- Modified mRNA (mod mRNA) Allows the Human Body to be a Bioreactor for Antibodies
- Nucleobase Modifications in mod mRNA Provide Multiple Benefits
- Delivery of mod mRNA Remains a Big Challenge
As indicated by the title, this blog is Part 2 of a two-part series on how mRNA therapeutics are entering the monoclonal antibody (mAb) field. Part 1 was posted on July 9, 2019, and it provides a historical perspective on the evolution of mAbs, the importance of which was recognized by the award of three Nobel Prizes in 1901 (the first ever Nobel), 1983, and 1987. Part 2 of this series will discuss how mod mRNA-encoded protein has become an appealing alternative for producing mAbs, over traditional hybridoma-based protein production formats.
The Human Body as Its Own Bioreactor for Antibodies
In Part 1, the reference to a 2019 review by Van Hoecke and Roose noted the complexities and problems associated with hybridoma-based production of mAbs. An elegant solution to circumvent the problems of complex production and purification processes, as well as aberrant posttranslational modifications of an Ab, is to deliver the genetic information of the mAb. Transient gene transfer aims to administer the Ab-encoding nucleotide sequences in DNA or mRNA form, instead of the Ab protein itself, directly to patients. This allows for the in situ production of an Ab in a cost and labor effective manner, potentially for a prolonged period of time. In striking contrast to proteins, which can have quite different physicochemical properties, the polyanionic nature of the sugar-phosphate backbone of DNA and mRNA leads to largely consistent physicochemical characteristics regardless of the encoded protein. Because of this, the production and purification process for DNA and mRNA is more straightforward.
The field of DNA-based therapeutics was sparked in 1990, when Wolff et al. demonstrated that naked plasmid DNA (pDNA) injected in the quadriceps of mice, resulted in the expression of the encoded protein, as depicted here. According to Van Hoecke and Roose, preclinical studies have shown that the delivery of DNA-encoded Abs can protect against infectious diseases, such as dengue virus and respiratory syncytial virus. However, a pDNA-based pharmaceutical for humans has not been brought to market yet, due to concerns regarding the potential integration of pDNA into the host genome, the fear of anti-DNA autoantibodies, and the site and type of vaccination.
Illustration of a bacterial plasmid, a relatively small double-stranded DNA molecule that is commonly used for cloning in bacteria. The cloning plasmids contain a site for a DNA insert (orange).
mRNA: A New and Appealing Therapeutic Platform
Synthetic mRNA as an Attractive Chemical Blueprint: The aforementioned limitations of pDNA, as well as the advent of synthesizing chemically modified mRNA (mod mRNA) to increase stability in vivo yet retain translatability, have led to a considerable interest in mRNA as a delivery vector for safer and more controlled Ab formation. Readers interested in mod mRNA can peruse my earlier blogs, and also take advantage of Chemical Modification of mRNA by Dr. Anton McCaffrey, Sr. Dir. R&D Biology, TriLink BioTechnologies, an excellent video presentation.
Nonetheless, in order to successfully replace the current protein Ab format, mRNA approaches will need to overcome some challenges, such as delivery and immunogenicity, according to Van Hoecke and Roose. As depicted here, the template for transcription of mRNA consists of five structural elements: (i) the optimized cap structure, (ii) the optimized 5′ untranslated region (UTR), (iii) the codon-optimized coding sequence, (iv) the optimized 3′ UTR and (v) the polyA tail. These structural elements are discussed on the TriLink webpage titled Anatomy of an mRNA.
Schematic representation of optimized mRNA. Taken from Van Van Hoecke and Roose J. Transl. Med. (2019) 17:54. © The Authors 2019 (Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium.
The TriLink Education webpage features a number of posters on translation-optimization strategies, as well as a poster titled Strategies to Minimize Innate Immune Stimulation to Maximize Messenger RNA Bioavailability. For maximal expression in cells or target organs, transfected mRNAs must avoid detection by pattern recognition receptors (PRRs). The function of PRRs is to sense pathogenic non-self RNAs, including improperly capped RNAs and double stranded RNA. PRR activation leads to cytokine production, translational arrest, and cell toxicity or death.
Mammalian mRNAs are modified post-transcriptionally to contain nucleotides with 2’-O-methyl residues, pseudouridine (Ψ) (triphosphate shown here; R = H), and N6-methyladenosine (m6A). These modifications can reduce the activation of PRRs, and they allow for maximal translation of the transfected mRNA. In this poster, TriLink R&D experimental results are presented for transcribed mRNA using several Ψ derivatives to explore the impact on translation and innate immune stimulation: N1-ethyl-Ψ, N1-fluoroethyl-Ψ, N1-propyl-Ψ, N1- isopropyl-Ψ and N1-methoxylmethyl-Ψ 5’-triphosphates. Among the conclusions drawn from this study, efficiency of transcription using U-depleted templates greatly improved quality and yield, while N1-substituted Ψ mRNAs show potential translational and immunological properties.
Improvements such as these have catapulted the entry of mRNA therapeutics in different fields of applications, as depicted here in overview form. According to Van Hoecke and Roose, the first field of entry was the therapeutic cancer vaccination field, in which a “lower safety bar” is needed. As the IVT mRNA was further optimized to reduce the inflammatory side effects, it entered a second field of application: prophylactic vaccination. In addition to these two areas, there is an interest in mRNA for protein replacement therapy. However, this is highly challenging as it requires targeted expression of the mRNA and repeated administration, in some cases systemically. These requirements imply a “higher safety bar,” and investigations are still in the preclinical stage. Gene editing strategies using mRNA are also in the early stages of development, according to Van Hoecke and Roose, who provide various references.
