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How mRNA therapy enters the field of monoclonal antibodies
How mRNA therapy enters the field of monoclonal antibodies. This article will focus on describing how mRNA drugs solve antibody and protein drug production, the pain points encountered in the delivery process, and the potential of mRNA drug delivery systems. At the same time, we will pay attention to the problems and difficulties faced by mRNA drugs in the process of realizing protein drug substitution.
Abstract: How mRNA therapy enters the field of monoclonal antibodies
In 1975, Milstein and Kohler invented the hybridoma technology for making monoclonal antibodies, which revolutionized the medical world. Since then, monoclonal antibodies have entered almost every field of biomedical research. At present, antibodies have been used as first-line therapy for a variety of highly different indications.
From autoimmune diseases to allergic asthma to cancer, there is no exception. However, due to the inherent production challenges and high development costs of protein drugs, it hinders the wider availability and implementation of antibody therapies.
For these reasons, it is imperative to find a more cost-effective and safe drug delivery system. In the past ten years, the research of mRNA medicine has made great progress, which can be used to express any meaningful protein in the body. Currently, a variety of clinical trials are proceeding in an orderly manner, aiming to explore the use of mRNA drugs and disease prevention and treatment.
Antibodies: from natural defense to treatment
The monoclonal antibody technology was founded in 1975. Since then, a steady stream of monoclonal antibody production has become possible, and the two scientists won the Nobel Prize in Physiology and Medicine in 1984. In 1986, the world’s first monoclonal antibody drug OKT3 was born.
However, it must be said that the first antibody drug must face the serious immunogenicity problem of mouse-derived antibodies. Fortunately, in the 1990s, molecular biology and recombinant monoclonal antibody technology had made great progress, which led to revolutionary progress in the monoclonal antibody industry, (Figure 1) fewer mouse sequences and the ultimate achievement of fully human antibodies. Today, antibodies are used as therapeutic drugs. As of November 2020, 97 antibody drugs have been approved by the FDA for listing. In 2019, antibody drug sales exceeded 140 billion U.S. dollars. In particular, therapeutic antibody drugs represented by immune checkpoints have brought revolutionary treatment options to cancer patients. Immune checkpoint antibodies have also become the most important and successful drugs for cancer treatment.
Although monoclonal antibody pharmaceutical companies have become the fastest-growing pharmaceutical companies, the necessary activities such as technology, supervision, and production strategies are still facing huge challenges in the production and preparation of huge clinical antibody demand. This huge challenge comes from the fact that most of the antibody drugs currently used for treatment are produced from mammalian cells, and a lot of purification work and formulation development work are required in the later stage.
In addition, monoclonal antibodies have a variety of post-translational modifications during the production process, including glycosylation, deamidation, oxidation, mismatches of free cysteine to form additional disulfide bonds, and N-terminal coke valleys. Cyclization structure formed by aminoacylation, C-terminal Lys deletion and other modifications. All of the above modifications strongly affect the activity and safety of antibodies. Therefore, the development and production of antibody drugs requires a series of strict characterization and quality control, and expensive late-stage development is required to enter the clinic.
Figure 1. Overview of the different forms of therapeutic monoclonal antibodies
The Age of Individuals as Bioreactors: Nucleic Acid Forms
An ingenious strategy that can circumvent the intricate post-translational modification of antibodies, expression and purification, and tedious and massive characterization and quality control in the later stage is to deliver the genetic information of the antibody drug itself. The genetic information of the expressed antibody is delivered to the patient in the form of DNA or mRNA instantaneously, which allows the patient to express in situ, eliminating all the tedious and time-consuming links in the preparation of antibody drugs.
Protein drugs are synthesized from 20 different amino acids, and different proteins have different physical and chemical properties. Therefore, different buffer systems, storage conditions, and formulation types need to be considered in the later stage. All these conditions must be optimized according to different antibody drugs. However, DNA and RNA consist of only four nucleotides, and their overall structure is a sugar-phosphate backbone with strong negative charges, and importantly, a high degree of physical and chemical consistency. The direct result is that there is no need to tailor the required expression and purification conditions for each antibody. Another major advantage of nucleic acid-encoded antibodies is that high-quality antibodies can be quickly designed and produced during the outbreak of infectious diseases, without the need for tedious production optimization conditions in the antibody production process.
