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Therapeutic Drug Monitoring of Anti-Thymocyte Globulin in Allogeneic Stem Cell Transplantation: Proof of Concept




doi: 10.3389/fphar.2022.828094.


eCollection 2022.

Affiliations

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J I Meesters-Ensing et al.


Front Pharmacol.


.

Abstract

Anti-thymocyte globulin (ATG), a polyclonal antibody, is used in allogeneic hematopoietic cell transplantation (HCT) to prevent graft-vs.-host-disease (GvHD) and graft failure (GF). Overexposure to ATG leads to poor early T-cell recovery, which is associated with viral infections and poor survival. Patients with severe inflammation are at high risk for GF and GvHD, and may have active infections warranting swift T-cell recovery. As ATG exposure may be critical in these patients, individualized dosing combined with therapeutic drug monitoring (TDM) may improve outcomes. We describe the individualized dosing approach, an optimal sampling scheme, the assay to measure the active fraction of ATG, and the workflow to perform TDM. Using a previously published population pharmacokinetic (PK) model, we determine the dose to reach optimal exposures associated with low GvHD and rejection, and at the same time promote T-cell recovery. Based on an optimal sampling scheme, peak and trough samples are taken during the first 3 days of once-daily dosing. The fraction of ATG able to bind to T-cells (active ATG) is analyzed using a bio-assay in which Jurkat cells are co-cultured with patient’s plasma and the binding is quantified using flow cytometry. TDM is performed based on these ATG concentrations on the third day of dosing; subsequent doses can be adjusted based on the expected area under the curve. We show that individualized ATG dosing with TDM is feasible. This approach is unique in the setting of antibody treatment and may result in better immune reconstitution post-HCT and subsequently better survival chances.


Keywords:

TDM (therapeutic drug monitoring); anti-thymocyte globulin (ATG); antibody; pediatrics–children; stem cell transplant (SCT).

Conflict of interest statement

RA, JJB, CAL, and SN received an unrestricted research grant from Sanofi. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures



FIGURE 1

Active ATG plasma concentration data. The graph shows aATG concentrations over time in pediatric patients (n = 267) during the whole course of ATG treatment up to 60 days after first dosing. ATG clearance varies extensively between patients. Adapted from Admiraal et al., Lancet Haematology 2017.


FIGURE 2


FIGURE 2

Overview of evaluated dosing regimens. Numbers represent the different dosing scenarios in Table 2.


FIGURE 3


FIGURE 3

Active ATG binding assay. Standard curve, QC, negative control, and 4- and 8-fold diluted patient plasma samples are incubated in a 96-well U-bottom plate with target Jurkat T-cells, to allow for binding of aATG. Detection of bound aATG is done by subsequent incubation with a Gt-α-Rb-Ig-biotin antibody and streptavidin-PE. Cells are then analyzed on a FACS Canto II flow cytometer by acquiring 70 µl sample at a flow rate of 3 μl/s and intermittent mixing, measuring 10.000 events within the live T-cell gate. The median fluorescence intensity (MFI) of PE measured is then used to extrapolate the concentration of aATG (in AU/ml) in the patient sample from the standard curve. FB, FACS buffer (PBS/1%HSA). This figure was created with Biorender.com.


FIGURE 4


FIGURE 4

Active ATG binding assay validation. Graph A and B show results for assay accuracy and precision expressed in %CV. Results are shown for Intra- and inter-assay variability results of (A) standard curve samples and (B) patient samples (data shown as mean +SEM). The LLoQ was set at 0.04 AU/ml (A). Graph (C) demonstrates that high patient sample dilutions (16-fold) show increased deviation above the threshold of 25% and should therefore be excluded from analysis (data shown as mean ± SEM). Dashed lines in graphs (A–C) represent maximal acceptable assay/sample variability expressed in %CV. Graph (D) demonstrates that both plasma and serum samples from the same patient show similar results in the aATG binding assay and can therefore both be used for analysis.

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