
==== Front
Mol Ther
Mol Ther
Molecular Therapy
1525-0016
1525-0024
American Society of Gene & Cell Therapy

S1525-0016(24)00338-1
10.1016/j.ymthe.2024.05.036
Original Article
In vivo mRNA expression of a multi-mechanistic mAb combination protects against Staphylococcus aureus infection
Tkaczyk Christine christine.tkaczyk@astrazeneca.com
1∗
Newton Michael 2
Patnaik Mun Mun 1
Thom George 3
Strain Martin 4
Gamson Adam 1
Daramola Olalekan 5
Murthy Andal 5
Douthwaite Julie 3
Stepanov Oleg 6
Boger Elin 7
Yang Haitao 8
Esser Mark T. 1
Lidwell Ashley 1
DiGiandomenico Antonio 1
Santos Luis 29
Sellman Bret R. 1
1 AstraZeneca, Early Vaccines & Immune Therapies, Gaithersburg, MD 20878, USA
2 AstraZeneca, BioPharmaceutical Development, BioPharmaceuticals R&D, Gaithersburg, MD 20878, USA
3 AstraZeneca, Discovery Sciences, BioPharmaceuticals R&D, Cambridge CB21 6GH, UK
4 AstraZeneca, Biologics Engineering, BioPharmaceuticals R&D, Cambridge CB216GH, UK
5 AstraZeneca, BioPharmaceutical Development, BioPharmaceuticals R&D, Cambridge CB21 6GH, UK
6 Clinical Pharmacology and Pharmacometrics, BioPharmaceuticals R&D, AstraZeneca, Cambridge CB2 8PA, UK
7 Drug Metabolism and Pharmacokinetics, Research and Early Development, Respirator & immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
8 Clinical Pharmacology and Pharmacometrics, BioPharmaceuticals R&D, AstraZeneca, Gaithersburg, MD 20878, USA
∗ Corresponding author: Christine Tkaczyk, AstraZeneca, Early Vaccines & Immune Therapies, Gaithersburg, MD 20878, USA. christine.tkaczyk@astrazeneca.com
9 Present address: Prime Medicine, Inc., Cambridge, MA, 02139, USA

31 5 2024
07 8 2024
31 5 2024
32 8 25052518
4 12 2023
29 5 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Single monoclonal antibodies (mAbs) can be expressed in vivo through gene delivery of their mRNA formulated with lipid nanoparticles (LNPs). However, delivery of a mAb combination could be challenging due to the risk of heavy and light variable chain mispairing. We evaluated the pharmacokinetics of a three mAb combination against Staphylococcus aureus first in single chain variable fragment scFv-Fc and then in immunoglobulin G 1 (IgG1) format in mice. Intravenous delivery of each mRNA/LNP or the trio (1 mg/kg each) induced functional antibody expression after 24 h (10–100 μg/mL) with 64%–78% cognate-chain paired IgG expression after 3 days, and an absence of non-cognate chain pairing for scFv-Fc. We did not observe reduced neutralizing activity for each mAb compared with the level of expression of chain-paired mAbs. Delivery of the trio mRNA protected mice in an S. aureus-induced dermonecrosis model. Intravenous administration of the three mRNA in non-human primates achieved peak serum IgG levels ranging between 2.9 and 13.7 μg/mL with a half-life of 11.8–15.4 days. These results suggest nucleic acid delivery of mAb combinations holds promise and may be a viable option to streamline the development of therapeutic antibodies.

Graphical abstract

Tkaczyk and colleagues demonstrated that the in vivo expression of three mAb combination against S. aureus via mRNA/LNPs resulted in minimal non-cognate chain pairing, serum expression within 24-h between 15 and 90 μg/mL in mice, and protection in a dermonecrosis model. Delivery of the three mRNA/LNPs results in mAb expression between 2.9 and 13.7 μg/mL in cynomolgus monkeys.

Keywords

in vivo expressed monoclonal antibodies
scFv-Fc
immunotherapy
mRNA/LNP
Staphylococcus aureus
bacteria pathogenesis
mouse disease models
monoclonal antibody combination
alpha toxin
clumping factor A
==== Body
pmcIntroduction

The clinical approval of the first monoclonal antibody (mAb) against CD3 in 1986 to prevent graft versus host disease extended the field of immunotherapy.1 There are now more than 100 mAbs approved to treat a variety of indications, including asthma, cancer, auto-immunity, and infectious disease,2 with others under investigation in late-stage clinical development.3 MAbs have the advantage of being highly specific and safe and provide immediate protection upon delivery, even in immune-compromised patients. However, manufacture from large bioreactors can be time intensive and costly due to challenges associated with expression, purification, aggregation, and post-translational modifications.4

The recent success with severe acute respiratory syndrome coronavirus 2 (SARs-CoV-2) mRNA vaccines highlights the opportunity of delivering gene-encoded biologics to people. This not only decreases development timelines, but could address some of the limitations associated with manufacturing recombinant mAbs by using the body as its own bioreactor to produce the mAb directly from nucleic acid sequences (e.g., DNA or mRNA).4,5 In this context, DNA is delivered to cells mechanically through electroporation or after packaging in a recombinant viral vector such as adeno-associated virus vectors (AAV). Recombinant AAV vectors are available in a variety of serotypes with tissue tropisms for tissue specific delivery.6 Both technologies have shown efficacy in pre-clinical models for cancer immunotherapy,7,8 and protection against viral9,10,11,12,13,14 and bacterial infections.15,16 However, in vivo pharmacokinetic (PK) studies showed that DNA delivery does not offer immediate immune protection with peak mAb expression generally observed a few days after injection.17 Alternatively, mRNA encapsulated in lipid nanoparticles (LNPs) offers the advantage to bypass DNA transcription in the nucleus and induces peak mAb expression in sera within 24 h after administration in preclinical models.18,19 Viable therapeutic mAb levels have been achieved in mouse sera upon intravenous administration of mRNA encoding both light chain (LC) and heavy chain (HC) sequences formulated in an LNP and was shown to provide protection against viral infection18,20 or a benefit in tumor regression.21

The mRNA/LNP technology also offers the potential to reduce the cost associated with the development and manufacturing of multiple mAbs. The unique amino acid sequence of each antibody dictates their physicochemical characteristics, which may result in formulation challenges to producing high yields as a recombinant mAb. While the manufacture and formulation of an mAb combination can be complex due to different isoelectric points and incompatibility of their respective formulation buffers, nucleic acid-encoded mAbs are conserved structures, and do not necessitate tailor-made formulations.4,22 In the event of an infection outbreak, a therapeutic mAb or mAb combination could be developed more quickly as mRNA for rapid deployment to a large population. However, little is known about the feasibility of delivering two or more antibodies together via mRNA. In particular, HC and LC mispairing may modify epitope recognition, which could result in a loss of binding activity and altered PKs.