The tipping point of a DNA-based treatment strategy was in 1990, Wolff et al. reported that plasmid DNA was injected into mice intramuscularly to express the target protein in situ. Different pre-clinical studies have proved that antibody expression genes can be delivered into the body for the purpose of preventing and treating infectious diseases, such as dengue virus, respiratory syncytial virus and chikungunya virus. Currently, some DNA drugs encoding antibodies are in clinical phase II-III. Despite this, there are no pDNA drugs on the market. The main reason is the worry that pDNA has the risk of integrating into the human genome and the production of anti-DNA autoantibodies. However, the facts have proved that these risks and impacts are extremely low. In addition, the site of inoculation is critical to the effectiveness of DNA vaccination. At present, the main clinical trial vaccination method is intramuscular injection. The effectiveness of intramuscular injection depends on the amount of inoculation. The amount of inoculation will increase local pressure, thereby increasing cell uptake, but at the same time it will also cause slight local tissue damage. These local tissue damages will stimulate and recruit professional antigen-presenting cells, causing the maturation of antigen-presenting cells. Therefore, intramuscular injection in rodents will produce a stronger immune response than large animals and humans.
Although many studies are still focusing on the progress of pDNA, given the limitations of traditional methods and the development of the latest in vitro stable transcription technology, mRNA delivery technology is more popular. In addition to safer drug properties (such as no risk of genomic integration), the transient expression of mRNA-encoded antibodies makes exposure more controllable and produces more protein during peak expression than naked pDNA. Because the mRNA vaccine lacks the original skeleton on the plasmid vector, there is no problem of inducing the production of anti-carrier antibodies. More importantly, RNA transcription is performed in vitro without any mammalian cells, so there is no risk of introducing foreign viruses. Secondly, using mRNA, the information encoded by the antibody is transmitted to the cytoplasm and directly translated into the encoded protein in the body. In this way, the risk of abnormal post-translational modifications inherent in protein delivery can be avoided. From a regulatory perspective, mRNA delivery has recently been listed by the EMA as an advanced therapeutic drug product (ATMP) and as a more precise gene therapy drug (GTMP).
All the above advantages highlight the great potential of IVT mRNA in remodeling antibody-mediated therapy. Nevertheless, to successfully replace current protein antibodies, mRNA methods need to transcend their own challenges, which are mainly at the level of delivery and immunogenicity.
mRNA is a promising therapeutic platform
Synthesis of mRNA has an attractive prospect
A few years ago, wolff et al. reported a strategy for expressing target proteins in IVT mRNA in vivo. Despite this promising discovery, it was thought that mRNA was particularly unstable. Under normal circumstances, unmodified IVT mRNA would be degraded by ubiquitous extracellular or ribonuclease. The instability of mRNA is that the idea of its application in treatment is full of challenges. In order to improve molecular stability and protein translation, the researchers made a series of modifications to the vector used to produce mRNA and the synthetic mRNA itself.
The mRNA in vitro transcription template is composed of five cis-acting structural elements. From 5’to 3′, they are:
- (1) optimized cap structure,
- (2) optimized 5’UTR,
- (3) optimized target gene sequence,
- ( 4) Optimized 3’UTR,
- (5) Optimized poly(A) tail.
These cis-acting structural elements are constantly being optimized to obtain better mRNA characteristics. Figure 2 is a schematic representation of the optimized mRNA. The poly-A-tail and cap structures are of great significance for the effective translation of mRNA and the stability of mRNA decay, while UTR controls the translation and half-life of mRNA. Finally, purification and incorporation of modified nucleosides, including 1-methylpseudouracil (m1ψ), such as high performance liquid chromatography, makes mRNA immunogenicity non-immunogenic and significantly improves translation efficiency.
Figure 2. Overview of the optimization of mRNA structure
IVT mRNA is a linear DNA template containing UTR’s optimized by phage promoters, optimized sequences, and synthesized in vitro using an RNA polymerase (T7, T3 or SP6) and mixed nucleotides. The cap structure and polyA tail can be added during the transcription process, but can also be added through enzyme action after IVT. The resulting product is purified to remove contaminants such as short transcripts or dsRNA. In this way, the in vitro synthesis of IVT mRNA is similar to the process of mRNA processing in eukaryotic cells.
