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Abstract BACKGROUND:
Risankizumab (RISA) is a humanized IgG1 kappa therapeutic monoclonal antibody (tmAb) that targets interleukin 23 (IL23). RISA is used in the treatment of moderate to severe plaque psoriasis, psoriatic arthritis, Crohn’s disease, and ulcerative colitis. Therapeutic drug monitoring (TDM) can be used to measure RISA serum concentrations, helping to identify thresholds associated with improved outcomes, determine target concentrations for patients or point to a loss of response. While loss of response is characterized by a return of symptoms, loss of response may be linked to either suboptimal RISA pharmacokinetics or the presence of anti-drug antibodies (ADA). Our goal is to show whether the presence of ADA in patient serum would interfere with RISA quantitation.
METHODS:
Anti-Risankizumab clones TZA058 and TZA059 (Bio-Rad; Hercules, CA), were spiked independently at concentrations of 39, 78, 156, 312, 625, and 1250 ng/mL into two pooled residual waste serum samples that were spiked with 5 and 75 µg/mL RISA. The ADA spiked samples were incubated overnight at 8°C. These ADA spikes were then run on the validated RISA quantitation assay and compared to the 5 and 75 µg/mL RISA spikes that were not spiked with any RISA ADA. Briefly, the RISA quantitation assay is a laboratory developed test employing immunopurification with magnetic beads coupled with IL23, followed by liquid chromatography and high-resolution mass spectrometry. Samples were prepared by adding IL23 coupled bead slurry, PBS, unknown/standard/QC, and stable isotopically labeled (SIL)-RISA (Promise Proteomics; Grenoble, France) to a 96-well filter plate (Sigma-Aldrich; St. Louis, MO). Samples were mixed at room temperature, then washed twice with PBS and twice with HPLC water. Samples were then eluted into a PCR plate using 5% acetic acid and reduced with 100 mM DTT in 1M ammonium bicarbonate. The enriched and reduced samples were analyzed using an Agilent 1290 Infinity II LC connected to an AB Sciex 7600 ZenoTOF mass spectrometer. A volume of 20 µL was injected onto the analytical column with separation being performed over a 4-minute gradient. The +11, +12, and +13 charge states were combined to give the XIC used for quantitation for both RISA and SIL-RISA.
RESULTS:
The ADA spikes made with clone TZA058 had an average percent difference of -13.4% (-6.2% to -29.9%) for the 5 µg/mL RISA pool and an average percent difference of -11.1% (-5.8% to -15.1%) for the 75 µg/mL RISA pool. The ADA spikes made from clone TZA059 had an average percent difference of -12.2% (-2.0 to -35.3%) for the 5 µg/mL RISA pool and an average percent difference of -12.6% (-6.4% to -16.9%) for the 75 µg/mL RISA pool. All ADA spikes quantitated within 20% difference from the spiked RISA pools, with exception to the 1250 ng/mL ADA in 5 µg/mL RISA for both the TZA058 and TZA059 clones, which quantitated at -29.9% and -35.3% difference respectively. Although the 1250 ng/mL ADA spikes saw a larger percentage difference from the 5 µg/mL RISA without ADA, the absolute difference was only 1.3 µg/mL for clone TZA058 and 1.5 µg/mL for clone TZA059, which is not a clinically significant drop in RISA concentration.
CONCLUSION:
We have shown that the presence of RISA ADA at high concentrations (1250 ng/mL) could potentially interfere with the drug quantitation assay by suppressing the RISA drug quantitation measurement specifically at low levels of RISA. In the example we’ve shown, this low bias may not be clinically significant. Although the development of RISA ADAs is considered rare (<1% of all patients), when RISA is below 10 µg/mL, it would be best practice to evaluate the presence of ADAs.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