Given the complexity of bacterial pathogenesis, multiple mAbs targeting several virulence factors are more likely to provide broad strain coverage and protect against a variety of Staphylococcus aureus disease indications.23 For these reasons, we generated and characterized a three mAb combination against the Gram-positive bacterium S. aureus targeting alpha toxin (AT), four bi-component leukotoxins (Leuks) (LukSF, LukED, HlgAB, and HlgCB) and surface-expressed clumping factor A (ClfA). Together, these three mAbs inhibit toxin-mediated cell lysis, target the bacteria for opsonophagocytic killing, and prevent bacterial agglutination to provide broad disease coverage against skin and soft tissue infections, pneumonia, and bacteremia against diverse clinical isolates.24,25,26,27

Herein, we evaluated the PKs and functional activity of this mAb trio when delivered as mRNA/LNP in mice. To minimize the likelihood of HC and LC mispairing, the mAb trio was first evaluated in an scFv-Fc format and then in immunoglobulin G (IgG) format. Twenty-four hours after administration, each antibody exhibited different serum peak serum concentration (Cmax) levels ranging between 10 and 22 μg/mL and 15 and 90 μg/mL in scFv-Fc and IgG formats, respectively; each mAb exhibited functional activity in both formats and the trio mAb provided protection in an S. aureus dermonecrosis model. Delivery of the trio IgG via mRNA to non-human primates (NHPs) resulted in serum levels ranging between 2.9 and 13.7 μg/mL with a terminal half-life between 11.8 and 15.4 days. Together these data suggest that native human IgG1 mAb combinations delivered via the mRNA platform could be a suitable approach to prevent serious bacterial infections.

Results

IgG conversion to scFv-Fc does not impact the in vitro functionality of the three S. aureus mAbs

We previously reported on a multi-mechanistic combination of three human IgG1 mAbs targeting six S. aureus virulence factors, AT, four bi-component Leuks, and ClfA virulence factors.25,27 Crystal structures of the antibody-antigen complex highlighted the contribution of both variable heavy (VH) and variable light (VL) domains in epitope binding and neutralizing activity28 and unpublished data. Since each IgG-encoding mRNA contains two separate sequences for the HCs and LCs (Figure S1A), co-delivery of the three mRNA could theoretically result in the expression of 45 different molecules if HC and LC mispairing occurred between the different IgGs (Figure S1B). This non-cognate pairing would potentially result in reduced binding and functional activity by the expressed mAbs. To reduce the likelihood for chain non-cognate pairing, we first designed mRNA encoding the three mAbs in an scFv-Fc format, where the two variable regions of the IgG are connected by a polypeptide linker fused to the CH2 and CH3 domains of the Fc constant domain (Figure S1C). Subsequently, co-delivery could only result in six homo- or hetero-dimers if mispairing were to occur (Figure S1D). However, the insertion of the polypeptide linker, and deletion of the CH1 domain may impair antibody activity, as previously reported.29 Hence, we compared the in vitro activity of each scFv-Fc with their parental IgG. Neutralizing activity of the anti-AT and the anti-Leuk mAbs was evaluated in a rabbit red blood cell hemolytic assay, respectively, in presence of AT or γ-hemolysin HlgAB, and the anti-ClfA mAb activity was measured in an immobilized fibrinogen binding assay, as described previously.27 Both the anti-AT or anti-Leuk mAbs exhibited a similar neutralizing half-maximal inhibitory concentration in scFv-Fc or IgG formats, while the anti-ClfA scFv-Fc showed a 1.6-fold decrease relative to the native IgG (Figure 1). Therefore, the conversion of each mAb from IgG to scFv-Fc did not dramatically impair their activity, and both formats were evaluated in vitro and in vivo using an mRNA/LNP platform.Figure 1 In vitro neutralizing activity of S. aureus mAbs in IgG or scFvFc format

(A) Neutralizing hemolytic activity of purified AT (10 ng/mL) on rabbit red blood cells (RBCs) in the presence of serial dilutions of anti-AT mAb in scFv-Fc or IgG format. (B) Neutralizing hemolytic activity of purified HlgAB (10 ng/mL) on rabbit RBC in presence of serial dilutions of anti-Leuk mAb in both formats. (C) Inhibition of ClfA binding to fibrinogen in presence of serial dilution of anti-ClfA mAb in both formats. Half-maximal inhibitory concentration (IC50) for each mAb was calculated with GraphPad Prism 9.4 software. X values were transformed using the equation X = Log(X) followed by nonlinear regression analyses using log(agonist) versus response (three parameters) where Y = Bottom + (Top − Bottom)/(1 + 10 ˄([LogEC50-X]).

mRNA encoding scFv-Fc or IgG induced expression of functional antibody in vitro and in vivo

The feasibility of expressing an mAb combination via mRNA/LNPs was first evaluated in vitro. Expi293F cells were transfected with mRNA coding for a single antibody or a combination of two or three mAbs, and expression evaluated after 48 h in culture supernatants. To determine the percent of cognate pairing resulting from each transfection condition, we quantified the levels of total antibodies and cognate chain-paired antibodies in supernatants. Total antibodies were measured by a bioluminescence interferometry (BLI) method with a protein A sensor, and cognate paired antibodies were quantified by antigen-based ELISA. Comparing the IgG concentration through both methods indicates that transfection with two or three mRNA-encoding IgG resulted in expression of 70%–99% cognate-paired IgG (Figure 2A). Quantification of single expressed IgG showed less than 10% difference by both methods, which could be attributed to different sensitivities between the assays (Figure 2A). However, because a duet mAb comprising the anti-AT and anti-Leuk arms still exhibits neutralizing activity as compared with parental IgG mAbs (Figure S2), we cannot rule out the formation of IgG expressing paired HC/LC only on one arm. A similar approach showed no difference in scFv-Fc expression by both methods confirming an absence of chain mispairing (Figure 2B). The neutralizing antibody titers were used as a second surrogate to quantify cognate chain pairing on at least one arm of the IgG. There was no difference between the level of expression by antigen-based ELISA and neutralizing activity when mRNA was transfected alone or in combination confirming that 70%–99% IgG were fully or partially chain-paired and functional (Figure S3A), and absence of chain mispairing for the expressed scFv-Fc antibodies (Figure S3B). Interestingly, transfection with scFv-Fc mRNA induced higher titers compared with mRNA for IgG (8 μg/mL vs. 3 μg/mL, respectively). This was not due to formation of scFv-Fc heterodimers since similar antibody levels were measured by BLI, antigen-based ELISA or neutralizing activity (Figures 2B and S3B). Since the scFv-Fc contains two identical chains, we can speculate that the intracellular machinery required more energy and translational factors for translating both mRNA-encoding VH and VL chains as compared with the scFv-Fc chain. In vitro expression of mRNA-encoded mAbs was confirmed in vivo. Intravenous administration of a single scFv-Fc (Figure S4A) or IgG-encoding mRNA (Figure S4B) at 2.5 or 0.5 mg/kg resulted in a dose-dependent expression of each antibody with a Cmax in serum at 24 h and a more rapid clearance of the scFv-Fc. Interestingly, we observed IgG-dependent differences in Cmax ranging from 15 to 95 μg/mL, suggesting that a particular mRNA sequence may dictate antibody expression in vivo. We cannot exclude the role of the mRNA secondary structure for each VH and VL in controlling the formulation stability, translation efficiency, or endosomal escape upon cellular uptake of the mRNA formulations.30Figure 2 In vitro expression of IgG or scFv-Fc induced by mRNA

