mRNA mediates passive vaccination against infectious agents, toxins, and tumors
2017; Springer Nature; Volume: 9; Issue: 10 Linguagem: Inglês
10.15252/emmm.201707678
ISSN1757-4684
AutoresMoritz Thran, Jean Mukherjee, Marion Pönisch, Katja Fiedler, Andreas Theß, Barbara L. Mui, Michael J. Hope, Ying K. Tam, Nigel Horscroft, Regina Heidenreich, Mariola Fotin‐Mleczek, Charles B. Shoemaker, Thomas Schlake,
Tópico(s)Bacteriophages and microbial interactions
ResumoResearch Article9 August 2017Open Access Source DataTransparent process mRNA mediates passive vaccination against infectious agents, toxins, and tumors Moritz Thran Moritz Thran CureVac AG, Tübingen, Germany Search for more papers by this author Jean Mukherjee Jean Mukherjee Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, MA, USA Search for more papers by this author Marion Pönisch Marion Pönisch CureVac AG, Tübingen, Germany Search for more papers by this author Katja Fiedler Katja Fiedler CureVac AG, Tübingen, Germany Search for more papers by this author Andreas Thess Andreas Thess CureVac AG, Tübingen, Germany Search for more papers by this author Barbara L Mui Barbara L Mui Acuitas Therapeutics, Vancouver, BC, Canada Search for more papers by this author Michael J Hope Michael J Hope Acuitas Therapeutics, Vancouver, BC, Canada Search for more papers by this author Ying K Tam Ying K Tam Acuitas Therapeutics, Vancouver, BC, Canada Search for more papers by this author Nigel Horscroft Nigel Horscroft CureVac AG, Tübingen, Germany Search for more papers by this author Regina Heidenreich Regina Heidenreich CureVac AG, Tübingen, Germany Search for more papers by this author Mariola Fotin-Mleczek Mariola Fotin-Mleczek CureVac AG, Tübingen, Germany Search for more papers by this author Charles B Shoemaker Charles B Shoemaker Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, MA, USA Search for more papers by this author Thomas Schlake Corresponding Author Thomas Schlake [email protected] orcid.org/0000-0001-6292-4055 CureVac AG, Tübingen, Germany Search for more papers by this author Moritz Thran Moritz Thran CureVac AG, Tübingen, Germany Search for more papers by this author Jean Mukherjee Jean Mukherjee Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, MA, USA Search for more papers by this author Marion Pönisch Marion Pönisch CureVac AG, Tübingen, Germany Search for more papers by this author Katja Fiedler Katja Fiedler CureVac AG, Tübingen, Germany Search for more papers by this author Andreas Thess Andreas Thess CureVac AG, Tübingen, Germany Search for more papers by this author Barbara L Mui Barbara L Mui Acuitas Therapeutics, Vancouver, BC, Canada Search for more papers by this author Michael J Hope Michael J Hope Acuitas Therapeutics, Vancouver, BC, Canada Search for more papers by this author Ying K Tam Ying K Tam Acuitas Therapeutics, Vancouver, BC, Canada Search for more papers by this author Nigel Horscroft Nigel Horscroft CureVac AG, Tübingen, Germany Search for more papers by this author Regina Heidenreich Regina Heidenreich CureVac AG, Tübingen, Germany Search for more papers by this author Mariola Fotin-Mleczek Mariola Fotin-Mleczek CureVac AG, Tübingen, Germany Search for more papers by this author Charles B Shoemaker Charles B Shoemaker Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, MA, USA Search for more papers by this author Thomas Schlake Corresponding Author Thomas Schlake [email protected] orcid.org/0000-0001-6292-4055 CureVac AG, Tübingen, Germany Search for more papers by this author Author Information Moritz Thran1, Jean Mukherjee2, Marion Pönisch1, Katja Fiedler1, Andreas Thess1, Barbara L Mui3, Michael J Hope3, Ying K Tam3, Nigel Horscroft1, Regina Heidenreich1, Mariola Fotin-Mleczek1, Charles B Shoemaker2 and Thomas Schlake *,1 1CureVac AG, Tübingen, Germany 2Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, MA, USA 3Acuitas Therapeutics, Vancouver, BC, Canada *Corresponding author. Tel: +49 7071 9883 1607; E-mail: [email protected] EMBO Mol Med (2017)9:1434-1447https://doi.org/10.15252/emmm.201707678 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA–lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long-lasting serum antibody titers in vivo, thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA-mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization. Synopsis As a transient carrier of information, exogenous mRNA is considered a possibly powerful tool to instruct a body to produce its own therapeutics. Antibody-encoding, sequence-optimized but otherwise unmodified mRNA in LNPs empowered mice to successfully fight various biological threats. Sequence-optimization enables efficient antibody expression from in vitro transcribed mRNA. Exogenous mRNA can instruct cells to produce various types