A series of modifications to the vector used to produce mRNA and the synthetic mRNA itself have improved the biological characteristics of IVT mRNA. These improvements have led to different application areas of mRNA therapy. The first field to enter is the field of therapeutic cancer vaccines, which requires a lower safety standard. With the further optimization of IVT mRNA and the further reduction of inflammatory side effects, IVT mRNA has entered the second application field, namely the field of vaccination.
There are different clinical studies passed and ongoing IVT mRNA in the field of cancer vaccination and vaccination. Outside of these two areas, people are interested in IVT mRNA as a protein replacement therapy. However, this is very challenging because it requires targeted expression of mRNA and repeated administration, in some cases even systemic administration. These requirements mean that the system must have high security, which is very challenging. So far, there is no clinical research on IVT mRNA in the field of protein replacement.
Until recently, mRNA has entered the field of gene editing to transiently express the required enzymes in cells. For example, there is a transient expression of zinc finger nucleases (ZFNs) using mRNA-based vectors, such as ZFNs, which can produce off-target effects at non-targeted chromosomal sites whose sequences are similar to the expected targets. Similarly, mRNA encoding transcriptional activator-like effector nucleases (TALENs) have been marketed. But in the field of genome editing, the latest tool (CRISPR) / CRISPR-associated (Cas) system. In addition, the system can also use mRNA encoding Cas cleavage protein. Figure 3 outlines mRNA-based therapy.
Origin of antibody expression based on mRNA platform
Hoerr et al. first proposed the concept of using mRNA to encode antibody protein in a patent application in 2008, and RNA encodes antibody (EP 2101823 B1). In March 2017, Pardi et al. published the first article on the feasibility of passive vaccination using mRNA. In this article, m1ψ contains the mRNA encoding the light and heavy chains of VRC01 (a broadly neutralizing antibody against HIV-1), which is administered systemically in the form of liposomal nanoparticles LNP. Pardi et al. also confirmed that intravenous injection of the liposome nanoparticles can efficiently express the target protein in the liver. A single intravenous injection of 30 μg of mRNA-LNP expressing VRC01 antibody, the level of VRC01 in serum reached a peak at 24 hours, and gradually decreased over time until the next injection on the 11th day. MRNA-LNP encoding VRC01 antibody is better than direct administration of VRC01 antibody in the prevention of HIV-1 in mouse models.
Figure 3. Overview of treatment strategies based on IVT mRNA
A few months later, Thran et al. respectively confirmed the feasibility of using mRNA for passive vaccination in three different disease models: as an anti-pathogen therapy (rabies model), as an antitoxin therapy (botulism model), and as an antitoxin therapy (botulism model). Anti-tumor therapy (lymphoma model).
Experiments have shown that a single injection of mRNA-LNP encoding monoclonal antibodies or camel heavy chain antibodies (VHHs) is sufficient to establish rapid, potent and durable serum antibody titers in the body. These high-titer antibodies can completely protect mice from virus attack or poisoning, and can eliminate tumor cells in mouse models. In addition, the researchers also studied the general tolerance of mRNA-LNP treatment. Although some circulating cytokines have short-term low-level increases, this weak increase does not prevent the expression of high protein. More importantly, the histopathology of the liver, the target organ of mRNA-LNPs, did not reveal any signs of abnormality or inflammation.
In the same period, the third researcher Stadler et al. reported on the preclinical study of mRNA for the construction of bispecific antibody BiTE. Shows a strong anti-tumor effect. Compared with monoclonal antibody drugs, traditional bispecific antibody drugs have a more complicated and tedious process of expression and purification. In order to solve these problems, Stadler et al. designed IVT-modified mRNA, encoding bispecific antibodies, called RiboMABs, against T cell receptor-related molecule CD3 and one of the three tumor-associated antigens (TAA).
