The effect of anti-thymocyte globulin (ATG) on the outcome of hematopoietic stem cell transplantation (SCT) is dependent on formulation, dose and exposure. However, ATG levels are not routinely measured and therapeutic levels are not well defined. In ex vivo T cell-deplete SCT, the potential effect of residual ATG has important implications on the timing of adoptive T cell transfer. Here we measured active rabbit ATG concentration using a flow cytometry-based method that can be implemented in any laboratory. Three adult patients received 6 mg/kg Thymoglobulin over 4 days, leading to peak plasma active ATG concentration of 20.8 ± 1.4 µg/mL, suggesting volume of distribution of 16–19 L. The half-life of active ATG was 6.1 ± 0.7 days and plasmapheresis at Day 25 ± 1 post-transplant reduced mean plasma concentration from 1.25 to 0.61 µg/mL. Total ATG and active ATG do not have a constant relationship because of differences in volumes of distribution and half-lives. Thymoglobulin can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro at concentrations as low as 0.03 µg/mL, with a log-linear relationship between ATG concentration and ADCC. Plasmapheresis can remove ATG but likely has modest biological impact when performed 4 weeks after 6 mg/kg ATG.
The use of anti-thymocyte globulin (ATG) for graft-versus-host disease (GVHD) prophylaxis in HLA-matched transplantation is a subject of some debate with therapeutic gains from reduced rates of GVHD being variably offset by increased risks of infection and leukemia relapse [1,2,3,4]. Its role is more defined in ex vivo T cell-deplete haploidentical stem cell transplantation (SCT), where it depletes recipient T cells that may otherwise mediate graft rejection and residual donor T cells in the graft that may mediate GVHD. The use of rabbit ATG at a dose of 6 mg/kg (Thymoglobulin) or 12–25 mg/kg (Fresenius, Grafalon, Neovii) results in high rates of engraftment and low rates of GVHD, although delayed immune reconstitution remains a challenge [5, 6]. Donor T cell add-back can accelerate immune reconstitution but is usually delayed to allow clearance of ATG. The half-life of total ATG is estimated at 30 days in solid organ transplant settings and 10 days in SCT settings [7,8,9]. However, measurement of total ATG by enzyme-linked immunosorbent assay (ELISA) overestimates the amount of biologically active ATG, which is the fraction that can bind human lymphocytes [10, 11]. Active ATG, measured by flow cytometry, has been recently shown to be correlated with clinical outcomes in SCT [10, 12, 13]. The estimated half-life of rabbit ATG (Thymoglobulin) is 6 days and patients who received 6 mg/kg of ATG are expected to take a median of 15 days (range 8–38) to achieve an ATG concentration below 1 μg/mL, the commonly accepted minimum therapeutic level [10, 14]. Plasmapheresis is sometimes used to promote clearance of ATG prior to T cell add-back but the efficacy of this procedure has not been directly measured. In this report, we describe the measurement of active and total ATG concentration in patients undergoing T cell-deplete haploidentical SCT and subsequent plasmapheresis prior to T cell add-back; and the correlation between ATG concentration and in vitro antibody-dependent cell-mediated cytotoxicity (ADCC).
Subjects and methods
The patients were enrolled on a phase I clinical study of T cell-deplete haploidentical SCT followed by add-back of safety-switch modified donor T cells . The conditioning regimen consisted of 8 Gy total body irradiation on Day −9, thiotepa (5 mg/kg/day) on Days −8 and −7, fludarabine (40 mg/m2/day) on Days −7 to −3, and rabbit ATG (Thymoglobulin, Sanofi-Genzyme; 1.5 mg/kg/day) on Days −5 to −2 . CD34-selected (Miltenyi Biotec, Germany) peripheral blood stem cells were infused on Day 0 without post-transplant immunosuppression, followed by add-back of inducible caspase-9 (iCasp9) transduced donor T cells on Day +25 or +26 [15,16,17,18]. Plasmapheresis to remove residual ATG prior to T cell add-back was performed by continuous flow centrifugal-based apheresis. Additional details for the study protocol have been described previously .