(A) IgG or (B) scFv-Fc were quantified respectively by protein A octet (▪) or antigen-specific ELISA (□) 48 h after transfection with mRNA encoding for a single, two or three antibodies. Data represent the mean values of duplicates ± SD. The percent of chain paired IgG was calculated as: 100 × ([paired IgG]/[total IgG]), and indicated for transfection with two or three mRNA-encoding IgG.

Trio scFv-Fc protect mice against S. aureus-induced dermonecrosis when delivered as mRNA

We next sought to evaluate the expression levels in vivo and protective activity of the mRNA encoded scFv-Fc combination. The mRNA/LNP formulations were delivered to mice intravenously individually or mixed at a 1:1:1 ratio (1 mg/kg each). Except for the anti-Leuk mAb at day 7 (p = 0.0421), there was no significant difference in serum expression levels for each scFv-Fc antibody delivered alone (1 mg/kg) or via the three mRNA mixture (3 mg/kg total) (Figure 3A). Serum mAb levels at 24 h, ranged between 6 and 32 μg/mL, depending on the antibody. Moreover, similar activities were detected for each scFv-Fc delivered as a single mRNA or as a mixture of three-mRNA (except anti-ClfA mAb at day 3; p = 0.0176), suggesting minimal chain mispairing (Figure 3B). Antibody activity was confirmed in the S. aureus-dermonecrosis model where animals were intravenously administered with the trio mRNA combination and intradermally infected with S. aureus 24 h later. In this model, the multi-mechanistic mAb combination, by neutralizing five toxins and targeting ClfA, allows the animals to mount an appropriate immune response consisting of abscess formation with early neutrophil recruitment to facilitate bacteria clearance by opsonophagocytic killing.27,31 One day after infection, we observed the formation of large skin blisters on negative control scFv-Fc-treated mice, which turned into dermonecrotic lesions by day 7 after challenge. In contrast, animals receiving the trio scFv-Fc exhibited small blisters, which turned into an abscess at day 7 (Figure S5). The skin lesion sizes for the trio scFv-Fc group were significantly smaller as compared with a negative control scFv-Fc at days 1 and 7 after inoculation (p < 0.0001) (Figure S5A) indicating the trio mRNA encoding mAbs mediated protective activity. However, since the scFv-Fc format confers co-expression of the VH and VL chains, we could not exclude the production of heterodimer antibodies with two target dualities (Figure S1D).Figure 3 In vivo PK of expression and neutralization of mRNA/LNP-induced single or trio scFv-Fc

Tg32FcRn mice (n = 4) were intravenously injected with mRNA/LNP-encoding for a single mAb or the trio mRNA/LNP (1 mg/kg each). MAb concentration (A) and neutralizing activity (B) were quantified in sera at indicated time point post injection. Data are represented as geometric means.

Trio IgG encoded by mRNA/LNPs exhibited some chain mispairing and protect mice against S. aureus-induced dermonecrosis when delivered as mRNA

We next tested the IgG expression kinetics and neutralizing activity in sera when the three mRNA/LNP-encoding IgG were intravenously injected at 1 mg/kg each (3 mg/kg total). Similar to in vitro expression, an absence of mispairing on one arm or both arms would result in comparable expression of total cognate-paired IgG and total IgG. Therefore, mouse sera were collected at indicated time points and the three IgG quantified by their respective antigen-based ELISA (Figure 4A) or by a total IgG ELISA (Figure 4B). The total IgG expression was overall higher over time when compared with the total cognate-paired IgG at any time point highlighting the formation of non-cognate pairing in vivo (Figure 4B). Intravenous delivery of the three mRNA/LNP resulted in 64%–78% expression of cognate-paired IgG after 3 days (Figure 4C). The concentrations of total IgG and paired IgG were similar 24 h after delivery (Figure 4B), suggesting minimal chain mispairing at this time point (Figure 4C). However, in vitro our methodology cannot rule out the formation of partially paired IgG where only one arm contained a paired HC/LC. We can speculate that the uptake of the three mRNA formulations by host cells first results in a high level of mRNA translation, promoting exclusively the formation of cognate paired IgG, which then reaches steady state expression by 2–3 days that later could promote mispairing of VH and VL. Furthermore, there was no significant difference between cognate chain-paired IgG levels and activity suggesting that all the chain-paired IgG were functional (Figure 4A). However, we cannot exclude that some IgG were only paired on one arm. The trio mRNA-encoded IgG also conferred protection against S. aureus-induced dermonecrosis (Figure 5) as measured by a significant decrease between trio IgG and negative control IgG of skin lesion sizes (Figure 5A) (p = 0.0002) and bacteria colony-forming units recovered from lesions (Figure 5C) (p = 0.0079). PK comparison of the trio IgG delivered intravenously as recombinant proteins (Figure 6A) or encoded by their respective mRNA (Figure 6B) showed comparable terminal half-life for the three mAbs expressed in a particular format (Figure 6C).Figure 4 In vivo PK of expression and neutralization of an mRNA/LNP-encoded trio IgG in mice

Tg32FcRn mice (n = 4) were intravenously injected with the three mRNA/LNP encoding the trio S. aureus IgG (1 mg/kg each). Mice were bled at the indicated time points and sera evaluated by (A) antigen-based ELISA for chain-paired IgG quantification (expression) or in vitro neutralizing assay (neutralization). Data are represented as mean values ± SD. (B) Expression of total IgG (⋅) and chain-paired IgG (▲). Total IgG were quantified by ELISA, and total chain paired IgG were calculated by adding the concentration of the three [IgG] quantified in (A). Data represent mean values ± SD. (C) PK of expression of the percent of chain paired IgG. For each mice, the percent of paired IgG was calculated as 100 × ([total paired IgG]/[total IgG]). Data represent the mean values of four mice. This experiment is representative of three separate experiments.

