Case Study 1.

In vitro immunogenicity assessment of new biologics (cytokine induction assay). Published in Cancer Research, 2019, 79(6):1239- 1251.

 

The nanobioconjugate (a potent anticancer drug candidate), along with the drug vehicle (PMLA), positive (PHA-M) and negative (PBS) controls, was tested for immunogenicity (cytokine release). Nanodrug was added in vitro to the whole blood samples from three healthy donors at therapeutic 1X dosage (based on the expected Cmax in human at the human equivalent to the mouse efficacy dose), or 3X dosage. Diluted blood was incubated with controls or nanomaterial for 24 hours. Using quantitative Meso Scale Discovery (MSD) assay, nine cytokines (TNFa, IFNgamma, IL12p70, IL1b, IL2, IL4, IL6, IL8, and IL10) in the donors' blood were simultaneously measured.

 

Results

A therapeutic 1X concentration and even 3X elevated concentration of the nanodrug did not significantly induce any of the nine cytokines in the donors' blood except for IL8 in the 3X elevated dose group. However, this IL8 level was still 40-fold lower than in the positive control group. These data showed that there is potentially low risk of the nanodrug containing antibodies and oligos to induce severe immunogenic response (“cytokine storm”). Other immunogenic in vitro tests (e.g., T4 lymphocyte immunogenic response in PBMC) are also available for testing of the new therapeutic proteins and other biologics.

Case Study 2.

Development and Evaluation (Quantitation) of the Anti-Drug Antibodies (ADA) Assay in the Dog Plasma.

 

The purpose of this study was to develop and qualify a method for detection of anti-drug antibodies (ADA) against therapeutic peptide with nanocarrier in dog plasma using a Meso Scale Diagnostics (MSD) Electrochemiluminescence Immunoassay. The qualification plan was approved by the Sponsor. MSD utilizes electrochemiluminescence detection to detect binding events on patterned arrays by converting electric energy into light energy. Electrochemiluminescence detection offers a unique combination of sensitivity, dynamic range and convenience.

The MSD assay measures ADA against the therapeutic nanomaterial in dog plasma in an MSD 96-well-standard plate. In brief, the assay employs a sandwich immunoassay format where wells were coated with the drug on the standard MSD plate containing high-binding electrodes. Samples in serial dilutions and a solution containing the labeled secondary detection antibody are added to the plate. The ADA in the sample binds to the drugs immobilized on the working electrode surface; recruitment of the labeled detection antibody to the ADA completes the sandwich. Then an MSD read buffer is added to the plate that provides the appropriate chemical environment for electrochemiluminescence and the plate is loaded into an MSD SECTOR instrument for the analysis, see Figure 1.

Figure 1. MSD detection using electrochemiluminescence (antibody capture is shown, in this assay drug capture was used). (Figure from MSD clinical immunology applications brochure, https://www.mesoscale.com/~/media/files/brochures/immunologybrochure.pdf).

The ADA from dog plasma will be detected using the immobilized Test Article (drug) for capture with anti-dog secondary antibody linked with sulfo-TAG streptavidin for development of the assay. The system must be qualified for different species (i.e. rat, dog, human). In order to qualify the ADA assay, we designed an assay utilizing ADA raised in rabbits immunized with the drug (the only available specific antibody to the drug), see Figure 2.

Figure 2. MSD detection using coated drug for capture, shown with rabbit polyclonal anti-drug Antibody and anti-rabbit secondary antibody, used for development and qualification of the assay.

Results

The method was qualified for accurately measuring induction of Ab to the test article in dog plasma. The screen cut-off point was determined for each dilution of plasma. The data showed good precision (%CV) and good accuracy (%RE) for both intra- and inter-assay calculations confirming acceptable selectivity of the assay.

The range of quantitation during sample analysis was 0.49 ng/L – 500 ng/mL. The limit of detection was 0.456 ng/mL. The lowest limit of qualification (LLOQ) was 0.98 ng/mL. The minimum required dilution (MRD) of samples was 1:50 (2% plasma) for this assay. This allow to use as low as 2.5 uL of plasma for ADA analysis. Using of this method confirmed that there were no ADA induced in dogs due to the drug administration.

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