Rybakova et al. have recently described the development of an IVT-mRNA system for the in vivo delivery of a widely used humanized anti-HER2 antibody, trastuzumab (aka Herceptin). They have also demonstrated its anticancer activity. The researchers engineered the IVT-mRNA sequence to maximize expression, later formulating the IVT-mRNA into lipid-based nanoparticles (LNPs) to protect the mRNA from degradation and enable efficient in vivo delivery. Systemic delivery of the optimized IVT-mRNA loaded into LNPs resulted in antibody serum concentrations of 45±8.6 μg/ml for 14 days post LNP injection. Further studies demonstrated an improved pharmacokinetic profile of the produced protein compared to injection of trastuzumab protein. Finally, treatment of tumor-bearing mice with trastuzumab IVT-mRNA LNPs selectively reduced volume of HER2-positive tumors and improved animal survival. Thus, the data from this study demonstrates that utilizing IVT-mRNA LNPs to express full size therapeutic antibodies in the liver can provide an effective strategy for cancer treatment, as well as an alternative to protein administration.
Emergence of mRNA as a Platform for mAb Production: The first peer-reviewed publication showing the feasibility of using mRNA for passive vaccination was published by Pardi et al. in Nature Communications. N1-methyl-Ψ-containing mRNAs encoding the light and heavy chains of VRC01, a broadly neutralizing antibody against HIV-1, were formulated in lipid nanoparticles (LNPs; reviewed by Cullis and Hope) and delivered systemically. The therapeutic capacity of these mRNA LNPs was shown in a prophylactic mouse model for HIV-1, and it outperformed the recombinant purified protein VRC01 mAb delivery.
The feasibility of using mRNA for passive vaccination was independently confirmed in a publication a few months later by Thran et al. in three different disease models: as an anti-pathogen therapy (rabies model), as an anti-toxin therapy (botulism model) and as an anti-tumor therapy (lymphoma model). Van Hoecke and Roose add that, in the same period, a third group entered the field of mRNA encoding antibodies. Stadler et al. reported preclinical data in Nature Medicine on a new class of drugs that instruct the body to create its own bispecific antibodies, called RiboMABs. These act by connecting human immune cells to tumor cells for highly efficient killing, and have demonstrated great promise as immunotherapy agents. A successful example in the clinic is blinatumomab, a form of bispecific T-cell engaging Abs or BiTE®, which is used to treat acute lymphoblastic leukemia. As depicted here, BiTE® targets two sites in order to cluster T-cells to lymphoma tumor cells.
Van Hoecke and Roose point out that most bispecific Ab formats suffer from the demanding procedures required for the production, purification, and formulation of the recombinant protein. In addition, the low serum half-life of bispecific antibodies (<2 h in patients), warrants a continuous infusion for therapy, hindering the development of new drugs in this class of therapeutics. Stadler et al. bypassed these limitations by engineering IVT mod mRNA RiboMABs, which should be easier to administer and require less frequent dosing than a conventional protein-based bispecific Ab.
Another major advantage is on the development side, as one can easily change the DNA, make the RNA from it, and compare it to other candidate antibody constructs. This quick procedure allows for the evaluation of different antibodies in a very short period of time. RiboMAB was formulated into LNPs and tested in xenograft mice harboring large ovarian tumors. Three weekly IV administrations of RiboMAB completely eliminated the cancer. This outcome was comparable to the effectiveness of the corresponding recombinant bispecific antibody, albeit the latter had to be administered three times as often to achieve the same degree of tumor eradication.
Challenges and Conclusions for mRNA as an Ab Platform: In my previous blog Deluge of mRNA Delivery Publications, I commented on how the strong interest in mRNA therapeutics has continued to drive increased numbers of delivery publications. Delivery, which is perhaps the overarching challenge for mRNA as an Ab platform, is discussed at length in the review by Van Hoecke and Roose, who delve into issues with formulation (or not), route and frequency of administration, and Ab serum titers. They also address unwanted immunogenicity and toxicity issues, which extend beyond the scope of this blog.
Among the conclusions offered by Van Hoecke and Roose, the following struck me as worth sharing:
- The fact that only a few of the more than 70 mAbs that have been licensed by regulatory agencies are directed against infectious diseases is “astonishing”. To the researchers, “this is stunning,” because mAbs represent a powerful alternative in combatting infectious diseases, especially for cases in which antibiotics fail or antivirals are not available,.
- Reasons for this, they say, “can be found in the money and time consuming production and purification processes, extensive downstream quality control, and the need for a cold supply chain. The higher costs related to protein-based mAbs are in striking contrast with the costs for most small molecule drugs or antibiotics. Use of IVT mod Abs is said to “have great potential as they are simple, fast and cost effective as it does not require complex and expensive laboratory infrastructures, with a generic production process for all mRNAs.”
- To successfully replace the current protein antibody format, “mRNA approaches will need to surpass their own challenges, which are mainly situated at the level of delivery and immunogenicity. But with the emergence of mRNA as a therapeutic and the growing research on this topic, mRNA therapeutics will likely further evolve and improve the coming years.”
I fully agree with this optimistic view of the future and, as usual, welcome your comments.