Compared with traditional protein-based bispecific antibodies, Ribomab is easier to use and requires a smaller dosage to produce Abs that achieve therapeutic effects. But the main advantage is that in terms of development, you can easily change the DNA to make the corresponding RNA and compare it with the candidate antibody structure. This rapid procedure can evaluate different antibodies in a short period of time.
A few micrograms of mRNA encoding RiboMABs can be injected intravenously to achieve rapid expression of antibodies in the liver and circulate into the blood. The level of RiboMABs in the blood reaches its peak within a few hours and can maintain a therapeutic level concentration for about a week. Experiments have shown that RiboMABs are tested in xenograft mouse models with larger ovarian tumors. The 3-week RiboMAB treatment completely eliminated the tumor, which was equivalent to the effectiveness of the corresponding recombinant bispecific antibody, although the latter required three uses to achieve the same degree of tumor eradication.
The research discussed above requires the liver to be used as a bioreactor to deliver mRNA and provide antibodies systematically. The difference is that Tiwari et al. reported local delivery of anti-respiratory syncytial virus (RSV) monoclonal antibody (palivizumab) and VHH mRNA. Because protection from respiratory syncytial virus infection only requires protective antibodies in the lungs, rather than the entire body, local delivery of mRNA encoding Ab is more advantageous.
The authors used secretory and membrane-anchored palvizumab or anti-rsv VHH encoded by naked mRNA to deliver them to the lungs through intratracheal aerosol. Studies have shown that using this delivery method, as many as 45% of lung cells show detectable antibody expression, resulting in RSV infection, secreted Ab can be maintained for 4 days, and anchored VHH can be maintained for 7 days. The virus infection is greatly reduced. More importantly, when naked mRNA is delivered through tracheal aerosol, no significant increase in cytokine expression was observed in the lung within 24 hours after treatment. Table 1 summarizes the preclinical studies of mRNA-encoded antibodies.
mRNA highlight therapy
IVT mRNA activates the immune system
IVT mRNA immunogenicity is the biggest obstacle to its preparation. Eukaryotic cells express different pattern recognition receptors (PRRs) to recognize the characteristic structures of infection. mRNA is recognized by PRRs, such as toll-like receptors (TLR) 3, 7, 8 and retinoic acid inducible gene I (RIG-I), leading to the expression of pro-inflammatory cytokines or the activation of inflammasomes. TLR3 and TLR7/8 recognize double-stranded and single-stranded RNA, respectively. Systemic delivery of conventional and unpurified IVT mRNA can activate the immune system and subsequently lead to the production of pro-inflammatory cytokines and type I interferons.
Compared with endogenous RNA, foreign RNA has (potentially) different base modification patterns. The incorporation of naturally occurring modified nucleosides can (partially) bypass PRR’s recognition of mRNA, thereby reducing immune stimulation and enhancing the expression of encoded proteins. For example, the activation of TLR 7 and 8 can be avoided by adding naturally modified nucleosides, such as pseudouracil, 2-thiouracil, 5-methylpyridine, N1-methylpseudouracil or 5-methylcytidine .
In addition, studies have shown that IVT mRNA containing pseudouracil and 2-thiouracil is not recognized by RIG-I and PKR. Previous studies on IVT mRNA showed that all uridine was replaced with pseudouridine, which is the most common naturally occurring nucleoside modification, indicating that the mRNA is not immunogenic. Research by Kormann et al. showed that combinatorial chemical modification of 2-thiouracil and 5-methylcytidine can effectively reduce the recognition of IVT mRNA by TLR3, 7, 8 and RIG-I on human PBMC cells. Recently, studies have shown that (m1ψ) mRNA modified by N1-methylpseudouracil can effectively escape innate immunity and increase expression in vivo and in vitro. In short, chemical modification of mRNA nucleosides is an important technology for regulating mRNA immunogenicity, and a lot of research is ongoing.
Unfortunately, modified nucleoside-containing RNA transcribed by phage RNA polymerase still maintains a low level of activation of the innate immune response pathway. The background activation of RNA receptors by nucleoside-modified RNA may be due to the fact that the modification does not completely inhibit the ability of RNA to activate and recognize receptors, or due to contaminants with activated structures in the presence of nucleoside modifications.