of antibodies. Optimized mRNA in lipid nanoparticles provides efficient antibody expression in hepatocytes upon intravenous administration. mRNA-mediated antibody expression can protect mice against toxins, viruses, and tumors. Introduction Today, vaccination is among the most effective medical treatments for humans and animals, saving millions of lives by inducing immunity against various pathogens. Protection afforded by currently licensed vaccines is primarily based on induction of a humoral immune response (Amanna & Slifka, 2011). It has been demonstrated that neutralizing antibodies are sufficient to prevent dissemination of many diseases within populations (Plotkin, 2010). Hence, passive immunization, by transferring either serum or purified antibodies, is a potent means to confer immediate protection against various threats. Clinical benefit was demonstrated for invasive bacterial infections (Casadevall & Scharff, 1994), for various viral diseases (Janeway, 1945; Hammon et al, 1953), as well as for anti-fungal therapy (Bugli et al, 2013), and polyclonal antibody products were licensed for several viruses. With the advent of antibiotics and vaccines, serum therapy was almost abandoned but retained as a niche treatment for toxins, venoms, and distinct viral infections. For instance, post-exposure treatment of rabies as recommended by the World Health Organization consists of the immediate treatment with anti-rabies immunoglobulins in combination with a rabies vaccine (Steele, 1988). As further example, botulism is treated with immune serum (Pediatrics, 1997). Antibody therapies were revolutionized by the development of the hybridoma technology for the production of monoclonal antibodies (mAbs) (Kohler & Milstein, 1975). Since then, more than thirty mAbs have been licensed, but only three of these mAbs are for infectious disease indications, respiratory syncytial virus, anthrax, and very recently rabies in India (Nagarajan et al, 2014). Nevertheless, there is ample evidence that it is possible to generate protective mAbs against many viruses and microorganisms (Teitelbaum et al, 1998; Nosanchuk et al, 2003; Both et al, 2013). Passive immunization declined as a treatment option for a variety of reasons. The primary reason has been the emergence of antibiotics and vaccines. In addition, both serum- and mAb-based therapeutics are costly to produce, store, and deliver as they require a cold supply chain and are often administered intravenously (Chames et al, 2009; Keizer et al, 2010). Moreover, passive immunization confers only a relatively short duration of protection compared to vaccines. However, there is a renewed interest in passive immunization. Emergence of microbial resistance to antibiotics has increased the demand for alternative therapies. Further, the discovery of broadly neutralizing antibodies may offer new therapeutic options for influenza and HIV (Burton et al, 2012). Finally, in contrast to vaccines, antibodies enable a rapid onset of protective immunity. Particularly in a pandemic, passive immunization can offer a critical advantage for blocking virus spread. As an alternative to the administration of recombinant antibodies, DNA-based approaches have been extensively investigated. Both plasmids as well as viral vectors (adenovirus, adeno-associated virus) have been used for passive immunization. Using viral vectors, sustained expression of low to mid microgram per milliliter levels was obtained in different models and conferred protection against influenza and RSV challenges (Lewis et al, 2002; Skaricic et al, 2008; Balazs et al, 2011). In general, however, DNA-based passive immunization suffers from the risk of genomic integration, potentially causing fatal mutations. Moreover, for safety reasons, transient vectors or regulated expression would be preferred for clinical use (Deal & Balazs, 2015). In addition, immunogenicity of virus particles has to be considered a main obstacle for the use of this type of vectors for DNA-mediated passive immunization. In contrast, mRNA may offer an attractive alternative for passive immunization. Following initial studies in the early 1990s demonstrating that exogenous mRNA can direct protein expression in vivo, mRNA has emerged as a promising drug platform technology in recent years (Wolff et al, 1990; Jirikowski et al, 1992). Several studies have demonstrated the utility of mRNA as the basis of vaccines in cancer immunotherapy as well as to promote prophylactic protection from infectious diseases (Hoerr et al, 2000; Fotin-Mleczek et al, 2011; Petsch et al, 2012; Kubler et al, 2015; Kranz et al, 2016). Conclusive data have also been presented for mRNA as a platform for protein (replacement) therapies (Kormann et al, 2011; Kariko et al, 2012; Zangi et