Date Published: February 6, 2020
Publisher: Springer International Publishing
Author(s): Mohsin El Amrani, Rick Admiraal, Lobke Willaert, Lysette J. C. Ebskamp-van Raaij, Amelia M. Lacna, C. Erik Hack, Alwin D. R. Huitema, Stefan Nierkens, Erik M. van Maarseveen.
The addition of rabbit anti-human thymocyte globulin (ATG) to the conditioning regimen prior to allogeneic hematopoietic cell transplantation has significantly reduced the risk of graft-versus-host disease (GvHD) and graft failure. However, ATG has a small therapeutic window. Overexposure of ATG post-HCT hampers T cell immune reconstitution and has been associated with increased relapse rates and viral reactivations, whereas underexposure has been associated with an increased incidence of GvHD, both of which lead to increased mortality. Therapeutic drug monitoring of T cell binding ATG plasma levels provides a means to optimize dosing for patients at high risk for graft failure to ensure timely T cell immune reconstitution and subsequently increase survival chances. This manuscript describes the first liquid chromatography tandem-mass spectrometry (LC-MS/MS) method to quantify the pharmacologically active fraction of polyclonal ATG in plasma. This was achieved through immunoaffinity purification of active ATG from plasma with Jurkat T cells. After the binding and washing, samples were eluted, denatured, and trypsin-digested. Signature peptides originating from the IgG constant chain were measured with LC-MS/MS. Critical method parameters were optimized, and the method was successfully validated following European Medicines Agency (EMA) guidelines. The method covered the therapeutic range of ATG and was validated at a lower limit of quantification (LLOQ) of 1 AU/mL with an overall CV and bias of 11.8% and − 2.5%, respectively. In conclusion, we developed a LC-MS/MS-based method to quantify active polyclonal rabbit ATG in human plasma. We suggest that this novel assay can be used to monitor and optimize dosing of ATG in clinical practice.
Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment strategy for both malignant and non-malignant life-threatening diseases such as leukemia, lymphoma, aplastic anemia, primary immune deficiencies, and inherited metabolic disorders (1). However, graft-versus-host disease (GvHD), with mortalities as high as 50%, poses a serious side effect with incidences ranging between 20 and 70%, depending on histocompatibility mismatches, the age of recipient, and the intensity of preparative regimes (2,3). GvHD is likely caused by the transplanted donor T cells recognizing major and minor histocompatibility complex proteins on the recipient antigen-presenting cells (3). Prophylaxis with polyclonal anti-thymocyte globulin (ATG) which targets different antigens expressed on, e.g., T cells, B cells, natural killer cells, and dendritic cells lead to depletion of these cells from the host blood and peripheral lymphoid tissues (4). Though ATG is used as a rescue therapy for acute rejection in solid organ transplantation, its main application is in hematology to treat and prevent acute and chronic GvHD following HCT in patients with hematologic cancers (4,5). However, the overall survival remains similar between ATG-treated and ATG-untreated patients due to increased risk of relapse and infections in ATG-treated patients (6–9). This outcome is defined by the delicate balance in timing of ATG exposures, where a high ATG exposure pre-HCT is associated with reduced GvHD and graft failures, but high exposures post-HCT are associated with increased relapse rates and reduced survival chances in patients with viral reactivations and GvHD. The presence of lytic levels of ATG post-HCT leads to poor reconstitution of donor T cells which in turn would limit graft versus tumor effect leading to increased risk of a relapse and a reduced control of viruses and regulation of GvH-activity. European Society of Bone Marrow transplantation (EBMT) has historically recommended dosing ATG at a rate of 7.5 mg/kg over 3 days starting from day − 3 pre-HCT in adults. Our group has shown that this dosing leads to overexposure in a majority of patients since dosing is close to graft infusion, and absolute lymphocyte counts (ALS), the most important determinant of ATG clearance at patient’s body weight exceeding 40 kg, is not taken into account (6,10). Furthermore, to allow for improved ATG exposure, an alternative dosing regimen starting at day − 9 and dosing for 4 days based on ALC values and weight has been proposed. However, optimum ATG dosing of high-risk patients such as those with chronic granulomatous disease (CGD) or hemophagocytic lymphohistiocytosis (HLH) remains difficult possibly because of large T cell pools in tissues. In these patients, therapeutic drug monitoring (TDM) of free T cell binding (active) ATG may improve CD4+ immune reconstitution and prevention of graft failure (11).
Here we describe the first LC-MS/MS method to quantify active ATG. LC-MS/MS allows for increased selectivity by directly measuring the signal intensity of LSVPTSEWQR and VVSTLPIAHQDWLR peptides, which originate from the constant chain and are exclusively present in rabbit IgG. The qualifier peptide VVS provided a means to check the results obtained from the quantifier peptide LSV as to ascertain that no isobaric interferences were present. Differences in results between VVS and LSV greater than > 15% are a sign of possible matrix interference and thus warrant further investigation. This method allows samples to be quantified for research purposes and routine therapeutic drug monitoring. Furthermore, due to its linear dynamic range 1–32 AU/mL, it allows quantification to be performed without the need of multiple sample dilutions as in the case with FACS methods. The assay does not depend on fluorescent tagged antibodies for detection and is easy to perform requiring only two washing steps after incubation. The use of 96-well plate format enables high-throughput analysis and the small sample volume required for analysis 10 μL is less invasive for the younger patients.