Peripheral blood was collected in serum-separation tubes, centrifuged for 10 min at 800 × g, and the serum stored at −80 °C. Peripheral blood mononuclear cells (PBMCs) for flow cytometric assays were obtained by density centrifugation of whole blood from healthy volunteers or buffy coats from the Australian Red Cross Blood Service. The use of healthy volunteers and buffy coats was approved by the QIMR Berghofer Human Research Ethics Committee (Project number P1443).
Preparation of ATG standards
Thymoglobulin ATG was reconstituted in phosphate-buffered saline (PBS) to 5 mg/mL and stored in aliquots at −80 °C. Standards for both flow cytometry-based active ATG and ELISA-based total ATG were prepared fresh by serial dilution of stock ATG in pooled human AB serum (Valley Biomedical, VA).
Measurement of active ATG
Active ATG was quantified by flow cytometry based on previously published method , with modifications. In brief, 50,000 PBMCs from healthy donors were incubated with 50 μL of 1% human IgG (Intragam P, CSL, Australia) for 30 min at 4 °C to minimize non-specific binding. The sample was centrifuged without wash and the supernatant removed. The cells were then incubated for 30 min at 4 °C with 50 μL of serum sample or ATG standard, both diluted 1:5 in PBS with 1% bovine serum albumin (BSA). The cells were washed twice in PBS with 1% BSA; stained with Alexa Fluor 647 (AF647) goat anti-rabbit IgG (H+L) (Life Technologies, Catalog #: A-21245) at 1:200 dilution, CD3 PE/Cy7 (clone UCHT1, Biolegend), and CD45 V500 (HI30, BD Pharmingen) for 30 min; washed; and analyzed immediately by flow cytometry (LSR Fortessa, BD Biosciences). Data were analyzed by FlowJo v9 (Ashland, OR) and the mean fluorescent intensity (MFI) of AF647 on CD3+ T cells were plotted against the concentration of ATG standards. The mass of active ATG cannot be readily measured, and for the purpose of this study, 1 mg of active ATG refers to the biologically active component of ATG that is present within 1 mg of freshly reconstituted ATG, which is used as the reference .
Measurement of total ATG
Total ATG was quantified by ELISA as previously described , with modifications. In brief, 96-well microplates were coated with 10 μg/mL goat-anti-rabbit IgG (H+L) (Jackson Immunoresearch) for 4 h at 37 °C, washed, and then incubated with ATG standards or serum samples for 2 h at room temperature. The plates were washed and incubated with horseradish peroxidase-conjugated mouse-anti-rabbit IgG (H+L) (Jackson Immunoresearch) for 1 h at room temperature. The plates were washed and incubated with 1-StepTM Ultra TMB-ELISA (Thermo Scientific) and read on BioTek microplate reader (BioTek Instruments) per the manufacturer’s protocol. Data were collected and analyzed by the Gen5TM software (BioTek Instruments). Purified rabbit IgG (Jackson Immunoresearch) was used as calibrators. Serum samples were diluted where indicated to ensure that their optical density fell within the range on the standard curve with the highest sensitivity.
To describe ATG concentrations, a one-compartment model was developed with the Nonparametric Adaptive Grid (NPAG) algorithm within the freely available Pmetrics package for R (Los Angeles, CA). Clearance was modeled as a first-order process. Discrimination between different models used comparison of the −2 log likelihood (−2LL) and assessment of goodness-of-fit models. The final model best described the data when volume of distribution (Vd) was normalized to a body weight of 70 kg. Clearance and Vd were then used to calculate drug half-life (t1/2) using the equation: t1/2 = 0.693 × Vd/CL. The amount of drug cleared by plasmapheresis was calculated using the equation: Amt (mg) = Cpre × Vd − Cpost × Vd, where Cpre and Cpost are concentrations immediately before and after plasmapheresis, respectively. The percentage of drug clearance was calculated following the equation: Percentage cleared = (Cpre − Cpost)/Cpre × 100%.
