptomic profiling, prediction of peptides and modeling of their binding to the MHC. Machine learning and deep learning methods also improve the prediction accuracy, by capturing intricate relationships between peptide sequences, antigen presentation, MHC processing, and TCR recognition. Neoantigen screening platforms based on AI can quickly identify very immunogenic neoepitopes that are ideal for designing personalized mRNA vaccines [68]. The technologies can greatly shorten the time needed to develop vaccines and enhance the identification of therapeutic targets with good clinical potential. AI also has the potential to forecast immunotherapy outcomes in patients and design multi-epitope vaccines from tumor mutational analysis. Advanced computational tools also enable identification of antigens which are not found in conventional analysis, such as low-frequency neoantigens, neoepitopes from splicing, and fusion antigens. The combination of artificial intelligence and high throughput sequencing technologies has thus helped to push towards precision oncology and personalised cancer treatment [69]. While there are several limitations, including algorithm variability, a small number of training datasets, and computational complexity, ongoing progress in AI, systems biology, and computational immunology will further enhance the accuracy of neoantigen predictions and the efficacy of the resulting therapies. These innovations will be important in the future for the advancement of mRNA-based cancer vaccines and future generation personalized immunotherapies [70].

5. Mechanism of Action Neoantigen mRNA Vaccines

5.1. Antigen Presentation Pathways

Neoantigen mRNA vaccines work by injecting synthetic mRNA that expresses tumor-specific neoantigens into host cells, mainly antigen-presenting cells (APCs), like dendritic cells. The mRNA is internalized by endocytosis and then translocated to the cytoplasm where the host ribosome translates it into neoantigenic proteins. Then these proteins are broken down into peptide fragments and displayed through major histocompatibility complex (MHC) pathways, which trigger adaptive immune responses. There are two major antigen presentation pathways: MHC class I pathway and MHC class II pathway [71]. Endogenously synthesized neoantigen proteins are broken down by proteasomes into short peptides which are taken up into the endoplasmic reticulum by transporter associated with antigen processing (TAP) proteins. The peptides bind to MHC class I proteins and are displayed on the surface of antigen-presenting cells, that then are recognized by the CD8-positive cytotoxic T lymphocytes. This route is critical for direct death of tumour cells. At the same time, the extracellular or phagocytosed neoantigen proteins can be degraded in the lysosomes and presented to CD4+ helper T cells by MHC class II molecules. Some DC cells can also have a cross-presentation function in which the extracellular antigens are presented by MHC class I molecules to make more effective activation of cytotoxic T cells [72]. In order to establish a powerful antitumor immunity, efficient antigen presentation is essential. The specificity of neoantigens allows for targeting of the malignant cells while sparing damage to healthy tissue. Various advanced platforms of mRNA vaccines and lipid nanoparticles for intracellular delivery and dendritic cell activation further improve the presentation of antigens. Thus, antigen presentation pathways are the basic immunological underpinning of the therapeutic effect of neoantigen mRNA vaccines in cancer immunotherapy [73].

5.2. Activation of CD4+ and CD8+ T Cells

A major mechanism by which neoantigen mRNA vaccines have anti-tumoral effects is by activating CD4+ helper T cells and CD8+ cytotoxic T lymphocytes. After dendritic cells and other antigen presenting cells present the antigens, naïve T lymphocytes spot specific neoantigen-derived peptide-MHC complexes via their T-cell receptors (TCRs), and activate and expand their number. The activation of CD8+ cytotoxic T lymphocytes is mainly through the recognition of neoantigen peptides presented on MHC class I molecules. These cells activate and expand into effector cytotoxic T cells that can directly kill tumor cells that express the same neoantigen [74]. Cytotoxic T cells kill malignant cells by releasing perforin and granzymes, by inducing apoptosis by Fas-Fas ligand interaction and by secreting inflammatory cytokines like interferon gamma (IFN-γ). Neoantigen peptides presented on MHC molecules of class II activate the CD4+ helper T cells. These cells have an important supportive function by secreting cytokines that stimulate cytoplasmic T-cell function, activate B-cells, and stimulate the maturation of antigen presenting cells. CD4+ T cells also help to maintain long-term immune responses and immune memory. There is a strong need to activate both CD4+ and CD8+ T-cell populations in order to give rise to a durable and effective tumor immunity. One of the major advantage of neoantigen mRNA vaccines is that they can carry multiple neoepitopes that will elicit a wide T cell response against the heterogeneous population of tumor cells. This immune activation is multifaceted, and promotes the activity of the immune system to eliminate tumors, while also decreasing the risk of immune escape mechanisms, such as antigen loss or antigen mutation [75].

