(W1430-02-10) Analysis of Anti-Drug Antibodies (ADA) in Multiple Animal Species from mRNA LNP Therapeutics Supporting Rare Diseases Demonstrated Low Immunogenicity of PEG-2000 by ECLA and ELISA Methodology

Purpose: mRNA lipid nanoparticles (LNPs) are being considered as a promising modality for the chronic treatment of rare diseases. Safety assessment of such compounds must constitute monitoring and characterization of immunogenicity, including the humoral response to PEG, a component of the LNP. We developed and qualified an electrochemiluminescence assay (ECLA), and a more sensitive enzyme-linked immunosorbent assay (ELISA) method, to measure anti-PEG-2000 antibodies in the serum or plasma of multiple animal species after repeat administration of an mRNA LNP drug.

Methods: The ECLA is an indirect immunogenicity assay where ADA, “total” anti-PEG antibodies (IgG and IgM antibodies), were detected using biotin-PEG-2000 directly immobilized to a streptavidin Mesoscale Discovery (SA-MSD) 96-well plate. Serum or plasma study samples of rat, dog, or monkey were collected. The corresponding rat or humanized mouse anti-PEG IgG and IgM surrogate positive controls (SPCs), negative controls (NCs) or cut point controls (CCs) were prepared in neat, pooled matrix. However, pre-assay sample treatment was required to reduce high assay background due to matrix interference. Neutralized samples after treatment with trifluoroacetic acid were incubated overnight with streptavidin magnetic beads coated with biotin-PEG-2000 following a solid phase extraction acid-dissociation (SPEAD) procedure. After acetic acid extraction from beads, samples were neutralized, diluted in assay buffer, 5-fold, for optimal detection by the anti-PEG-2000 ECLA. Anti-PEG antibodies (i.e. dog ADA and rat SPC) bound to PEG were detected with respective species-specific primary antibodies followed by a ruthenylated secondary detection antibody whose ECL signal was detected by the MSD plate reader (Figure 1A). The presence of ADA was determined by comparing the normalized signal in the sample or control to a statistically derived assay cut point. The ELISA is a direct immunogenicity assay where total ADA are detected using biotin-PEG-2000 directly immobilized on a clear streptavidin coated (SA) 96-well plate. Serum samples of mouse, rat, or dog were collected. The corresponding controls were prepared in neat, pooled serum. The study samples and controls were diluted 50-fold in assay buffer for optimal detection by the anti-PEG-2000 ELISA. The anti-PEG antibodies bound to the SA plate were detected with goat anti-mouse, rat or dog IgG/IgM conjugated to horseradish peroxidase (detection reagent), followed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The absorbance signal generated was read by a SpectraMax M3 plate reader at 450 nm (Figure 1B and C). The presence of ADA was determined by comparing the normalized signal in the sample or control to a statistically derived assay cut point. In all cases, titer values were determined by serial dilution of confirmed positive samples in diluted matrix with its corresponding titer cut point following the three-tiered immunogenicity testing approach.

Results: Comparison of assay performance and anti-PEG-2000 titer study data from all the species revealed similarities and consistencies supporting our conclusion. Performance of the assays met industry and guidance recommendations for GLP and non-GLP studies: precision, sensitivity, specificity, MRD, matrix interference, drug tolerance, selectivity, titration linearity and stability. Precision of high and low SPCs and NCs ranged from 0.0% to 23.5% and 2.5% to 17.2% by ECLA or ELISA, respectively. Screening and confirmatory sensitivity were reported: 102 ng/mL, 69.8 ng/mL and 23.8 ng/mL, 34.2 ng/mL by ECLA or ELISA, respectively (Figure 2). Results are the representation of anti-PEG titers across multiple studies, species, and various study designs. Anti-PEG-2000 titer values of two rat, and two monkey studies ranged from 1:5 to >1:405 and < 1:15 to 1:1215 by ECLA, respectively. Anti-PEG-2000 titer values of two mouse and two rat studies ranged from < 1:50 to 1:3200 and < 1:50 to 1:200 by ELISA, respectively. Results of three dog studies (two with disease-model) by ECLA versus one study with wild-type dogs by ELISA exhibited titer ranges of < 1:5 to 1:193 and < 1:50 to 1:25600, respectively (Figure 3).

Conclusion: A faster assay run, reduced sample volume and improved screening sensitivity are distinct advantages that the direct ELISA has over the SPEAD indirect ECLA method. The assay performance between the methods was similar and met guidance and industry best practices. The majority of titers whether determined by ECLA or ELISA in mouse, rat, disease-model dogs or monkey remained consistent within a range of < 1:5 to 1:3200. The wild-type dogs were exposed longer to certain test compounds and showed higher titer by ELISA. Our reported anti-PEG titer data suggest that regardless of study designs or methods, the immunogenicity of PEG-2000 incorporated in LNPs is low.

Figure 1. Example of ECLA and ELISA methods. A) ECLA format with SPEAD bead sample preparation for multi-species (rat and dog). B) ELISA format for rat species. C) ELISA format for dog species.


Figure 2. Example of assay performance of the indirect ECLA and direct ELISA methods in rat serum.

Figure 3: Summary of titer results from non-clinical studies across multiple species. Anti-PEG-2000 titer results from non-clinical studies across multiple species, all subjects were dosed biweekly with exceptions.
* Dog species overall had a broader range of titer compared to mouse, rat and monkey, with one dog in particular