al, 2013; Thess et al, 2015; Balmayor et al, 2016). Physiological responses elicited in primates and domestic pigs treated with an mRNA-encoded hormone have suggested the feasibility of mRNA for large animal therapies (Thess et al, 2015). Compared to DNA-based approaches, mRNA benefits from fewer safety issues due to its non-integrative and transient nature, the latter of which contributes to better and/or easier control of protein expression, but may be disadvantageous, if prolonged bioavailability is desired or even required. In contrast to the manufacturing of many recombinant antibodies, mRNA has cost advantages, since different sequences, and as a consequence proteins, can be produced by a generic process. Thus, mRNA-based antibodies for passive immunization may be produced at competitive costs even in cases where the product is only required in emergencies such as an influenza pandemic. In the present study, we set out to evaluate the use of mRNA for passive immunization. To this end, we focused on two indications for which passive immunization is relevant today, rabies and botulism, that can be considered prototypes for anti-pathogen and anti-toxin therapies, respectively. To demonstrate the broad applicability of mRNA technology, we further used different antibody formats. In both disease models, high levels of in vivo serum expression were obtained that conferred full protection in pre- and post-exposure scenarios. The flexibility of the mRNA technology was further illustrated by complementary data from additional indications, including the demonstration of therapeutic efficacy in a tumor model. Thus, our work establishes the foundation for development of novel passive immunization therapies. Results In vitro expression and functionality of mRNA-encoded antibodies The intended goal of this study was to seek proof-of-principal data demonstrating the feasibility of passive immunization by means of prophylactic and therapeutic mRNA treatments encoding antibodies. Efficacious treatment with therapeutic proteins depends on both timely delivery and achieving protective levels. To demonstrate that mRNA meets these requirements, two representative and clinically relevant disease models were chosen. The first model, rabies, is an invariably fatal disease that demands rapid administration of rabies immunoglobulins in post-exposure scenarios to prevent encephalitis and finally death of infected individuals. In 2003, a cocktail of mAbs was developed which protected Syrian hamsters from lethal challenges with a highly virulent rabies virus strain in post-exposure scenarios (Prosniak et al, 2003). For the present study, we selected the monoclonal antibody S057, also well known as CR57, one component of the published mAb cocktail, because it provides broad neutralization of a variety of rabies virus strains via binding to glycoprotein G. The second model, botulism, is a rare but often fatal toxin-mediated illness that occurs following ingestion of food-borne bacterial spores or via pre-formed toxin. This latter exposure route is of concern as botulinum toxin is the most potent toxin known, is quite stable, has a history of use as a bioweapon, and is thus classified as a Category A bioterror agent. Due to the potency and rapid onset of symptoms, exposure to botulinum toxin demands immediate anti-toxin therapy which is currently an antiserum (Pediatrics, 1997). To demonstrate the flexibility of mRNA-mediated antibody expression, we selected a different antibody format, a camelid heavy-chain-only VH domain (VHH)-based neutralizing agent (VNA). In general, VNAs offer some advantages over classical antibodies such as ease to engineer, improved heat and pH stability, and better tissue penetration (Hamers-Casterman et al, 1993; van der Linden et al, 1999; Tillib et al, 2013). The use of parenterally administered VNAs that potently neutralize botulinum neurotoxin serotype A (BoNT/A) has been previously reported (Mukherjee et al, 2014). To further test the broad applicability of an mRNA-based passive immunization platform, results from the two main models were complemented by data with additional antibodies of the IgG mAb or VNA type, including an anti-tumor application. Since the major objective of passive immunization is protection mediated by achieving high serum titers, we chose the liver as the target organ for mRNA-mediated protein expression. To provide prolonged mRNA stability and efficient protein translation, a previously approved format for liver expression harboring crucial regulatory mRNA elements was used (Fig 1A and D; Thess et al, 2015). For VNA expression, we resorted to a recently developed design for prolonged serum half-life (Mukherjee et al, 2014). Basically, VNA sequences included two fused VHHs complemented