Natural killer (NK) cells were isolated from fresh PBMCs by immunomagnetic depletion of non-NK cells per the manufacturer’s instructions (NK Cell Isolation Kit, Miltenyi Biotec, Germany). The resulting NK cell fraction (purity >93%) and non-NK cell fraction (NK cells <1%, T cells >95% in CD19–CD14– compartment) were used as effector and target cells, respectively, in carboxyfluorescein succinimidyl ester (CFSE)-based ADCC and complement-dependent cytotoxicity assay . In brief, CFSE-labeled target cells were incubated with ATG at 20 µg/mL (peak active ATG concentration in vivo) and at doubling dilutions from 2.0 to 0.008 µg/mL for 1 h at room temperature, washed twice, and then incubated overnight at 37 °C 5% CO2 with NK cells for ADCC or fresh autologous plasma (50% v/v) for complement-dependent cytotoxicity, both supplemented with 50 U/mL interleukin-2. Cells were stained with the Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies), CD19 PerCP/Cy5.5 (HIB19, Biolegend), CD14 Pacific Blue (HCD14, Biolegend), CD56 PE (HCD56, Biolegend), CD16 Alexa Fluor 700 (3G8, Biolegend), CD3 PE/Cy7, CD45RA APC/Cy7 (HI100, Biolegend), and CD62L APC (DREG-56, Biolegend) and analyzed immediately by flow cytometry (LSR Fortessa, BD Biosciences).
Data are presented as mean ± standard deviation. Ordinary least-squares fit methods were used for curve fitting (Prism 7, GraphPad).
Serum ATG assays
The measurement of active ATG by flow cytometry is highly sensitive and has a large dynamic range. We used sequential gating to obtain the MFI of anti-rabbit IgG-AF647 within the T cell population (Fig. 1a, b). Log(MFI) and Log(ATG concentration) are linearly correlated (R2 > 0.99) across a 4-log dynamic range from 0.008 to 100 μg/mL (Fig. 1c). In line with previous publications, the measurement of total ATG by ELISA was 2 logs less sensitive, with a limit of detection of 1 μg/mL and a 2-log dynamic range (Fig. 1d) [19, 21].
Pharmacokinetics of ATG
We measured active and total ATG concentrations in 3 adult patients who each received a total of 6 mg/kg Thymoglobulin between Days −6 and −2 of transplant conditioning (Fig. 2a). The patient characteristics and ATG pharmacokinetics are presented in Table 1. Active ATG has a larger volume of distribution (Vd) than total ATG: mean 17.1 L versus 5.4 L, which corresponds to lower peak concentration of active ATG compared to total ATG: mean 20.8 µg/mL versus 85.9 µg/mL. The half-life (t1/2) of active ATG was approximately half that of total ATG (mean 6.1 versus 11.5 days), which were in line with previous reports [8, 10, 12]. As a consequence of the different half-lives, the ratio of active-to-total ATG concentration was not constant but decreased with time (Fig. 2b), resulting in a log-linear relationship between total and active ATG concentration (Fig. 2c).
All 3 patients underwent plasmapheresis on Day +25 or +26 post-transplant in preparation for the adoptive transfer of iCasp9 gene-modified T cells. The effect of plasmapheresis on active and total ATG concentration is shown in Fig. 2d. Plasmapheresis at 4 weeks after completion of ATG administration reduced the concentration of active and total ATG by 50.6 ± 6.4% and 66.8 ± 2.9%, respectively (Fig. 2e). Applying the formula: Amt removed (mg) = Cpre × Vd − Cpost × Vd, this was estimated to represent the removal of 10.9 ± 3.2 and 84.7 ± 14.8 mg of active and total ATG, respectively. Of note, there was no significant rebound of serum active ATG concentration. Clinically, rapid and sustained engraftment of gene-modified T cells was observed in all three patients .