Figure 5 Trio mRNA/LNP encoding trio IgG provided protection in S. aureus mouse dermonecrosis

Tg32 human FcRn mice (n = 5) were intravenously injected with the three mRNA/LNP encoding the trio S. aureus IgG (1 mg/kg each) or a negative control scFv-Fc (3 mg/kg). Mice were infected 24 h later by intradermal injection with S. aureus strain SF8300. (A) Lesion sizes were measured after 1 and 7 days. Data are represented as geometric means. Statistical difference for lesion sizes between AAV-delivered scFv-Fc negative control and S. aureus scFv-Fc trio were analyzed with an unpaired Student t test and considered statistically different if p < 0.05. (B) Representative picture of three mice at day 1 and day 7. (C) Bacteria CFUs were enumerated on total skin lesions at day 7. CFUs are represented as geometric mean values. Differences between groups was analyzed with s unpaired Student t test and difference considered statistically different if p < 0.05. This experiment is representative of two separate experiments.

Figure 6 Serum PK profile of trio IgG encoded by mRNA or delivered as recombinant proteins

Tg32 human FcRn mice (n = 4) were intravenously infused with (A) the three recombinant IgG (10 mg/kg each) or (B) the mRNA/LNP encoding IgG (1 mg/kg each) or sera samples were collected at indicated timepoints and antibody concentration in sera evaluated in their respective antigen-based ELISA. Data are represented as mean values ± SD. (C) Mean serum concentration of four mice per group were used to estimate the PK parameters using a non-compartmental approach.

Trio IgG-mRNA expression in cynomolgus monkeys

We next evaluated IgG expression levels from the mRNA/LNP-encoded mAb combination in NHPs. The mRNA/LNP formulations were scaled up for the NHP study and initially tested in mice to ensure quality. There was no major difference observed in mAb expression compared with previous batches as co-delivery of the three formulations (Figures 4 and S6). The cynomolgus monkeys were infused intravenously with a mixture of 0.7 mg/kg or 0.4 mg/kg each of the three mRNA-IgG formulations (respectively, 2.1 mg/kg and 1.2 mg/kg total). We previously determined that 2 mg/kg was the maximum tolerated dose in cynomolgus monkeys (not shown). Serum was collected at indicated time points. Both doses were well tolerated; no major changes were observed upon physical examination, in body weight or food consumption from day −1 to the end of the study. IL-6 and MCP-1 serum levels were elevated 6 h after infusion (9.7× and 15.9× for the 0.4 mg/kg and 0.7 mg/kg groups, respectively), but returned to baseline levels by 24 h (not shown). For all mAbs, a higher IgG expression was observed following the higher dose of 0.7 mg/kg as compared with 0.4 mg/kg. Each mAb showed a dose response depending on the amount of mRNA/LNP infused. The levels of anti-AT and anti-ClfA mAbs in the 0.4 mg/kg group reached a Cmax around 0.4 μg/mL between 2 and 5 days and were below the limit of detection after 7 days (not shown), while the anti-Leuk mAb reached a sustained level of 4 μg/mL after 5 days (Figure 7A) and was detectable up to day 21 (not shown). The trio IgG in the 0.7 mg/kg mRNA group reached the Cmax after 5–7 days (Figure 7B). The average Cmax was 2.91 ± 1.26, 13.7 ± 4.52 and 4.07 ± 1.28 μg/mL for anti-AT, anti-Leuk and anti-ClfA mAbs, respectively. The IgG terminal half-life ranged between 7.8 and 18 days (Table 1). Different serum exposure was observed for each of the three mAbs with the greatest area under the curve for anti-Leuk mAb (Table 1). The concentrations were detectable until a maximum of 49 days, 77 days, and 105 days for anti-ClfA, anti-AT, and anti-Leuk mAbs, respectively (Table S1).Figure 7 PKs of the mRNA/LNP encoded-trio IgG in NHPs

Serum IgG PK for trio IgG. NHP (n = 3) were intravenously infused with each IgG mRNA/LNP at 0.4 mg/kg (A) or 0.7 mg/kg (B). Antibody concentrations in sera were quantified by ELISA at indicated timepoints. Data are represented as mean values ± SD. Limit of detection (LOD) was 0.234 μg/mL for all three mAbs.

Table 1 PK parameters of trio IgG mRNA/LNP at 0.7 mg/kg in NHPs

mAb	T½ (day)	Tmax (day)	Cmax (μg/mL)	AUCtlast (day∗μg/mL)	tlast (day)	AUC0-inf (day∗μg/mL)	
AT	14.3 ± 5.72	5	2.91 ± 1.26	71.9 ± 47.5	53.7 ± 29.1	78.1 ± 49.3	
Leuk	15.4 ± 1.07	5	13.7 ± 4.52	95.7 ± 8.08	95.6 ± 7.0	372 ± 82.1	
ClfA	11.8 ± 1.39	5	4.07 ± 1.28	71.4 ± 20.1	46.7 ± 4.04	77.4 ± 21.4	
Mean values ± SD of three animals.

AUC0-inf, area under the concentration-time curve from day 0 to infinity; AUCtlast, area under the curve between first time of dosing and time for last observable concentration; Cmax, maximum observed concentration after dosing; T½, terminal half-life; tlast, time after dosing at which the last quantifiable concentration was observed; Tmax, time after dosing at which the maximum concentration is observed.

Sera were evaluated in the respective neutralizing assays for anti-AT, ClfA and Leuk mAbs. Animals showed pre-existing neutralizing antibody titers for AT and HlgCB above mAb levels detected during the study. Consequently, it was not possible to determine the neutralizing activity of the anti-AT and Leuk mAbs in these samples. The pre-existing ClfA neutralizing antibody levels were minimal, and anti-ClfA neutralizing titers induced from the mRNA/LNP were detected. No chain swapping was observed for the anti-ClfA mAb (Figure S7).

Discussion

Progress in gene editing technology, nucleic acid engineering and lipid-nanoparticle formulations have resulted in new clinical applications in the field of in vivo expressed biologics (IVEBs), where the body is used as its own bioreactor to deliver therapeutic vaccines32,33 or mAbs.17 DNA- or RNA-encoded antigen vaccines elicit a polyclonal and protective antibody response upon processing by the immune system of the induced proteins. Nucleic acid-based vaccines are faster to manufacture than traditional adjuvanted subunit vaccines and demonstrated a strong clinical benefit during the coronavirus disease 2019 pandemic.34 This recent success of mRNA-based vaccines suggests that there may be future opportunities for nucleic acid delivery of other therapeutics such as mAbs. However, the path for the clinical development of an mAb via IVEB is more complex. A successful formulation would result in rapid and prolonged serum IgG levels at concentrations required to effectively treat or prevent disease. Although in vivo expressed mAb technologies have demonstrated promise for a single mAb, both in preclinical studies and in people,4,17,35,36 the success for delivering a mAb combination is limited due to HC and LC mispairing resulting in inactive IgG molecules.29,37 To test this hypothesis, we utilized a three mAb combination targeting six different S. aureus virulence factors with demonstrated efficacy in multiple disease models.24,25,26,27 Using these three antibody sequences, we demonstrate for the first time the feasibility of delivering a mAb combination intravenously via the mRNA/LNP platform as scFv-Fc and IgG.

