Will mRNA vaccines become the new hope of cancer immunotherapy?
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Will mRNA vaccines become the new hope of cancer immunotherapy?
Tumor immunotherapy aims to activate the host’s anti-tumor immunity, form a tumor suppressor microenvironment, and ultimately eliminate tumors and improve the overall survival rate of patients. Tumor vaccine is a promising anti-tumor immunotherapy.
Vaccines against tumor-associated antigens ( TAAs ) or tumor-specific antigens ( TSAs ) can specifically attack and destroy malignant tumor cells with high expression of these antigens, and achieve sustained tumor killing through immune memory.
Therefore, compared with other types of immunotherapy, cancer vaccines can theoretically provide specific, safe and well-tolerated therapeutic effects.
mRNA – based nucleic acid vaccines were proposed more than 30 years ago, and mRNA vaccines have many advantages over traditional vaccines: Unlike some viral vaccines, mRNA does not integrate into the genome, avoiding concerns about insertional mutations; mRNA vaccines can be Manufactured in a cell-free manner, enabling fast, cost-effective, and efficient production.
Furthermore, a single mRNA vaccine can encode multiple antigens, enhance immune responses against adaptive pathogens, and be able to target multiple microbial or viral variants in a single formulation.
mRNA vaccines are an important class of cancer vaccines that can encode and express TAA, TSA and their related cytokines.
mRNA cancer vaccines can stimulate humoral and cellular immunity, improving the adaptability of these vaccines to different diseases and patients. Currently, a variety of mRNA cancer vaccines are being developed in the clinic, bringing a new dawn to cancer immunotherapy.
mRNA vaccine preparation strategy
In vitro transcribed ( IVT ) mRNA mimics the structure of endogenous mRNA and has five sections ranging from 5ʹ to 3ʹ including: 5ʹcap, 5ʹ untranslated region ( UTR ), an antigen-encoding open reading frame, 3ʹUTR and a PolyA tail.
Like native eukaryotic mRNAs, the 5′ cap structure contains a 7-methylguanosine nucleoside, which sterically protects the mRNA from exonuclease degradation and cooperates with translation initiation factor proteins , recruits ribosomes to initiate translation.
The length of the PolyA tail indirectly regulates mRNA translation and half-life.
The 5′ and 3′ UTRs flanking the coding region regulate mRNA translation, half-life and subcellular localization, and native UTRs from highly expressed genes such as α- and β-globin genes are the first choice for synthetic mRNAs.
In addition, UTRs can be optimized according to cell type, such as by removing miRNA-binding sites and AU-rich regions in the 3′ UTR to minimize mRNA degradation.
The open reading frame of an mRNA vaccine is the most critical component. Although the open reading frame is not as malleable as the non-coding regions, codon optimization can be used to increase translation without changing the protein sequence.
For example, CureVac AG found that human mRNA codons rarely have an A or U in the third position, thus replacing the A or U in the third position of the open reading frame with a G or C. and used this optimized strategy for its SARS-CoV-2 vaccine, CVnCoV, which is currently in Phase III trials.
To maximize translation, mRNA sequences often contain modified nucleosides, such as pseudouridine, N1-methylpseudouridine, or other nucleoside analogs.
The use of modified nucleosides, especially modified uridines, prevents recognition by pattern recognition receptors, ensuring that the translation process produces sufficient levels of protein.
Both Moderna and Pfizer–BioNTech SARS-CoV-2 vaccines contain nucleoside modifications.
Another strategy to avoid detection of pattern recognition receptors, pioneered by CureVac, uses sequence engineering and codon optimization to deplete uridine by increasing the GC content of vaccine mRNAs.
In addition to improvements in mRNA sequences, significant progress has been made in streamlining mRNA production. Synthetic mRNAs used clinically are transcribed in vitro from DNA plasmids using bacteriophage RNA polymerase T7 ( T3 and SP6 polymerases can also be used ).
Additionally, purification steps are simplified using CleanCap’s co-transcriptional capping technology.
mRNA delivery system
Since mRNAs are large ( 10 4 –10 6 Da ) and negatively charged, they cannot pass through the anionic lipid bilayer of the cell membrane.
Furthermore, in vivo, it is engulfed by cells of the innate immune system and degraded by nucleases.
Therefore, in vivo applications require the use of mRNA delivery vectors that transfect immune cells without causing toxicity or unnecessary immunogenicity.
At present, many solutions based on innovative materials have been developed for this purpose.
Lipid Nanoparticles (LNPs)
Lipid nanoparticles are the most clinically advanced mRNA carriers. As of June 2021, all SARS-CoV-2 mRNA vaccines under development or approved for clinical use use LNPs.