Correlation between ATG concentration and in vitro cytotoxicity
The generally accepted therapeutic level for rabbit ATG is 1 μg/mL [10, 12, 13], which is based on in vitro studies performed on a specific batch of polyclonal rabbit ATG nearly two decades ago . It is unlikely that the ATG at that time was bioequivalent to current commercial formulations, which themselves are neither bioequivalent nor interchangeable [2, 11, 22]. In order to understand the biological effect of plasmapheresis, we performed in vitro cytotoxicity assays at clinically relevant concentrations of Thymoglobulin ATG. There was no detectable in vitro complement-mediated killing at ATG concentrations up to 20 µg/mL (data not shown). In contrast, Thymoglobulin ATG-mediated T cell killing via ADCC pathway was detectable at concentrations as low as 0.03 µg/mL (Fig. 3a, b), with preferential loss of CD45RA(–)CD62L(+) central memory T cell subset (Fig. 3c). The percentage killing correlated with log[ATG] from 0.03 µg/mL through to 0.5 µg/mL. Higher concentrations of ATG were associated with a paradoxical reduction in in vitro ADCC, likely as a result of Fc saturation of FCγRIII on NK cells , as evidenced by a reduction in CD16 (FCγRIII) antibody staining on NK cells (Fig. 3di, ii). Despite ATG binding, only limited bystander killing of NK cells was observed in vitro (Fig. 3diii). Indeed all 3 patients demonstrated rapid NK cell reconstitution by Day 21 of transplantation (Fig. 3e).
ATG is widely used as part of the conditioning regimen to improve engraftment and reduce the incidence of GVHD. In T cell-replete SCT, active ATG exposure has been associated with transplant outcome: higher exposure was associated with increased relapse rate and lower exposure was associated with increased non-relapse mortality . In ex vivo T cell-deplete SCT, an additional consideration is the effect of residual ATG on T cell add-back strategies, which are increasingly used to help accelerate the reconstitution of pathogen- and leukemia-specific T cells. Residual ATG can adversely impact the engraftment and persistence of adoptively transferred T cells but unnecessary delay in T cell transfer is also undesirable given the narrow window of therapeutic opportunity. Plasmapheresis is used by some groups to promote ATG clearance prior to T cell add-back but the clinical efficacy of this procedure has not been studied.
Thymoglobulin ATG consists of purified IgG from the serum of immunized rabbits. The amount of ATG stated on the vial refers to total rabbit IgG and is the amount measured by ELISA. Indeed, the ELISA standard curve for Thymoglobulin was tightly correlated with that of purified rabbit IgG calibrators (data not shown). Flow cytometry measures the amount of biologically active ATG that can bind human T cells. However, the absolute mass of bound ATG cannot be readily determined, and active ATG is expressed either as an arbitrary unit [12, 13] or relative to the amount present in freshly reconstituted total ATG . For clarity, we have adopted the latter approach, where 1 mg of active ATG refers to the amount of biologically active ATG present in 1 mg of freshly reconstituted Thymoglobulin ATG. Consistent with previous reports [10, 19], active ATG was cleared more rapidly than total ATG, such that the active form constitutes a progressively lower proportion of total ATG with time. We show that plasmapheresis can indeed remove circulating active ATG but it likely has a relatively modest impact on ADCC because of the log-linear relationship between ATG concentration and ADCC. We show that freshly reconstituted Thymoglobulin ATG can mediate ADCC in vitro at concentrations as low as 0.03 µg/mL, which is far lower than the previously reported therapeutic level of 1 µg/mL. However, the correlation between in vitro and in vivo ADCC requires further investigation as all 3 patients in our study had successful engraftment of adoptively transferred T cells despite having plasma concentrations of active ATG >0.03 µg/mL at the time of T cell infusion. Additional studies are required to define the impact of different ATG cut-off on the engraftment, survival, and expansion of adoptively transferred T cells. The flow cytometry-based assay for measuring active ATG described here is readily implementable in most laboratories and should facilitate further studies in this area.
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This study was supported by a Project Grant (APP1053135) from the National Health and Medical Research Council (NH&MRC, Australia), QIMR Berghofer Ride To Conquer Cancer Flagship Award and Royal Brisbane, and Women’s Hospital Foundation. S-KT was supported by an NH&MRC Early Career Fellowship (APP1054786) and GRH was supported by a QLD Health Senior Clinical Research Fellowship.
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The authors declare that they have no conflict of interest.
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Zhang, P., Curley, C.I., Mudie, K. et al. Effect of plasmapheresis on ATG (Thymoglobulin) clearance prior to adoptive T cell transfer. Bone Marrow Transplant 54, 2110–2116 (2019). https://doi.org/10.1038/s41409-019-0505-5
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