by an albumin-binding peptide to increase serum persistence and epitope tags for protein detection (Fig 1A). In order to provide maximal flexibility for the expression of IgG mAbs in the present study, we decided to encode heavy and light chain on separate mRNA molecules (Fig 1D). In B cells, solitary heavy chains are retained at the endoplasmic reticulum by the chaperone binding immunoglobulin protein (BiP) (Hendershot et al, 1987). Heavy-chain retention for mRNA-mediated expression was confirmed in overexpressing non-B cells, including a hepatic cell line that may mimic in vivo conditions in the liver, our target organ for in vivo delivery (Appendix Fig S1). In contrast, light chains were also secreted in the absence of heavy chains. Since well-matched expression of heavy and light chain is critical for obtaining a maximum of functional antibody, we titrated the ratio between mRNAs encoding heavy and light chain in vitro. High expression was obtained over a broad range of molar ratios (heavy:light chain) (Appendix Fig S2). According to these results, a ratio of approximately 1.5:1 was subsequently used through the study. Figure 1. In vitro expression of mRNA-encoded antibodies mRNA design to encode heavy-chain-only VH domain (VHH)-based neutralizing agents (VNAs). VNAs were supplemented by affinity E-tags (Tag) and an albumin-binding peptide (ABP). Western blot analysis of VNA-BoNTA expression in cell lysates and supernatants (SN) after mRNA transfection of BHK cells. Equal amounts of three replicates were pooled and loaded on denaturing SDS–PAGE. Staining for β-actin was used as loading control. Quantification of VNA levels by antigen-specific ELISA of supernatants from transfected BHK cells. Titers were determined in triplicate. Supernatants from cells transfected with an irrelevant VNA were used as mock control. mRNA design to encode heavy- and light-chain molecules. Western blot analysis showing expression of heavy and light chains in cell lysates and accumulation in supernatants after mRNA transfection of BHK cells. Equal amounts of three replicates were pooled and loaded on denaturing SDS–PAGE. Signals correspond to heavy chain (HC), light chain (LC) and, as loading control, tubulin. Quantification of mAb levels in supernatants of BHK cells by IgG-specific ELISA. Titers were determined in triplicate. Data information: For mock transfection, an mRNA encoding eGFP was used. UTR, untranslated region; SP, signal peptide; A(n), poly(A)-tail. Results in (C and F) are expressed as means ± SD. Source data are available online for this figure. Source Data for Figure 1 [emmm201707678-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Constructs for IgG mAbs against rabies glycoprotein G (S057), influenza B hemagglutinin (CR8033; Dreyfus et al, 2012), HIV gp120 (VRC01; Wu et al, 2010), and human CD20 (rituximab) all revealed strong in vitro expression upon mRNA transfection of BHK cells as measured by Western blot analysis and IgG-specific ELISA (Fig 1E and F, and Appendix Fig S3A–F). Likewise, expression of VNAs against botulinum neurotoxin serotype A (VNA-BoNTA) and Shiga toxin 2 (Stx2) from E. coli (O157:H7) (VNA-Stx2) was readily detected by Western blot and antigen-specific ELISA (Fig 1B and C, and Appendix Fig S3G and H). In vitro antibody mRNA half-life in primary mouse hepatocytes was approximately 15 h (Appendix Fig S4A). Functionality of antibodies secreted into cell supernatants after mRNA transfection was analyzed by various means. Specific antigen binding of mAbs against rabies, influenza, and human CD20 was verified by staining of antigen-expressing target cells (Fig 2C–E and Appendix Fig S5). Specific activity of mRNA-derived anti-HIV mAb was demonstrated to be comparable to the respective recombinant protein by measuring the inhibition of virus entry into reporter cells (Fig 2F and Appendix Fig S6). VNA-BoNTA functionality was confirmed by a SNAP25 cleavage inhibition assay demonstrating that supernatants containing mRNA-encoded VNA-BoNTA had neutralizing potency comparable to a recombinant VNA-BoNTA protein standard (Fig 2A). Finally, supernatants containing mRNA-encoded VNA-Stx2 proved capable to protect cell viability in the presence of Shiga toxin with potency comparable to recombinant VNA-Stx2 (Fig 2B). Taken together, mRNA proved to be an efficient means to express diverse antibodies and antibody formats in vitro. Figure 2. Evaluation of antibody function in vitro A. BoNT/A toxin was incubated either with different doses of recombinant VNA, spiked into supernatant of mock-transfected BHK cells, or different dilutions of supernatant of BHK cells transfected with mRNA encoding VNA-BoNTA before SNAP25 cleavage assay. In this assay, BoNT/A cleaves SNAP25 which can be inhibited by VNA