LNPs offer many benefits for mRNA delivery, including formulation simplicity, modularity, biocompatibility, and large mRNA payload capacity.
In addition to RNA drugs, LNPs typically include four components, ionizable lipids, cholesterol, helper phospholipids, and pegylated lipids, which together encapsulate and protect fragile mRNAs.
Ionizable lipids form nanoparticles with mRNA in an acidic buffer, which positively charges the lipid and attracts the RNA.
Furthermore, they are positively charged in the acidic environment of endosomes, which facilitates their fusion with the endosomal membrane, releasing them into the cytoplasm.
DODAP and DODMA were the first ionizable lipids for RNA delivery. The efficacy of DODMA was improved by design, resulting in DLin-MC3-DMA.
This is the first FDA-approved drug formulation to use an ionizable lipid: the siRNA drug patisiran ( Onpattro ).
In addition to the efficient and safe delivery of siRNA, DLin-MC3-DMA was also used for mRNA delivery.
Currently, many groups in academia and industry use combinatorial reaction protocols to synthesize potential delivery materials, and this approach yields a number of potent lipids, including C12-200, 503O13, 306Oi10, OF-02, TT3, 5A2-SC8 , SM-102 ( for the Moderna vaccine mRNA-1273 against SARS-CoV-2 ) and ALC-0315 ( for the Pfizer vaccine BNT162b2 ).
In addition to the quest to improve efficacy, there is an increasing focus on improving the specificity of drugs, especially for vaccines and immunotherapies.
The lipid 11-A-M58, which contains a polycyclic adamantane tail, and the lipid 93-O17S59, which contains a cyclic imidazole head, have been designed to target T cells in vivo. Although the mechanism remains unclear, the cyclic groups of these lipids are critical for targeting T cells.
Although ionized lipids are arguably the most important components of LNPs, three other lipid components ( cholesterol, helper lipids, and pegylated lipids ) also contribute to nanoparticle formation and function.
Cholesterol, a naturally occurring lipid, enhances nanoparticle stability by filling the voids between lipids and facilitates fusion with endosomal membranes during uptake into cells.
Helper lipids regulate nanoparticle fluidity and enhance efficacy by promoting lipid phase transitions that facilitate membrane-endosome fusion. The choice of the optimal helper lipid depends on the ionizable lipid material and the RNA carrier.
For example, for lipid-like materials, saturated helper lipids ( such as DSPC ) are best for the delivery of short RNAs ( such as siRNA ), while unsaturated lipids ( such as DOPE ) are best for the delivery of mRNAs. DSPC has been used in FDA-approved SARS-CoV-2 vaccines mRNA-1273 and BNT162b2.
The pegylated lipid component of LNPs is composed of polyethylene glycol ( PEG ) combined with anchor lipids such as DMPE or DMG .
Hydrophilic PEG can stabilize LNPs, modulate nanoparticle size by limiting lipid fusion, and increase nanoparticle half-life by reducing nonspecific interactions with macrophages.
Both mRNA-1273 and BNT162b2 SARS-CoV-2 vaccines contain pegylated lipids.
Polymers and Polymer Nanoparticles
Although not as clinically advanced as LNPs, polymers have similar advantages to lipids for efficient mRNA delivery. Cationic polymers condense nucleic acids into complexes of different shapes and sizes that can enter cells by endocytosis.
Polyethyleneimine is the most widely studied nucleic acid delivery polymer. Despite its excellent efficacy, its toxicity limits applications due to its high charge density.
In addition, several less toxic biodegradable polymers have been developed. For example, poly( β-aminoester ) excels in mRNA delivery, especially to the lung.
Recently, a new class of lipid-containing polymers, called charge-altered releasable transporters ( CARTs ), have been developed, which can effectively target T cells, which are very difficult to manipulate.
Therefore, CARTs are extremely Attractive delivery material with great potential in mRNA vaccines and gene therapy.
Other delivery systems
In addition to lipid and polymer carriers, peptides can also deliver mRNA into cells thanks to cationic or amphiphilic amine groups ( such as arginine ) in their backbone and side chains, which The base electrostatically binds to the mRNA and forms a nanocomplex.
For example, membrane fusion cell-penetrating peptides containing a repeating arginine-alanine-leucine-alanine ( RALA ) motif.
The arginine-rich protamine peptide, which is positively charged at neutral pH, can also concentrate mRNA and facilitate its delivery.
The complex of protamine with mRNA activates the Toll-like receptor ( TLR7, TLR8 ) pathway that recognizes single-stranded mRNA, therefore, it can also be used as an adjuvant for vaccine or immunotherapy applications.