5.3. Induction of Adaptive Immune Responses

The mRNA vaccines are specifically engineered to generate strong adaptive immune responses that selectively target cancer cells, a property called neoantigen. Adaptive immunity is a specific, long lasting immunity mediated by T lymphocytes and B lymphocytes. After vaccination, antigen-presenting cells (APCs) process and present neoantigens to T cells, which trigger concerted cellular and humoral immune responses [76]. Neoantigen-based cancer vaccines rely on cellular immunity more than anything else. Activated CD8+ cytotoxic T lymphocytes enter the tumor tissue and kill the tumor cells presenting neoantigen derived peptides. At the same time, activated CD4+ helper T cells through their cytokine production and their ability to promote the growth and survival of cytotoxic T cells, further promote immune activation. Besides cellular responses, neoantigen mRNA vaccines can also activate humoral immunity by B-cell activation and antibody production. While the role of antibodies in cancer immunotherapy is less pronounced than in vaccines for infectious diseases, they can play a role in the eradication of tumors, such as by stimulating antibody-dependent cellular cytotoxicity and improving antigen presentation [77]. The great promise of neoantigen vaccines is that they can induce extremely individualized adaptive immune responses against the mutational landscape of a single tumor. Adaptive immunity induced by the vaccines is highly specific and also has minimal autoimmune toxicity, as the neoantigens are tumor specific. In addition, mRNA vaccine platforms have intrinsic adjuvant activity, which is able to activate innate immune pathways, such as the Toll-like receptors and interferon pathway. This natural immunity activation has a beneficial effect on antigen presentation and immune activation of adaptive immunity. Thus, personalized identification of neoantigens coupled with mRNA vaccine technology is a major breakthrough in precision oncology and personalized cancer immunotherapy [78].

5.4. Memory T-Cell Formation

The generation of memory T cells is an important component of successful cancer therapeutics given the need for longterm immune surveillance against recurrence and metastasis. After activation and expansion, some of the antigen-specific T lymphocytes become long-lived memory T cells that can respond speedily to subsequent exposure to the same neoantigens. Neoantigen mRNA vaccines induce generation of central memory T cells and effector memory T cells. The central memory T cells are mostly in the lymphoid organs and have high proliferation ability; they will proliferate rapidly after re-exposure to antigen [79]. Effector memory T cells patrol in peripheral tissues, and offer rapid response cytotoxic activities against re occurring tumor cells. Several factors, such as the persistence of the antigen, cytokine signaling, activation of dendritic cells, and the quality of the initial T cell activation, all contribute to the formation of memory T cells. Cytokines, including interleukin-7 (IL-7) and interleukin-15 (IL-15) are crucial in the survival and activity of memory T cells. Neoantigen-specific memory T cells can be long-lived and remain in the system long after therapy to confer long-lasting antitumor immunity. One of the most significant functions of an immune memory that must be considered in cancer treatment is the ability to keep a trace of residual malignant cells dormant after the initial treatment, and then re-trigger the immune system to attack them, causing reappearance of the disease [80]. These memory T cells, which have been generated by neoantigen vaccination, can quickly identify and kill these remaining tumour cells before they cause much disease. The property of neoantigen mRNA vaccines to generate immune memory is different from many standard therapeutics, which typically result in short-term tumor control, but not long-term immunity. This means that the generation of memory T-cells is regarded as a key factor in long-term successful responses and the long-term efficacy of cancer immunotherapies [81].

5.5. Tumor microenvironment modulation

The tumor microenvironment (TME) is a very complex and dynamic network of cells, including tumor cells, stromal cells, immune cells, blood vessels, cytokines, and extracellular matrix components. Many cancers create a tumor microenvironment that is immunosuppressive and therefore suppresses effective anti-tumor immune responses and contributes to tumor progression. One of the most important roles of neoantigen mRNA vaccines is to modulate the tumor microenvironment to promote immune-mediated tumor destruction [82]. One of the main mechanisms is the infiltration of activated cytotoxic T lymphocytes into tumor tissues. Vaccine elicited neoantigen-specific T cells home to the tumour and release cytokines, including interferon gamma and tumour necrosis factor alpha, which have inflammatory properties. These cytokines amplify antigen presentation, suppress the proliferation of tumours, and attract other immune effector cells. Neoantigen vaccines may also decrease the immunosuppressive function of regulatory T cells, myeloid derived suppressor cells and tumor associated macrophages in the TME. Vaccination can also alter the tumor microenvironment from an immunosuppressive to immunostimulatory state by changing the cytokine patterns and inducing pro-inflammatory signaling pathways that are beneficial to anti-tumor immunity. Moreover, combination therapies with neoantigen mRNA vaccines and immune checkpoint inhibitors are shown to be synergistic in overcoming tumor-induced immune suppression [83]. The combination of checkpoint blockade and neoantigen vaccines yields superior therapeutic response, with the checkpoint blockade boosting T-cell activity and the neoantigen vaccines supplying highly specific immune targets. The immune microenvironment of solid tumors is especially relevant because immune exclusion factors, as well as inhibitory signaling pathways, can hinder treatment efficacy. Current studies are dedicated to further developing vaccine formulation, delivery systems and multi-component vaccines to enhance immune infiltration and address the mechanisms of tumor-mediated resistance. The advantages of neoantigen mRNA vaccines are that they induce specific immune responses and actively modify the tumor microenvironment to promote long-term and powerful anti-tumor immunity, highlighting the potential of mRNA vaccines in contemporary precision oncology [84].

Fig.3: Workflow of personalized mRNA cancer vaccine