binding to the toxin. Samples from cleavage assay were loaded on denaturing SDS–PAGE and BoNT/A-mediated cleavage of endogenous SNAP25 was analyzed. B. Cell-based toxicity assay to demonstrate the function of RNA-encoded VNA-Stx2. Supernatants from BHK cells transfected with VNA-encoding mRNA were analyzed in a dilutions series (11 nM initial concentration). Serial dilutions of supernatants from mock-transfected cells served as negative control. Recombinant VNA-Stx2 was spiked into mock-transfected medium at an initial concentration of 10 nM. C, D. Binding of mRNA-encoded anti-rabies mAb (C) or anti-influenza B mAb (D) expressed in BHK cell supernatants to antigen-positive or antigen-negative HeLa cells. Depicted is the median of phycoerythrin (PE) fluorescence of all living cells. Fluorescence was measured in triplicate. For mock transfections, supernatants of eGFP-mRNA transfected cells were used. E. Binding of mRNA-encoded rituximab expressed in BHK cells to Raji cells. Depicted is the median of phycoerythrin (PE) fluorescence of all living cells. Fluorescence was measured in triplicate. For mock transfections, supernatants of eGFP-mRNA transfected cells were used. F. Supernatants of BHK cells transfected with mRNA encoding VRC01 or untransfected BHK cells were subjected to a Magi R5-Tropic Antiviral Assay. Depicted are the mAb concentrations that produced either 50% (IC50) or 90% inhibition (IC90) of virus entry. Recombinant VRC01 mAb or TAK779 inhibitor was used as positive controls. Individual inhibition curves are shown in Appendix Fig S5. Data information: Results in (C, D and E) are expressed as median ± SD. Download figure Download PowerPoint Expression of mRNA-encoded antibodies in vivo Following the in vitro findings, it was explored whether protective and long-lasting serum antibody titers can be obtained in mice. Since affinity to its cognate antigen is crucial for the concentration at which a specific antibody is protective, required concentrations depend on the antibody of interest. Potential bottlenecks of reaching protective serum levels may include the mRNA delivery to and/or uptake into target cells, ribosome occupancy of mRNAs, and secretion of assembled antibodies into the blood stream. While a proven mRNA format was used to ensure efficient protein expression (see above), hepatocytes were chosen as target cells to secure efficient secretion. In order to maximize protein expression from liver, a potent lipid nanoparticle (LNP) formulation was applied. It has been specifically developed for delivery into the liver and has previously been shown to be very effective in that respect, thus providing robust protein expression (Pardi et al, 2015; Thess et al, 2015). In line with current clinical antibody applications, this LNP formulation is usually administered intravenously. Hence, this route was used throughout the present study, although for instance, the development of a therapy for rabies will most likely require intramuscular injection. First, the dose–response relationship upon administration of mRNA-LNP was determined for two humanized mAbs by analyzing mouse sera with an IgG-specific ELISA. A single injection of increasing mRNA-LNP doses induced rising serum levels of antibody without reaching peak levels up to the highest doses tested in this study (Fig 3A and Appendix Fig S7A). The highest levels observed were in the low two-digit μg/ml range for both antibodies. For the anti-rabies mAb, the dose–response relationship was confirmed by analyzing virus neutralization titers which were comparable to those from an earlier active immunization study and above the protective level of 0.5 IU/ml as defined for humans at all mRNA doses tested but the lowest (Fig 3B; Schnee et al, 2016). Hence, as for in vitro expression, mRNA-mediated mAb expression in vivo efficiently produced functional antibodies. While maximum expression levels observed for VNA-Stx2 were almost the same as for the mAbs according to an antigen-specific ELISA of sera, VNA-BoNTA gave rise to about 20-fold higher protein serum titers (Fig 3E and Appendix Fig S7B–D). We hypothesize that this difference can be attributed to the fact that we did not apply individually optimized mRNAs but a fixed mRNA design. Interestingly, there appears to be more than a strictly linear increase of serum titers with mRNA doses for mAbs. However, this effect is not related to the necessity of correctly assembling heavy and light chains and the separation of both on distinct mRNA molecules, since the nonlinearity is equal or even more pronounced for the single-chain VNA constructs. Figure 3. In vivo expression characteristics of antibody-encoding mRNA Quantification of mAb titers in mice by IgG-specific ELISA of sera obtained 24 h after a single