Co-administration of an effector antibody enhances the half-life and therapeutic potential of RNA-encoded nanobodies – Scientific Reports

Protein design

VNA frameworks have been described previously³⁷. In brief, VNAs contained a murine Ig kappa signal peptide (P01661, Uniprot), one epitopic O-tag (DELGPRLMGK), V_HH domains targeting epitopes of C. difficile toxins A and B¹¹, another O-tag and a carboxyl-terminal murine ABP⁵⁷. VNAs lacking the ABP contained one E-tag epitope (GAPVPYPDPLEPR) and one HA-tag epitope (YPYDVPDYA). The O-tag epitope is a modified OLLAS tag⁴⁰ in which several amino acids at the amino end of the original OLLAS tag were found to be unnecessary for strong mAb binding and eliminated. The recombinant rat OLLAS mAb (used as the EfAb in this study) has been reported previously⁴⁰.

The design of the RNA-encoded EfAb was based on the variable regions of the rat EfAb⁴⁰ fused to a human heavy chain IgG1 framework (IGHG1*03 / G1m17,1; K120; E12/M14; derived from P01857, Uniprot) and to a kappa light chain (P01834, Uniprot). The signal peptides for heavy and light chain were derived from P01750 (Uniprot) and Q6P5S8 (Uniprot), respectively. All sequences can be found in Supplement Table 1. The sequence of SO57 antibody has been described previously³⁷. The non-translated version of GFP lacked a start codon and included several stop codons.

RNA design

The design and synthesis of mRNA sequences have been described previously^{58, 59}. In brief, mRNAs contained a 5’ UTR, the open reading frame, a 3’ UTR and a poly-A sequence followed by a C30 stretch and a histone stem loop and involved unmodified nucleotides. Following in vitro transcription, mRNAs were enzymatically capped and 2′-O-methylated using vaccinia virus capping and 2’-O-Methyltransferase enzymes (CELLSCRIPT) and enzymatically adenylated using A-Plus Poly(A) Polymerase (CELLSCRIPT). All mRNAs were purified by RP-HPLC as described previously⁵⁹. A detailed protocol has been described elsewhere⁶⁰.

RNA formulation

In general, lipid nanoparticle formulation was performed with Acuitas therapeutics LNP technology and has been described previously⁶¹. In brief, the LNPs were generally composed of an ionizable amino lipid, phospholipid, cholesterol, and a PEGylated lipid. mRNAs encoding IgG were mixed at a molar ratio of 1.2:1 (HC:LC, EfAB) or 1.5:1 (HC:LC, SO57) before LNP formulation. For injections, mRNA-LNP was diluted in phosphate-buffered saline pH 7.4 (PBS). mRNAs-LNP encoding VNA-TcdB lacking ABP contained SM-102 as ionizable amino lipid.

Cell transfections

Transfection of mRNAs into cells has been described previously³⁷. In brief, baby hamster kidney cells (BHK, ATCC) were cultivated, transfected, and maintained in conditioned medium (RPMI 1640, 10% FCS, 1% Penicillin / Streptomycin, 1% L-Glutamine, Lonza). Supernatants were harvested and cell lysates were prepared at either 24 or 48 h post transfection.

Protein purification

The production and purification of recombinant proteins has been described previously⁶².

Western blot analysis

Cell lysates and supernatants were loaded on 12% criterion TGX gels according to manufacturer’s instructions (Bio-Rad). Following SDS-PAGE, samples were transferred onto a 0.22 µm nitrocellulose membrane using the criterion blotting system (Bio-Rad). All washing and incubation steps have been described previously³⁷. For detection of O-tagged VNAs, the following antibodies have been used: OLLAS Epitope Tag Antibody (L2) from rat (Novusbio, 1:1,000) and a Rabbit anti-α/β tubulin (New England Biolabs, 1:1000) as primary antibodies, and Goat anti-Rat IgG (H + L) IRDye 800 CW (LI-COR, 1:15,000) as well as a Goat anti-Rabbit IgG (H + L) IRDye 680 RD (LI-COR, 1:15,000) as secondary antibodies. Primary antibodies were incubated for two hours. Secondary antibodies were incubated for one hour. For detection of RNA-encoded EfAb the following antibodies have been used: A Rabbit anti-tubulin UNLB (Cell Signaling Technology, 1:15,000) as primary antibody, a Goat anti-human IgG CW800 (LI-COR, 1:15,000) as well as a Goat anti-rabbit RD680 (LI-COR, 1:15,000) as secondary antibodies. Primary and secondary antibodies have been incubated for one hour. Protein detection and image processing were carried out in an Odyssey CLx^® Imaging system and Image Studio version 5.2.5 (LI-COR) according to manufacturer’s recommendations.

ELISA

The general procedure of sandwich ELISA based detection and has been described previously⁵. In brief, all ELISAs were performed using serial fivefold dilutions of supernatants, sera, and standards. Microplates were coated overnight at 4°C and blocked for a period of one to two hours. Diluted samples as well as purified and quantified standards were incubated at room temperature for one hour followed by incubation of detection Abs for one hour. Plates were washed three times in between incubation steps. Finally, 3,3=,5,5=-tetramethylbenzidine (TMB, Sigma) was added, and reactions were terminated with 1M sulfuric acid at times optimized for each condition. Plates were measured at 450 nm with 570 nm correction in a BioTek Synergy plate reader. Antibodies for detection of VNAs, EfAb and ADA are summarized in Supplement Table S2. Serum VNA concentration quantification was performed as previously described¹⁸.The quantification of human IgG1 (RNA-EfAb) from cell culture supernatants has been described previously³⁷. The quantification of VNA-TcdB lacking ABP was performed according to³⁷ and involved a Rabbit anti-E-tag antibody for capture (Bethyl, 1:500) and a biotinylated Rabbit anti-HA-tag antibody (Abcam, 1:15,000) for detection.