It is well known that RNA transcribed by phage polymerase in vitro contains a variety of contaminants, including short RNA and double-stranded RNA produced by synthesis failure, rna produced by self-complementary 3’extension, RNA primer transcribed from RNA template and RNA-dependent RNA Polymerase activity. For example, dsRNA activates RIG-I, MDA5, PKR and 2′-5′ oligoadenylate synthase. High-performance liquid chromatography (HPLC) purification removes dsRNA and other contaminants in in vitro transcribed RNA, and produces higher levels without releasing type I IFNs or TNF-α and not significantly inducing genes related to the activation of RNA recognition receptors. translation.
In addition, RIG-I, IFIT1 and MDA receptors can distinguish different cap structures. Decades of studies have confirmed that m7G cap, as a unique molecular module, can recruit intracellular proteins and mediate cap-related biological functions, such as pre-mRNA processing, nuclear export, and cap-dependent protein synthesis. Until recently, the role of cap 2’O methylation as an identifier for self-RNA, distinguishing it from foreign RNA, and assisting in the innate immune response to foreign RNA, has become clear. These new findings emphasize the importance of proper cap structure in the synthesis of functional mRNA, and point out that the search for better cap structure can be continued. mRNA can also be removed with phosphatase to remove the uncapped 3’triphosphate ends.
Although IVT mRNA has the above-mentioned adaptability, the problems of ADA (anti-drug antibody) response and transient cytokines have not been solved, which hinders the clinical applicability of mRNA, especially when multiple administrations are required. The above two questions.
However, the inherent immunogenicity of mRNA is a double-edged sword. On the one hand, the (systemic) delivery of conventional and unpurified IVT mRNA can activate the immune system, which subsequently leads to the expression of unwanted (systemic) pro-inflammatory cytokines and type I interferons (IFNs). This inherent immunostimulatory activity can directly interfere with the target treatment effect, for example, in gen replacement therapy, because the occurrence of innate immunity reduces the expression of the target protein.
But on the other hand, in some application fields, such as preventive vaccination, the inflammatory cytokines produced by the recognition of mRNA may increase the effectiveness of the triggered immune response and make mRNA become its own adjuvant. The intrinsic adjuvant properties of mRNA appear to be based primarily on its ability to stimulate type I IFNs. The effect of type I interferons on T cell immunity can be beneficial or harmful, depending on their induction kinetics, strength and anatomical distribution. Studies have shown that type I interferon has a significant stimulating effect on intravenous injection, but has a significant inhibitory effect on local injection. In addition, the application of mRNA faces a relative confusion. The ubiquitous RNA enzymes affect the extracellular half-life of mRNA.
The development of mRNA therapy faces the same challenge as any nucleic acid, that is, the problem of delivery. As a negatively charged high molecular weight molecule, mRNA is inherently unsuitable for passing through the cell membrane and reaching its target location, the cytoplasm. The difficulty of efficient delivery of RNA has severely hindered the application of RNA in drug development. For this reason, a variety of methods have been developed, including optimized injection strategies, gene gun-based drug delivery, protamine complexes, RNA adjuvants, and delivery of RNA encapsulated in nanoparticles composed of polymers and liposomes .
In theory, exogenous RNA needs to pass through the lipid bilayer of the cell membrane, be internalized by the target cell, and be translated into a functional antigen. Naked mRNA is taken up spontaneously by many different types of cells. Most types of cells do internalize mRNA through various internalization pathways, resulting in mRNA being trapped in the acid lysosome compartment for degradation. Immune cell dendritic cells (DCs), which specialize in antigen presentation and nucleic acid induction, seem to be an exception to this rule, because it has been reported that dendritic cells can be expressed at a reasonable efficiency when administered in lymph nodes or tumors. Naked mRNA.
How DCs transport mRNA to the cytoplasm remains unsolved, but this seems to involve the transport of a large molecule to the cytoplasm, similar to the internalization of protein antigens in DCs. Studies have shown that the uptake of naked RNA by immature dendritic cells is an activation process, including scavenger receptor-mediated endocytosis and micropinocytosis. Both of these pathways lead to endolysosome localization, and only a small part of the complete RNA eventually enters the cytoplasm. To solve this problem, various forms of mRNA have been designed to enter the antigen-presenting cells and increase the amount of RNA that enters the cytoplasm after absorption. Most of the developed methods are based on the form of nanoparticles, such as liposomes, polymers and peptides.