One concern for delivering an mAb combination through in vivo expressed mAb platform is that the nucleic acids coding each mAb could enter the same cell and result in heavy and light non-cognate chain pairing, which would reduce mAb binding activity. While the VH complementary determining region 3 is essential in the epitope recognition,38 the role of the cognate LC should not be neglected. The VL may either interact directly with the antigen and engage its own CDR residues for epitope binding or indirectly modulate the VH conformation and binding residue exposure through hydrophobic and disulfide bound interaction.39,40 Intuitively, this mispairing could be reduced by injecting the respective IgG-encoded nucleic acid formulations at different sites intramuscularly or engineered multi-specific antibodies comprising two or more covalently linked antibody variable domains.41,42 Recently, an anti-SARS-CoV-2 duo mAb cocktail (EVUSHELD) encoded by two individual DNA plasmids delivered intramuscularly at separate sites by electroporation retained full activity and provided protection in preclinical challenge models.37 Alternatively, intravenous delivery of nucleic acid-encoding an anti-Clostridioides difficile tetra-specific antibody formed with four different single chain VH domain of HC only antibody (VHH) linked by glycine-serine linkers,43,44 or intramuscular delivery of a DNA-encoded bispecific mAb against Pseudomonas aeruginosa16,45 induced antibody expression level in sera resulting in disease protection in mice. Four AAV-based therapies have been clinically approved for gene replacement therapy for treatment of hemophilia B, macular degeneration or neurological disorder indications.46,47,48,49 Although AAV-encoded mAbs induced circulating levels of more than 100 μg/mL and demonstrated protection against HIV infection in preclinical models, the platform showed immunogenicity issues and did not progress beyond phase I clinical trials.50 Additionally, a strong anti-drug antibody (ADA) response associated to some AAV serotypes resulted in accelerated antibody clearance in cynomolgus monkeys.51 Finally, most of the population possess neutralizing antibody titers against multiple AAV serotypes52 which may induce ADA and immunogenicity after redosing, accelerate clearance, and be associated with life-threatening severe toxicity.53,54 Collectively, these clinical observations suggest that the AAV-encoded platform may not be the safest and most efficient option to deliver therapeutic mAbs clinically. Although mRNA-encoded protein-based vaccines provide a protective response after intramuscular administration, the highest levels of mAb expression in sera are achieved preferentially after intravenous infusion with the current mRNA/LNP technology by primarily targeting hepatocytes.4 To de-risk our approach, we initially tested co-delivery via tail vein of three S. aureus mAbs in the scFv-Fc format. In this format, chain mispairing was not observed in vitro, and the scFv-Fc combination provided strong protection in a mouse dermonecrosis model. Delivery of the three antibodies in IgG format resulted after 3 days in expression in 64%–78% of cognate chained-paired IgG with a prolonged PK and higher expression levels in sera compared with scFv-Fc and similar protection in vivo.

A major challenge for delivering an mAb combination via mRNA/LNP platform is reaching similar circulating mAb levels required for clinical efficacy. Dose titrations of recombinant mAbs in preclinical models along with PK studies enable the selection of human dose ranges for further clinical development and help determine expression levels to achieve through nucleic acid delivery. S. aureus is a commensal bacterium causing a broad range of diseases upon penetration in the body through skin barrier damage or through an indwelling device.55 S. aureus modulates expression of its virulence factors in response to its growth phase and its environment.56 The trio mAb targeting AT, ClfA, and Leuks promotes disease coverage in multiple preclinical models and showed protection at different doses depending on the disease model. The minimum doses of mAb combination required for protective efficacy in S. aureus-induced dermonecrosis, lethal bacteremia, and lethal pneumonia are, respectively, 20 μg/mL, 150 μg/mL, and 300 μg/mL.24,57 The mRNA-encoded three mAb combination reached peak serum levels between 15 and 90 μg/mL in mice and conferred protection in a dermonecrosis model. Therefore, to provide protection against other S. aureus induced-diseases, the mRNA platform may require optimization including the selection of a new ionizable lipid or refining the lipid composition for increased transfection efficiency58 or mRNA engineering to improve intracellular stability and increase half-life.59 The current data also highlighted the potential importance of the mRNA secondary structure on mAb expression. Although each mRNA was optimized similarly, the PKs of expression of the anti-Leuk mAb was higher than the two other mAbs in mice and cynomolgus monkeys. The secondary structure of mRNA may control the formulation stability and half-life, ultimately resulting in enhanced protein expression.60 Hence, each mRNA may require specific optimization by employing powerful algorithms for both structural stability and codon usage.61 Although demonstrating an acceptable safety profile, intravenous infusion of NHPs with the three mRNA-encoding mAbs induced antibody levels ranging between 2.9 and 13.7 μg/mL. These data confirmed previous observations that antibody levels induced in rodents do not necessarily translate to similar levels in NHPs for a particular mRNA-LNP formulation.20 Therefore, even if a mRNA-encoded mAb reached a target sera concentration conferring protection in pre-clinical models,62 there is little evidence that this platform can provide the therapeutic concentration required for protection in humans. To our knowledge, the mRNA/LNP platform was tested only once in humans for the expression of an anti-Chikungunya virus mAb, and achieved therapeutically relevant neutralizing titers (1 μg/mL) in sera over 16 weeks.35 However, the levels observed in humans were lower than those observed in macaques,20,35 highlighting the difficulty of translating results from animals into people. Since anti-infective mAb therapeutic serum concentration are specific to their respective targets, we cannot exclude that different mAbs against other pathogens could be successfully delivered via the mRNA platform. Engineering an mAb to increase its potency and lower its protective concentration, increasing the likelihood of success by mRNA delivery. The situation is even more complex for an mAb combination where different expression levels between mAbs were observed in vitro and in vivo for each mAb. Since preclinical models for the S. aureus mAb trio were developed with an mAb combination comprising the same dose of each antibody,25,27 a therapeutic and commercially viable in vivo-expressed mAb approach for such a combination may require fine-tuning the mRNA/LNP dose or selecting mAb sequences early in the discovery process optimized for nucleic acid delivery. Currently, mAb sequence cannot be used to predict expression levels in vivo, which is an opportunity for future research to further streamline development timelines for nucleic acid encoded mAbs. Together our data demonstrate the feasibility of delivering a combination of several therapeutic mAbs through mRNA/LNP platform and opens new perspectives for further clinical development.