CureVac AG is evaluating RNActive, a protamine-containing delivery platform, for clinical trials in melanoma, prostate cancer and non-small cell lung cancer.
Finally, mRNA can also be delivered by cationic nanoemulsions based on squalene, which consist of oily squalene cores.
Some squalene formulations, such as Novartis’ MF59, are used as adjuvants in FDA-approved influenza vaccines.
MF59 causes cells at the injection site to secrete chemokines, thereby recruiting antigen-presenting cells, inducing monocytes to differentiate into dendritic cells, and enhancing antigen uptake by antigen-presenting cells.
The mechanism by which squalene-based cationic nanoemulsions escape from endosomes and deliver mRNA into the cytoplasm is unclear.
Immunization of mRNA vaccines
Following injection of an mRNA cancer vaccine, the protein encoded by the mRNA is synthesized by the ribosome and then post-translationally modified to produce a properly folded functional protein, which is presented to the immune system.
This process is similar to the natural process of RNA virus infection and successive induction of protective immune responses. Entry of exogenous mRNA into the cytoplasm results in a similar response to endogenous mRNA.
Induce innate immune response
The host immune system activates the innate immune response through the detection of pathogen-associated molecular patterns ( PAMPs ) by PRRs.
Following vaccine injection, mRNA and delivery system components will be recognized as foreign substances through a series of PRRs, leading to the activation of Toll-like receptors ( TLRs ), such as TLR3, TLR7 and TLR8, which are mainly expressed on antigen presenting cells ( APCs ).
Exogenous IVT mRNA is recognized by various PRRs in the cell membrane, endosome, and cytoplasm, and plays a role in stimulating endogenous immune responses.
The immunogenicity of IVT mRNA is mainly mediated by TLR7 and TLR8. TLR7 is expressed by B cells, macrophages, and dendritic cells and mediates the recognition of single-stranded RNA ( ssRNA ). In the cytoplasm, some other PRRs can sense different types of RNA, such as dsRNA and ssRNA.
In non-immune cells, the cytoplasmic retinoic acid-inducible gene I-like receptor ( RLR ) and melanoma differentiation-related gene 5 ( MDA5 ) sense exogenous mRNA and regulate cytokine and chemokine production, leading to the recruitment of innate immune cells to the mRNA injection site.
Induce adaptive immune response
Following mRNA vaccination, the encoded protein is translated and presented to the immune system and stimulates adaptive immunity.
The mRNA-encoded proteins are taken up by APCs through endocytosis or phagocytosis, and they may form phagocytic vesicles or endosomes containing antigenic proteins that are presented by the major histocompatibility complex on dendritic cells.
mRNA cancer vaccines typically encode TAAs that are expressed on cancer cells.
These TAAs can be further divided into tissue differentiation antigens ( such as MART-1 ), tumor germline antigens ( such as NY-ESO-1 or MAGE-3 ), normal proteins overexpressed by tumor cells ( such as EGFR, MUC1, Her2/neu ) , viral proteins ( such as EBV, HPV ) and tumor-specific mutant antigens ( such as MUM-1, β-catenin or CDK4 ).
In addition, somatic mutations may generate neoantigen epitopes, and mRNA vaccines encoding neoantigens are considered to be the best candidates for cancer vaccines.
In addition to inducing T-cell immunity, mRNA vaccines also induce neutralizing antibodies.
Follicular helper T cells ( Tfh ) are not only critical for germinal center development, but also drive immunoglobulin-like transformation, affinity maturation, and durable B-cell memory responses.
These cells can be activated by the mRNA vaccine to produce sufficient numbers of potent and durable neutralizing antibodies.
Clinical Development of mRNA Cancer Vaccines
Vaccines based on IVT mRNA are being progressively developed for a variety of tumor treatments.
Currently, mRNA cancer vaccines can be classified into cancer vaccines encoding TAA, TSA, cytokines and antibodies according to the final product type.
mRNA vaccine encoding TAAs
NY-ESO-1, tyrosinase, MAGE-A3, MAGE-C2, and TPTE have been used as TAAs for melanoma mRNA vaccines in multiple clinical trials.
BNT111 is a BioNTech FixVac platform to develop cancer vaccines that utilizes a fixed combination of TAAs designed to trigger a robust and precise immune response against cancer.
In late 2021, the FDA granted Fast Track designation for BNT111, a novel cancer immunotherapy for advanced melanoma, which is currently being studied in two clinical trials.
An interim analysis of a phase I trial ( NCT02410733 ) reported that BNT111 is an effective immunotherapy.
A subsequent phase II trial ( NCT04526899 ) investigated the safety and efficacy of BNT111 alone or in combination with the anti-PD-1 antibody cemiplimab.