According to research by Paunovska et al., not all intravenously injected mRNA liposome nanoparticles have the same protein expression level, which mainly depends on the form of ionized liposomes and the form of lipidated preparations. Second, as described in Jain’s paper, the UTR sequence determines the degree of specificity of RNA organization and cells. A recent study by Fenton et al. reported that lipid nanoparticles selectively target B cells in the spleen.
However, in most cell types, effective mRNA expression requires the mRNA to be encapsulated in a nano-expression vector to help the cell uptake and mediate the escape of mRNA from endosomes into the cytoplasm. For complex mRNAs with negative charges, cationic lipids are very suitable because the two components interact spontaneously to form liposome complexes.
There are two main benefits of liposome delivery of mRNA.
- First, mRNA is concentrated and aggregated into particles within the microsomes, which are then taken up by specialized antigen-presenting cells.
- Second, in the condensed state, mRNA is not susceptible to degradation mediated by intracellular and extracellular enzymes.
In order to develop a safe and powerful carrier suitable for mRNA delivery, the mRNA field is currently making full use of the vast knowledge and experience gained during the clinical development of small interfering RNA (siRNA). An efficient delivery system of siRNA can be achieved by encapsulating it in LNPs. The first RNAi LNP drug product (Patisiran) was approved by the FDA in August 2018, and there are other projects in the later clinical stage. Lipid nanoparticles are composed of four different lipids with special functions, which are mixed with RNA in different proportions under acidic conditions. Ionized lipids are one of the most critical components of these LNPs, and are responsible for the complexation of mRNA and the release of mRNA in vivo through charge interaction.
Compared with the early cationic lipids with permanent positive charges, the new generation of lipids contains amine groups that are positively charged under acidic pH conditions, and are electrically neutral under physiological pH conditions, thereby reducing toxicity and improving Efficiency. In addition to ionizable lipids, LNPs usually contain cholesterol, an auxiliary lipid, and polyethylene glycol lipids.
Although LNPs are promising carriers, to realize the clinical application of LNPs mRNA, there is still a major safety barrier to be resolved. So far, clinical data on the safety and effectiveness of LNPs mRNA are very limited, and ongoing staging studies are still limited to local (intramuscular, intratumor) delivery of LNP-packaged mRNA.
After obtaining encouraging results in rodents and non-human primates, Moderna initiated a low-dose first-in-human (100µg) evaluation of the safety and immunogenicity of the HA influenza vaccine mRNA LNP (clinical trial) NCT03076385). Interim results were reported in early 2017, demonstrating that the vaccine induced sufficient immunogenicity and acceptable tolerance. However, even at this low dose, in most and 3/23 subjects showed mild and moderate local reactivity or systemic side effects. Although these data support further research on mRNA LNP therapy, studies still show that clinical safety is still the biggest obstacle to the development of mRNA drugs, especially when high-dose (mg/Kg) systemic administration is repeated, this safety problem will still arise In systems where mRNA encodes antibody drugs.
The toxicity of mRNA LNPs can be diverse, including immune-related toxic events, and cytotoxicity caused by liver lipid accumulation. Similar to other nanomedicines, LNPs have been reported to activate the complement system, which has the risk of triggering allergic reactions, called complement activation-related pseudoallergies (CAPPA). In addition, due to stability reasons, LNPs usually contain PEG-modified lipids, which can easily activate splenic B cells to produce anti-PEG antibodies. The anti-PEG antibody not only mediates allergic reactions during the second administration, but is also the basis of accelerated blood clearance (ABC).
The mediated LNPs can be quickly cleared by macrophages, and cause each subsequent administration The mRNA expression level gradually decreased. The degree of anti-PEG antibody production is induced and regulated by two factors:
(1) the immunogenicity of the mRNA,
(2) the length of the fatty acid chain that constitutes the fat modified by PEG.