Materials and methods

Antibodies

The three anti-S. aureus mAbs were human IgG1 isotypes and contained an N3Y mutation (L432C/H433S/N434Y/Y436L/T437C) on their Fc region for half-life extension.63 The anti-AT mAb (MEDI4893∗ or LC10) was generated from Velocimmune humanized mice {Tkaczyk, 2012 #35}. The anti-ClfA (SAR114) and the anti-Leuk (SAN177) mAbs were generated from tonsillar human B memory cells using the Antigen-specific Memory B cell Repertoire Analysis (Ambra) technology as described previously.24,27 R347 is a human IgG1 anti-pg120 of HIV and used as an isotype control (c-IgG).24 The duet mAb comprising the HC/LC of the anti-AT mAb on one arm and of the anti-Leuk mAb on the other arm was constructed, as previously reported.64

scFv-Fc constructs

Each mAb was initially converted into a scFv molecule where the VL and VH chains were linked using a (G4S)2 linker. Synthetic scFv gene fragments were synthesized by Geneart and then cloned into a mammalian expression vector in frame with human Fc with the N3Y modification for half-life extension.63 Clones of the scFv-Fc were confirmed by Sanger sequencing. The scFv-Fcs were transiently expressed using HEK293 suspension cells and purified from conditioned media using MabSelect SuRe column chromatography and polished over a Superdex HiLoad column to remove aggregates.

mRNA synthesis

Each mRNA vector comprises a 5′ cap structure incorporated through the inclusion of a cap analogue during mRNA synthesis by a T7 polymerase-driven in vitro transcription (IVT) reaction; a 5′ untranslated region sequence from the human hydroxysteroid 17beta dehydrogenase 4 gene; a signal sequence from the human kappa Ab LC; an open reading frame (respectively coding for scFv-Fc, IgG HCs or LCs); a 3′ untranslated region sequence taken from the human albumin gene; a polyA tail. The mRNA molecules are prepared as mRNA where uridine is replaced by 5methoxyuridine to minimize recognition of the mRNA by the innate immune system in vivo. The mRNA vectors were prepared from corresponding DNA plasmids which contain the entire mRNA cassettes as described above, along with a T7 polymerase promoter sequence upstream of the mRNA cassette to allow synthesis of mRNA using IVT. The DNA template is linearized by a restriction digestion before IVT, to ensure all mRNA molecules terminate directly after the polyA tail, referred to as runoff transcription.

LNP formulation

The formulations were prepared using a NanoAssemblr Benchtop microfluidic mixing system (Precision Nanosystems) at a 3:1 aqueous:ethanol flow rate ratio and total flow rate of 12 mL/min. The mRNA is prepared in a 50 mM citrate buffer, pH 3.0, while the lipids are prepared in ethanol. The LNPs were mixed at a 50:10:38.5:1.5 M ratios of the ionizable lipid: distearoylphosphatidylcholine (sourced from NOF): cholesterol (Sigma): 1,2-Dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG 2,000, sourced from NOF). All LNPs were prepared at an approximate N/P ratio of 4.7. For the IgG encoding constructs the mRNA is mixed at a 1:1 M ratio of HC:LC expressing mRNA. LNP formulations are dialyzed against 1× PBS, pH 7.2 for 2–4 h, and then dialyzed against 1× PBS, 10% (w/v) sucrose, pH 7.2 overnight (∼18 h) using dialysis cassettes at 4°C. The LNPs were then concentrated using Vivaspin centrifugal filters (Cytiva) and passed through a 0.22-μm filter and stored at −80°C. The mRNA-LNP formulation was and thawed at room temperature (RT) before use. All formulations were characterized by dynamic light scattering for particle size (Malvern Zetasizer Nano-ZS), RiboGreen assay for RNA encapsulation and concentration, pH, osmolality (Advanced Instruments OsmoPRO), and endotoxin (Charles River Endosafe Nexgen-PTS).

In vitro transfection with mRNA encoding scFv-Fc or IgG mAb

HEK-Expi293F cells (ATCC) were cultured in RPMI 10% fetal bovine serum (FBS) (VWR) and plated in a 24-well plate (VWR) in suspension at 3 × 106 cells/mL in 3 mL/well (9 × 106 cells/well). Transfection was performed with Lipofectamine 2000 (VWR) at 4:1 ratio (4 μL lipofectamine per 1 μL mRNA). Cells were transfected with mRNA encoding for a single, two or three scFv-Fc or IgG at 300 ng per construct. For IgG transfection, 200 ng HC mRNA was combined with 100 ng LC mRNA. As a filler, mRNA encoding for eGFP was added as required to reach a 900 ng total mRNA per well. For the scFv-Fc transfection, mRNA for scFv-Fc and eGFP were mixed separately with lipofectamine, and then combined in each well. For the IgG, HC and LC mRNA transcripts of each mAb were first combined before mixing with lipofectamine. Cell supernatants were harvested 48 h after transfection and frozen at −80°C for quantification of antibody expression and neutralizing activity in their respective assay.

Quantification of total IgG or scFv-Fc secreted in vitro by Bio-Layer Interferometry

Total IgG or scFv-Fc were quantified in each well after 48-h transfection using a BLI platform on an Octet RH96 (Sartorius AG, Germany) with protein A biosensors. Purified IgG and scFv-Fc were diluted in Expi293 expression medium (RMPI 10% FBS) to generate standards and quality control (QC) samples. Standards were 3-fold serially starting at 100 μg/mL, and test samples were diluted 2-fold. Sample dilutions, equilibration and neutralization steps were all performed in RMPI 10% FBS, with 10 mM glycine pH 1.7 used for regeneration steps. Sensors were equilibrated for 10 min before pre-conditioning with three cycles of regeneration and neutralization (30 s each). Each sample binding step (120 s) was followed by another three cycles of regeneration and neutralization. All steps were performed at 30°C and with a 400 rpm shaking. The data were analyzed using Octet Analysis Studio software.

Quantification of IgG or scFv-Fc by antigen-specific ELISA

In vitro expressed mAbs was quantified by its respective antigen-specific ELISA in each well after 48 h transfection. Plates were washed between each incubation with PBS 0.1% Tween 20 (Sigma). MaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with either AT (1.5 μg/mL), ClfA (2 μg/mL), or HlgB (2 μg/mL) in 0.2 M carbonate/bicarbonate buffer, washed and blocked for 1 h at RT with PBS 5% BSA. Plates were incubated with cell culture supernatant diluted in PBS. Respective purified IgG or scFv-Fc mAbs were used as standards. After 1.5 h incubation at RT on a 200 rpm rotating shaking, plates were incubated for 30 min with 50 μL horseradish peroxidase (HRP)-conjugated goat anti-human IgG diluted 1:10,000 (Jackson Immuno-Research) and then 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL). The reaction was stopped after 10 min with 100 μL 0.2 M H2SO4. The optical density at 450 nm (OD450) was measured with a spectrophotometer (Molecular Devices). For wells transfected with two or three mRNA, the total concentration of in vitro expressed antibody was obtained by adding the concentrations of each antibody measured by their antigen-based ELISA.