intravenous injection of increasing amounts of mRNA-LNP encoding anti-rabies mAb. Each group consisted of five animals. Same sera as in (A) were subjected to a fluorescent antibody virus neutralization (FAVN) assay to determine anti-rabies virus neutralizing titers (VNTs in international units). Quantification of mAb titers by IgG-specific ELISA at various times after a single intravenous injection of 40 μg of mRNA-LNP encoding anti-rabies mAb. Five cohorts of eight mice each were alternately bled to enable the given sampling schedule. Quantification of anti-rabies and anti-influenza B mAb titers by IgG-specific ELISA 1 or 24 days post-injection of 40 μg mRNA-LNP. Each group comprised 12 mice. Quantification of VNA-BoNTA titers by antigen-specific ELISA of sera at various times after a single injection of 40 μg of mRNA-LNP encoding VNA-BoNTA. For each sampling, a separate cohort of mice, each comprising five animals, was used. Data information: Individual measurements as well as means are given for each dose and time, respectively. Download figure Download PowerPoint Next, we addressed the kinetics of protein availability as a further important parameter for any antibody therapy. In this context, two fundamental questions are of importance: How quickly are therapeutically effective serum titers reached and how long do they persist? The ramp-up time of antibody serum titers is determined by the kinetics of delivery and uptake of mRNA-LNP into hepatocytes, protein translation, and secretion. Serum half-life of antibodies is affected by both mRNA and protein half-life. In the case of mAbs, the latter is primarily determined by their Fc region. In the absence of such a domain, the half-life of the VNAs used here is substantially extended by the attached albumin-binding peptide (Dennis et al, 2002) and have a half-life of about 1 day in mice (Mukherjee et al, 2014), significantly shorter than the half-life typical for mAbs. For the anti-rabies mAb, serum titers were readily detectable 2 h after treatment and peaked 6–12 h post-injection (Fig 3C). During the following days, titers slowly decreased with a half-life of approximately 1 week. While this half-life remained unchanged in about 50% of the animals during the observation period of 4 weeks, the remaining mice revealed an accelerated drop of antibody serum titers from day 10 after treatment on. This bipartite kinetics was confirmed in a second independent study. While about half of the animals showed serum half-lives of approximately 1 week for the 24-day observation period, the others were characterized by substantially lower serum titers at the end of the study (Fig 3D). In contrast, mice that received mRNA coding for anti-influenza mAb all revealed the same high serum levels after 24 days (Fig 3D). The estimated serum half-life of this antibody was also about 1 week. For VNA-BoNTA and VNA-Stx2, considerable titers were measured as early as 2 h post-injection (Fig 3E and Appendix Fig S7B). Peak serum levels were reached 6–24 h after administration. Thus, early kinetics appeared similar for mAbs and VNAs. However, serum titers dropped much faster for VNAs than for mAbs after day 2. For both VNAs, serum half-lives were calculated to be in the range of 24–36 h which is in line with a previous report (Mukherjee et al, 2014). To arrive at an estimation for mRNA half-life in vivo in order to assess its contribution to protein bioavailability, we utilized mRNA-LNPs encoding the short-lived hormone erythropoietin, suggesting a half-life of roughly 15 h (Appendix Fig S4B), which is in good accordance with in vitro measurements. To get insights into potential reasons for the accelerated serum clearance of anti-rabies antibody in some animals, we investigated the emergence of ADA (anti-drug antibody) responses. Obviously, the individual clearance rates strongly correlated with the development of a humoral response against the anti-rabies antibody (Appendix Fig S8), thus perfectly explaining the divergent kinetics. In addition, mice were analyzed for general tolerability of mRNA-LNP treatment. We did not observe any adverse events throughout the present studies. There was a transient weak increase of some cytokines in circulation (Appendix Table S1), which obviously did not hamper high protein expression. Of note, equal or even higher levels were considered unproblematic in a recent study on modified mRNA encoding factor IX (Ramaswamy et al, 2017). Histopathology of liver, the target organ of mRNA-LNPs, did not reveal any signs of abnormality or inflammation (Appendix Fig S9). In summary, we could demonstrate that mRNA-LNP enables a rapid and strong expression of mRNA-encoded mAbs and VNAs. Moreover, sustained protein ava
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