As mentioned above, non-purified, unmodified mRNA can be recognized by a variety of different RNA recognition receptors, leading to the secretion and release of inflammatory factors and promoting the differentiation and maturation of different PEG-recognizing B cells to produce antibodies. High-purity innate silenced mRNA avoids these adjuvant effects, thereby limiting the activation of B cells. The second major determinant of PEG immunogenicity is the length of the PEG-lipid fatty acid chain used to stabilize LNPs.
Cullis’s research team proved that C18 polyethylene glycol lipids (such as distearoylglycerol) can be very stably incorporated into LNPs, while C14 polyethylene glycol lipids can quickly dissociate from LNP in the blood. The binding of this highly stable C18 PEG-lipid to LNPs expands and strengthens the recognition of PEG moieties by splenic B cells, and finally C18 PEG LNPs increase the titer of anti-PEG antibodies. Combining innate silenced mRNA and C14 PEG-based LNPs seems to be a very successful method to avoid allergic reactions and blood clearance effects. Recent preclinical studies have proven this, at least in relatively low doses and rodents.
RNA as an antibody platform specific challenges and prospects
So far, there have been few preclinical studies on mRNA encoding antibodies, mainly in small rodents. Therefore, this expression and effectiveness need to be verified in larger animals and ultimately in humans. Earlier studies on another type of secreted protein, erythropoietin, showed that the findings in mouse models can be applied to large animals, such as domestic pigs and even primates. These results give us hope that the data of mRNA-encoded antibodies in the mouse model may also be analogous to humans.
The feasibility of monoclonal antibody pharmaceuticals follows three principles: (1) Serum antibody titer increases rapidly after injection, (2) Serum antibody is high enough for protection and treatment, and (3) Serum antibody half-life is long enough.
The increase and level of monoclonal antibodies depends on the formulation and delivery route of the drug. Most of the mRNA delivery methods that have been developed are based on the formation of nanoparticles, such as the use of liposomes, polymers, and peptides. Pardi et al. found that mRNA encapsulated in lipid nanoparticles can produce high levels of protein for a long period of time when administered through multiple routes. So far, only lipid nanoparticle vectors have been used to encode Ab mRNA.
In addition to the form of mRNA, the delivery system also affects the titer of serum antibodies. There are three main delivery routes for mRNA vaccines currently studied: local delivery (eg, intrapulmonary, intradermal and subcutaneous), targeted delivery (lymph nodes) or systemic delivery (venous). Recent studies have shown that antibody titers can be detected on the first day after intravenous injection of mRNA encoding antibodies. So far, only studies by Tiwari et al. have shown data on local delivery of intratracheal aerosols to the lungs.
Except for these two methods of administration, no other routes of administration have been detected with mRNA encoding Abs. Given the approved treatment regimens, it is very important to target mRNA delivery to the organ of interest to minimize the amount of drug required to cause systemic toxicity, anti-antibody immune response, and reach therapeutic levels. Moreover, targeted delivery may also reduce the amount of mRNA reaching the target cell, thereby reducing the antibody encoded by the cell.
The therapeutic doses of recombinant antibodies currently used are usually very high. So far, it is not clear whether these high doses can be achieved by the administration of encoding mRNA. Despite this, there are still people who are optimistic that mRNA is superior to recombinant antibodies for use as therapeutic drugs. First of all, because the antibody is expressed in situ, it reduces the high local protein concentration required to achieve the therapeutic effect. Second, based on the doses tested so far, no saturation or dose limiting toxicity of mRNA-mediated antibodies has been detected. Furthermore, optimizing the targeted mRNA and further improving the formula may further greatly improve the efficacy.
The serum half-life of the Ab encoded by mRNA depends on the half-life of the Ab itself on the one hand, and on the mRNA encoding the Ab on the other hand. More specifically, the half-life of the first stage is determined by the half-life of mRNA and protein, while the half-life of the second stage is almost entirely determined by the characteristics of the protein. This means that the half-life of short-lived proteins can significantly benefit from mRNA expression.
For longevity proteins, using the mRNA expression platform has no significant effect on the duration of the therapeutic effect, but the mRNA half-life does contribute to peak expression. In addition, the size of the antibody molecule limits its applicability in mRNA format. For example, the commonly used IgG subtype is in the range of 150 kDa. In addition, antibodies are complex multi-domain proteins that must be assembled in the correct way.