A similar assay was used to determine the PK of each mAb in mouse sera. Standards, QC and sera samples were prediluted in mouse sera, and pre-pared in assay buffer with a 1:100 minimum required dilution.

Human IgG PK in mouse sera by total IgG ELISA

Maxisorp plates were coated overnight at 4°C with 100 μL of sheep anti-human IgG (Bethyl Laboratories) diluted at 1 μg/mL in 0.2 M carbonate/bicarbonate. Three washes with PBS 0.1% were performed between each incubation. Plates were blocked with PBS 5%BSA for 1.5 h at RT. Purified IgG standards, QC samples and sera samples (pre-diluted in mouse sera) were prepared in assay buffer with a 1:100 minimum required dilution and incubated for 1.5 h at RT on a 200 rpm shaking. The IgG bound was detected with 100 μL HRP-conjugated goat anti-human IgG (Bethyl Laboratories) at 1:15,000 dilution in assay buffer. After 0.5-h incubation at RT, 100 μL TMB was added, and reaction stopped with 100 μL H2SO4. The OD450 was measured with a spectrophotometer. The standard curve, composed of 11 concentrations, was generated using a four-parameter logistic curve fit [y = ((A − D)/(1+(x/C)ˆB)) + D without weighting in SoftMax Pro (SMP) software, version 5.4.5 The concentration of total antibody in serum samples was determined relative to the standard curve.

AT and HlgAB hemolytic assay on rabbit red blood cells

The neutralizing activity of the anti-AT and anti-Leuk in cell culture supernatant was quantified in a rabbit red blood cell hemolytic assay respectively in presence of 10 ng/mL of AT or γ-hemolysin HlgAB as described.27 Supernatant dilutions (1:200 and 1:50) in culture media (RPMI, 10%FBS) were incubated with AT or HlgAB for 2 h at RT with 450 rpm shaking at a 1:1 ratio in a U bottom plate (Thermo Fisher Scientific). Serial dilutions in culture media of anti-AT or anti-Leuk mAbs in IgG or scFv-Fc were used as standards. Non-specific human IgG or scFv-Fc R347 was used as negative control. Two hundred microliters were then transferred in duplicate to another U-bottom plate and incubated with 50 μL of washed rabbit RBC at 5× 106/mL (Peel Freeze) for 2 h at 37°C. Plates were then centrifuged at 1,200 rpm for 3 min, and 50 μL supernatant was transferred to new plates. The OD450 was measured with a spectrophotometer. The % of inhibition of AT or HlgAB-hemolysis was calculated as: 100 –(100∗[ODtoxin+mAb]/[ODtoxin alone]).

The functionality of in vivo-expressed anti-AT or anti-Leuk scFvFc antibodies was quantified from mouse sera with a similar assay. Recombinant scFv-Fc or IgG were used as standards and diluted in PBS 0.5% BSA. Mouse sera were diluted at a minimum dilution of 1:200 in PBS 0.5% BSA. For each assay, the standards, samples and QC were analyzed in duplicates. The standard curve, comprised of 11 concentrations, was generated using a four-parameter logistic curve fit [y = ((A − D)/(1+(x/C)ˆB)) + D without weighting in SoftMax Pro (SMP) software, version 5.4.5 The concentration of total antibody in serum samples was determined relative to the standard curve.

Fibrinogen-ClfA binding assay

The neutralizing activity of in vitro-expressed anti-ClfA mAb was evaluated in a fibrinogen-ClfA binding assay as described.27 Between each incubation, plates were washed three times with PBS 0.1% Tween 20. Maxisorp plates were coated overnight at 4°C with 2 μg/mL of fibrinogen (Enzyme Research Laboratories) diluted in PBS. The next day, plates were blocked for 1 h at RT with 200 μL/well of casein (Thermo Fisher Scientific). In parallel, a mix of 50 μL transfection supernatant (diluted 1:2 or 1:4 in RPMI 10% FBS) and 50 μL AviTag ClfA221-559 (2 μg/mL) were incubated in a U-bottom plate for 30 min. Purified anti-ClfA mAb IgG or scFv-Fc were used to generate standards and QC samples. The mixture was then incubated on the fibrinogen-coated plates for 1.5 h at RT under 250 rpm shaking. Plates were then incubated for 30 min at RT on shaking with 100 μL of HRP-conjugated streptavidin (1:20,000; GE Healthcare). The bound ClfA was detected by adding 100 μL of TMB substrate, and the reaction stopped after 10 min with 100 μL of H2SO4. The OD450 was measured with a spectrophotometer. The % of inhibition of ClfA/fibrinogen binding was calculated as: 100 –(100∗[ODClfA+mAb]/[ODClfA alone]).

The anti-ClfA mAb expressed in mouse sera was quantified with a similar assay. Standards and QC were diluted in PBS 0.5% BSA. Mouse sera minimum required dilution was 1:25. For each assay, standards, samples and QC were analyzed in duplicates. The standard curve, comprised of 11 concentrations, was generated using a 4-parameter logistic curve fit [y = ((A − D)/(1+(x/C)ˆB)) + D without weighting in SoftMax Pro (SMP) software, version 5.4.5 The concentration of total antibody in serum samples was determined relative to the standard curve.

Mice

All animal studies were approved by the AstraZeneca Institutional Animal Care and Use Committee and were conducted in an Association for Accreditation and Assessment Laboratory Animal Care (AAALAC)-accredited facility in compliance with U.S. regulations governing the housing and use of animals. Human FcRn transgenic mice B6.Cg-FcgrtPrkdc Tg32Dcr/DcrJ (Tg32FcRn) were obtained from Jackson Laboratories.

Mouse injections with mRNA/LNP

Mice Tg32FcRn (8–9 weeks old) were injected intravenously in the tail vein with mRNA/LNP encoding a single mAb at indicated dose diluted in 200 μL of PBS. For delivery of the trio mAbs the three individual mRNA/LNP were thawed, and then mixed in a tube and injected intravenously together in the tail vein in 200 μL of PBS. Mice were bled via the sub-mandibular route at indicated time points, and sera evaluated for antibody concentration and neutralizing activity.

Bacteria

Community acquired-methicillin resistant S. aureus (CA-MRSA) USA300 strain SF8300 was kindly provided by Dr B. Diep (University of California San Francisco). Bacteria were grown to mid-log phase to an OD600 of 0.8 in trypticase soy broth (TSB, VWR International), washed twice in ice-cold PBS (Invitrogen, Carlsbad, CA), and frozen in 10% glycerol-TSB aliquots. Challenge inoculums were prepared from one frozen vial for each experiment, diluted in ice-cold PBS pH7.2 (VWR International, Swedesboro, NJ), and placed on ice until inoculation.