To quantify human IgG1 (SO57) from M. fascicularis serum, a human therapeutic IgG1 ELISA kit (Cayman) was used according to manufacturer’s instructions. To measure the binding affinity of murine ABP to albumin, ELISA plates were either coated with murine or porcine albumin and serial dilutions of recombinant VNA-BoNTA containing an E-tag epitope and a carboxyl terminal murine ABP were added. An anti-E-tag antibody conjugated to horseradish peroxidase was used for detection.

In vitro neutralization assays

The procedure has been reported previously^{5, 17}. In brief, Vero cells (ATCC) were incubated with serial dilutions of conditioned medium from BHK cells, untransfected contols or transfected with mRNA encoding VNAs, and toxins were added to all wells for a period of 24 h. A blinded researcher assessed the percentage of cells that were rounded in each well, and this estimate was plotted as a function of the dilution of conditioned medium present within each well. .

Animal experiments

Treatment and care of all animals used in experiments followed institutional animal care and use committee guidelines. Mouse and piglet studies were conducted at Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine (North Grafton, USA). All protocols describing animal use were approved by the Tufts University Institutional Animal Care Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council, USA. Six- to eight-week-old female outbred CD-1 mice (Charles River Labs, Wilmington, USA) were randomized based on body weight and organized in five mice per group or cohort. All injections were done intravenously in a volume of 100 µl into the tail vein. For dose finding studies, groups of mice received single intravenous injections of serially diluted mRNA-LNPs. Blood was harvested at 24 h post injection by either submandibular or retro-orbital bleeding and serum was prepared. In PK studies, for each time point, a separate cohort of mice was used. For co-administration of different mRNA-LNPs, a mixture of diluted mRNA-LNPs was prepared prior to co-injection. For co-administration of mRNA-LNP and recombinant EfAb, both components were pre-diluted in PBS to an intermediate twofold concentration, and mixed 1:1 prior intravenous co-injection.

Toxins were administered intraperitoneally at either 25 ng (early challenge, 24 h post mRNA-LNP treatment) or 50 ng (late challenge, 14 days post mRNA-LNP treatment) per mouse. Following intoxication, mice were monitored for signs and symptoms of toxemia (including lethargy, depression, anorexia, dehydration, ruffled coat, and hunched posture) to determine the clinical score (Supplement Table 3). Mice exhibiting signs of severe illness (lethargy, difficulty with ambulation, lack of responsiveness to tactile stimulation, wasting) or weight loss ≥ 15% body weight were humanely euthanized according to IACUC recommendations.

Gnotobiotic piglets were derived via caesarean section and maintained in sterile isolators for the duration of the experiment⁶³. In general, groups of two piglets were used if not stated differently. mRNA-LNPs were injected intravenously at a total dose of 1 mg/kg into the jugular veins. For co-administration of different mRNA-LNPs, a mixture of diluted mRNA-LNPs was prepared prior to injection. Mixtures either contained mRNA-LNPs encoding VNA-TcdB together with mRNA-LNPs encoding EfAb or together with mRNA-LNPs containing a non-coding RNA (non-translated GFP). Blood was harvested by routine methods and serum was prepared.

PK studies involving VNA-TcdB lacking ABP were conducted at Preclinics GmbH (Potsdam, Germany). Six- to eight-week-old female Balb/c mice were injected via the tail vein (100 µl) and blood was obtained by retro-orbital bleeding. The reference of the animal welfare approval is 2347-14-2018 Ä 5 approved by the animal welfare commission of the State of Brandenburg (LAVG—Landesamt für Arbeitsschutz, Verbraucherschutz und Gesundheit). The study protocol was reviewed by the internal council of Preclinics GmbH.

Cynomolgus monkeys (2.5–3 years and 2.5–3.1 kg) were housed and experiments were performed at Envigo CRS, S.A.U (Barcelona, Spain; now part of Covance). Animals received a single bolus injection of LNP-formulated mRNA (0.2 or 1 mg/kg) encoding SO57 in phosphate buffered saline pH 7.4. The total volume for intravenous injections was 1 ml/kg. Blood was collected from the femoral vein and serum was prepared. The study was approved by local authorities and the reference number was WS50NL.

All animal studies complied with the ARRIVE guidelines.

Computational and statistical analysis

Data were analyzed and plotted using GraphPad Prism software version 9.3.1. Serum titers of O-tagged VNAs, recombinant and RNA-encoded EfAb in mice and piglets were generated by EC50 quantification using BioTek Gen5 software. Serum titers of VNAs containing an HA-/E-tag combination in mice as well as serum titers of SO57 antibody in M. fascicularis were calculated by non-linear regression analysis using Graphpad Prism software version 9.3.1. Serum titers were either depicted as whisker plots showing min to max levels or as connected curves with error bars representing the SD of the mean. Graphs showing OD levels [A450] of in vitro expressed VNAs show superimposed symbols (mean with SD) with connecting lines. Data associated with the analysis of ADA are represented as OD levels [A450] of individual animals. Clinical scores of challenged animals are represented as curves with error bars representing the SEM of the mean. In vitro expression data of RNA-encoded EfAb are depicted as floating bars showing min to max values. Correlation analysis of titers and ADA in mice was done using simple linear regression.

The statistical significance of differences between groups when comparing titers was examined using an ordinary one-way ANOVA followed by Šídák’s multiple comparison post test (Graphpad Prism software version 9.3.1). All titer data were log10-transformed for the statistical analysis. Each P value was multiplicity adjusted to account for multiple comparisons. Differences were considered significant at adjusted P values of < 0.05.

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• Source: https://www.nature.com/articles/s41598-023-41092-7