In order to overcome the size and stability limitations of mAbs encoded by mRNA, a lot of research has been conducted on heavy chain antibodies (VHH) and small non-antibody scaffold proteins. There are two types of non-antibody scaffolds, namely (i) domain-sized compounds (6 to 20 kDa) such as DARPinsalphabodies, and (ii) bound peptides (2-4 kDa). Different stents are currently in the stages of academic research, preclinical and clinical development, and have shown great potential in terms of affinity, target neutralization, and stability. However, non-antibody scaffolds also face their own challenges, in which serum half-life and tissue penetration are the most important.
Particularly worthy of investigation is the effectiveness of mRNA encoding on non-antibody scaffolds, because the mRNA platform can have a positive effect on serum half-life, as described above.
The therapeutic antibody industry is staged a scene of exciting competitions on a global scale. Competition and cooperation coexist, with opportunities and challenges. Antibodies have shown unique effects in the fields of anti-infection, cancer treatment, respiratory diseases, metabolism, and cardiovascular diseases. The time, money and purification of antibody production, and the complicated downstream process have to make people try more possibilities.
In order to avoid complicated production and purification processes and abnormal post-translational modifications of protein-based monoclonal antibodies, alternative methods for rapid production, management and testing of monoclonal antibodies are currently being explored. Nucleic acid therapy has great potential because it is simple, fast, and cost-effective, does not require complex and expensive laboratory infrastructure, and all mRNA has a common production process. Recent advances in mRNA, including improvements in in vitro transcription, have increased interest in the therapeutic potential of this biomolecule.
Unlike DNA, mRNA only needs to reach the cytoplasm to induce protein expression, and there is no obvious risk of insertion mutation. Compared with protein-based platforms, mRNA-based Ab therapy platforms have different advantages. First, the expression of Ab encoded by mRNA can be detected within a few days. Second, the mRNA form is easier to produce intracellular antibodies. Third, since protein is composed of 20 different amino acids, the physical and chemical properties of different proteins are different, which means that the storage buffer and formula of each protein need to be optimized separately. mRNA only uses four nucleosides, resulting in its structure basically having the same physicochemical characteristics, regardless of the physicochemical properties of the protein it encodes.
A strategy based on mRNA encoding an antibody has been reported, and this strategy has gradually matured over the past few decades. . So far, the applicability of the mRNA platform for antibody therapy has been studied in the context of antitoxins, infectious diseases and oncology. Although initial reports indicated that mRNA is an emerging antibody gene transfer platform, the further development of mRNA-based monoclonal antibodies is limited by the need for safe and effective delivery systems. In addition, mRNA can only produce post-translational modifications of the antibody in its natural state. The strategy of increasing the serum half-life of antibodies by modification cannot be realized on the platform of mRNA encoding antibodies.
For passive immunity, very high safety is required. In the past few decades, people have made various optimizations of IVT mRNA to avoid harmful immune activation and cytokine induction of cellular RNA recognition receptors. Although the adaptability to IVT mRNA is described above, the ADA (anti-drug antibody) response and the appearance of transient cytokines can still be detected, thus hindering the clinical applicability of mRNA drugs, especially when multiple large doses are required At the time of administration. It is worth noting that when nanoparticles are repeatedly administered, they can also induce pseudoallergic complement activation. Since mice are not sensitive to induced complement activation pseudoallergies, more careful analysis of the formulation is required.
In short, mRNA encoding antibodies is a viable treatment option, which can avoid complicated production and purification processes and the inherent abnormal post-translational modifications of protein mAbs. Nevertheless, to successfully replace the current protein antibody format, mRNA methods need to surpass their own challenges, which are mainly drug delivery and mRNA immunogenicity levels. However, with the emergence of mRNA as a treatment method and the continuous growth of research on this topic, mRNA therapy is likely to be further developed and improved in the next few years.
How mRNA therapy enters the field of monoclonal antibodies
Lien Van Hoecke, Kenny Roose. How mRNA therapeutics are entering the monoclonal antibody field.J Transl Med. 2019 Feb 22;17(1):54.
(source:internet, reference only)