Protection in murine S. aureus dermonecrosis model

Dermonecrosis was induced by inoculating mice intradermally with S. aureus as described previously.31 Briefly, mice (n = 4/group) were shaved on their back and the mRNA/LNP formulations encoding the3 mAb combination or an irrelevant mAb (c-IgG or scFv-Fc) were delivered to Tg32FcRn mice (8 to 9 weeks old) by intravenous injection. Mice were then inoculated intradermally with S. aureus. Briefly CA-MRSA, USA300, 5 × 107 colony-forming unit (CFU)/mouse in 50 μL PBS 24 h later. Lesions were measured after 1 and 7 days. Data are representative as geometric means. Statistical difference for lesion sizes between mAb negative control and S. aureus mAb trio were analyzed with an unpaired Student t test and considered statistically different if p < 0.05.

Expression and functional activity of IgG combination in cynomolgus monkeys after infusion of mRNA/LNP

Studies in cynomolgus monkeys were conducted at LabCorp Laboratories. LabCorp is fully accredited by the AAALAC. All procedures were in compliance with applicable animal welfare acts and were approved by the local Institutional Animal Care and Use Committee. The cynomolgus monkeys used in the study were 29- to 37-month-old females, weighing 1.8–3 kg at the time of dosing. Animals were group-housed in European guideline (ETS 123) compliant pens (≤3 animals/pen) and enriched with bedding materials. Animals were individually housed during the study procedures. Environmental controls were set to maintain a temperature range of 20°C–26°C, a relative humidity range of 30%–70%, eight or more air changes/hour, and a 12-h light/12-h dark cycle. The light/dark cycle was interrupted for study-related activities. Any variations to these conditions had no effect on the study outcome. Water was provided ad libitum. Animals were given cage-enrichment devices and fruits, vegetable, or dietary enrichment. They were offered Certified Primate Diet #5048 (PMI Nutrition International Certified LabDiet) one to two times daily, except during study procedures. Dose formulations were administered once by intravenous infusion via the right or left saphenous vein using a motorized infusion pump set at 60 min at a dose of 5 mL/kg. Body weight, food consumption, and clinical signs and parameters were evaluated throughout the study.

Collection of serum samples from NHPs

Blood samples (approximately 1 mL) were collected via the femoral vein during the pre-dose phase (1 day before study initiation), then at 6, 24, and 48 h after infusion from each animal, and then on days 5, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, and 105. Blood was drawn into serum separator tubes (with clot activator), allowed to clot at RT, and centrifuged within 1 h of collection. Serum was harvested into four aliquots and stored in a −80°C freezer.

Human IgG PK in NHP sera

The NHP sera were analyzed by ELISA to quantify the three S aureus mAbs. Plates were washed three times with PBS 01.% Tween 20 between each incubation. Anti-AT and anti-ClfA IgG concentrations were quantified using two specific pair of anti-idiotype mouse mAbs. Briefly, standard curves and QCstandards were prepared in blank pooled cynomolgus monkey serum and using purified anti-AT and anti-ClfA IgG as reference standard ranging from 0.234 to 15 μg/mL. Maxisorp plates were first incubated overnight with anti-idiotype clones 1E10.2 or 7A2.1 at 4°C. Standards, QC, and test samples were then incubated for 90 min at RT, lates were next incubated with their respective biotin-conjugated anti-idiotype clones 6B9.23 or 7A2.1 for 1 h at RT. Follow washes and 1 h incubation at RT with streptavidin-HRP (diluted 1:10,000), . antibody binding was detected by addition of 100 μL TMB substrate, and reaction stopped with 100 μL 0.2M H2SO4. The OD450 was read on a spectrophotometer. Anti-Leuk mAb was quantified with an HlgB specific ELISA. Maxisorp plates were coated with 1.5 μg/mL of HlgB overnight at 4°C. Calibration curve standards and QC standards were prepared in blank pooled cynomolgus monkey serum. Purified anti-Leuk IgG was used as standard curve ranging from 0.234 to 15 μg/mL. Standards, controls, and test samples were incubated for 90 min at RT on HlgB-coated plates. After washes, plates were incubated for 1 h at RT with HRP-conjugated goat anti-human IgG (1:10,000). Antibody binding was detected with 100 μLTMB, and reaction stopped with 100 μL H2SO4. The OD450 was read on a spectrophotometer. For each assay, all standards, QCs, and samples were analyzed in duplicate wells. The standard curve, composed of 11 concentrations, was generated using a four-parameter logistic curve fit [y = ((A − D)/(1+(x/C)ˆB)) + D without weighting in SoftMax Pro (SMP) software, version 5.4.5 The concentration of total antibody in serum samples was determined relative to the standard curve.

PK estimation and half-life of human mAbs in NHP sera

Sera concentration data from three female cynomolgus monkeys per group dosed with the mRNA/LNP formulations was used to estimate PK parameters. Parameters were estimated using noncompartmental analysis in Phoenix WinNonlin version 8.1 (Certara). PK parameters were estimated for each dose event.

Statistics

GraphPad Prism (GraphPad Software) was used to perform statistical analysis in mouse studies. Statistical differences skin lesions or CFU between mRNA-encoded control mAb or S. aureus trio mAbs at day 1 or day 7 were analyzed with an unpaired Student’s t test. Statistical difference between a particular scFv-Fc expression or neutralizing activity in sera at day 1, day 3, or day 7 induced by a single mRNA or the trio mRNA were analyzed with an unpaired Student’s t test.

Data and code availability

All data needed to evaluate the conclusions in the paper are available in the main text or the Supplementary materials. All raw data in the paper are available upon request.

Supplemental information

Document S1. Figures S1–S7 and Table S1

Document S2. Article plus supplemental information

Acknowledgments

The authors thank Susan Wilson for generating the recombinants scFv-Fc; Emily Minton and Michael Ford for technical assistance with animal procedures, and Rui Ma and Xiuling Li for generating the anti-AT/Leuk duet mAb.

This research was developed with funding from the Defense Advanced Research Projects Agency (10.13039/100000185 DARPA ). The views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the 10.13039/100000005 Department of Defense or the U.S. Government

Author contributions

Conceptualization: C.T., L.S., L.D., G.T., and J.D.; methodology: M.P., M.N., A.G., C.T., and A.E.K.; investigation: M.P., A.S., S.H., A.M., G.T., E.B., H.Y., and O.S.; visualization: C.T. and M.P.; writing – original draft: C.T. and B.R.S.; writing-review and editing: C.T., B.R.S., M.N., M.T.E., and A.D.

Declaration of interests

The authors declare a conflict of interest. This work was funded by AstraZeneca. C.T., M.N., M.P., G.T., L.D., A.G., A.M., J.D., S.H., M.T.E., A.E.K., A.D., and B.R.S. are employees of AstraZeneca and may hold AstraZeneca stock. L.S. was employee of AstraZeneca and may hold AstraZeneca stock.

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2024.05.036.
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