CV9201 and CV9202 are two mRNA vaccines against target antigens expressed in non-small cell lung cancer.
Clinical trials ( NCT00923312, NCT01915524, NCT03164772 ) showed that both vaccines were well tolerated and immune responses were detectable after treatment, supporting further clinical studies in non-small cell lung cancer.
mRNA vaccine encoding TSA
mRNA vaccines encoding multiple mutated antigens are ideal for the treatment of mutation-induced malignancies, and this type of cancer vaccine has undergone the most clinical trials.
With the development of next-generation sequencing technology, personalized mRNA cancer vaccines encoding mutated antigens can be produced, and such personalized cancer vaccines hold very attractive prospects.
Two personalized mRNA cancer vaccines are currently in Phase II clinical trials, Moderna’s mRNA-4157 and BioNTech’s BNT122 ( RO7198457 ).
mRNA-4157 specifically screens and encodes 20-34 neoantigens on a single mRNA molecule, depending on the patient’s cancer mutation.
Interim data from a Phase 1 trial ( NCT03313778 ) showed that mRNA-4157 monotherapy or in combination with the PD-1 inhibitor Keytruda was well tolerated at all doses tested and elicited neoantigen-specific T cell responses.
The vaccine is currently in a Phase II clinical trial in melanoma patients ( NCT03897881 ).
BioNTech’s BNT122 has explored the efficacy and safety of combination therapy with PD-L1 antibodies, with Phase II clinical trials in melanoma, non-small cell lung cancer and colorectal cancer initiated in the first half of 2021.
mRNA encodes an immunostimulator
Theoretically, mRNA cancer vaccines can encode any protein, including immune stimulators, which can reshape the tumor immune microenvironment ( TIME ) and overcome tumor immune tolerance, which has become an important direction of mRNA tumor vaccine research。
In the past few years, clinical trials of cancer vaccines with cytokine-encoding mRNAs have been conducted.
Currently, there are only seven product candidates in clinical trials, and they belong to Moderna and BioNTech.
In 2017, Moderna developed the first immunostimulant expressing mRNA encoding ( mRNA-2416 ), which encodes OX40L, and a Phase 1 clinical trial ( NCT03323398 ) evaluated the safety and resistance of mRNA-2416 alone and in combination with durvalumab acceptability.
Available data suggest that mRNA-2416 monotherapy is tolerable at all dose levels, with no reported dose-limiting toxicities and most associated adverse events of grade 1 or 2.
More importantly, broad pro-inflammatory activity and beneficial changes were observed during the time of PD-L1 upregulation.
Currently, intratumoral injection of mRNA-2416 has entered a phase II clinical trial in advanced ovarian cancer.
Another mRNA vaccine, mRNA-2752 , encodes OX40L/IL23/IL36γ, and early clinical results show that mRNA-2752, in combination with the anti-PD-L1 antibody durvalumab, was well tolerated at all doses and showed anti-PD-L1 Signs of tumor activity.
In addition, BioNTech has developed BNT131, encoding IL-12sc, IL-15sushi, GM-CSF and IFNα , as monotherapy or in combination with cemiplimab in patients with advanced solid tumors.
Over the decades, advances in mRNA design and nucleic acid delivery technology, coupled with the discovery of neoantigen targets, have made mRNA vaccines a powerful tool in the fight against cancer.
The advantages of mRNA vaccines are reflected in the ability of mRNA cancer vaccines to encode multiple antigens simultaneously, as well as the potential for non-integration, high degradation, and no insertional mutagenesis.
The mRNA produced by IVT is free of cellular and pathogenic viral components and has no potential for infection; most mRNA vaccines tested in ongoing clinical trials are generally well tolerated with few injection site-specific immune responses.
Rapid production is another advantage of mRNA cancer vaccines, and the maturity of mRNA manufacturing technology allows novel vaccines to be produced in a short period of time. The recent discovery and identification of neoantigens has facilitated the development of personalized vaccine treatments.
Several clinical studies conducted by BioNTech and Moderna have demonstrated robust anti-tumor immunity using a personalized vaccine in several clinical trials for the treatment of multiple solid tumors, ushering in a new era of therapeutic tumor vaccines.
There is reason to believe that mRNA therapy will have the potential to transform modern medicine’s approach to vaccination and cancer immunotherapy.
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2. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat RevDrug Discov. 2021 Aug 25 : 1–22.
2. Recent advances in mRNA vaccine technology. Curr Opin Immunol. 2020 Aug;65:14-20.
Will mRNA vaccines become the new hope of cancer immunotherapy?
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