(T0930-06-33) Troubleshooting the Lack of Neutralizing Antibody Activity during Development of an Enzyme-Based nAb Assay

Purpose: To support clinical development of an mRNA-LNP therapeutic where the mRNA-translated protein is a functional enzyme, a neutralizing antibody (nAb) assay was developed to monitor neutralizing activity of any anti-enzyme antibodies in human serum. The method measures substrate conversion to product through a fluorescent endpoint assay. In the presence of nAbs, substrate conversion is blocked and a decrease in signal is observed. Due to large amounts of endogenous substrate and product within human sera, the SPEAD method was used to isolate anti-enzyme antibodies. Commercial and in-house produced enzyme-specific antibodies were screened for inhibition of substrate depletion. Three antibody candidates demonstrated some neutralization activity, but the method was not sensitive enough to measure clinically relevant levels of nAb. Numerous optimizations were attempted, including adjustments to biotin concentration, conjugation ratios, acid types, and different volumes of acid and base. However, nAb activity showed mixed results and high variability, suggesting acid dissociation might be incompatible with the enzyme assay. An orthogonal indirect Electrochemiluminescent (ECL) assay was developed to investigate issues with acid dissociation and to more quickly evaluate variables involved with the acid dissociation method. This method allowed for rapid interrogation of antibody performance under various acid dissociation conditions while consuming significantly less antibody than the nAb assay counterpart.

Methods: Neutralizing Antibody Assay: Solid Phase Extraction and Acid Dissociation (SPEAD) Treatment: The positive control is co-incubated with biotinylated enzyme, and subsequently added to a streptavidin-coated plate. Acid was added to release the nAb from the streptavidin-coated plate and neutralized with base in a non-binding plate. nAb Assay: The SPEAD treated samples were incubated with enzyme, substrate, and co-factors. Samples were then analyzed in a fluorescent endpoint assay to determine the amount of substrate depletion. Orthogonal Assay to Investigate nAb Assay: Direct Ligand-Binding Assay: Streptavadin-coated ELISA plates were coated with biotinylated enzyme. After a 1-hr incubation followed by a wash step, nAb candidates were added to the plate and allowed to bind captured enzyme. After a wash step to remove unbound antibody, wells were treated with acid and acidified material was neutralized in a dilution plate containing base. Neutralized samples were then transferred to an MSD plates that were coated with biotin-enzyme. Non-acidified and acidified-neutralized antibodies were also added directly to the MSD plate as controls. Samples and controls were incubated for 1 hour followed by a wash step. Ruthenylated detection antibody was then added to all wells and the plate was read using an MSD plate reader. All results were normalized to non-acid treated controls which consisted of measuring direct binding of antibody to MSD-plate-bound enzyme without acidification. Acetic acid concentrations ranging from 20 mM to 70 mM were evaluated in this format.

Results: Positive controls screened in the neutralizing antibody assay, after pre-treatment using the SPEAD method, did not show significant inhibition of enzyme activity. Using the direct ligand binding assay the impact of acid treatment and neutralization of positive control was evaluated. When samples were acid treated and neutralized in the presence of biotinylated enzyme, a significant decrease in binding of the positive control was observed. However, when positive controls samples were acid treated and neutralized prior to addition of biotinylated enzyme, no decrease in binding was observed. These results indicate that the enzyme is irreversibly damaged during acid-treatment.

Conclusion: A SPEAD-based neutralizing antibody assay was in development but had issues due to poor neutralizing antibody positive control performance. An orthogonal assay was developed to evaluate issues with the acid dissociation process. We observed that acidification and neutralization had no impact on antibody performance, but that the enzyme was irreversibly damaged during acid treatment. For these reasons alternative methods for depletion of endogenous enzyme substrate and product were pursued.

Acknowledgements: All co-authors work for Moderna, Inc. and hold shares with the company.