Anti-thymocyte globulin (ATG) is widely used to prevent graft-versus-host disease (GVHD) after allogeneic haematopoietic stem cell transplantation (alloSCT). ATG depletes donor T cells in vivo, reducing acute and chronic GVHD, but excessive depletion risks impairing graft-versus-tumour effects and donor immune engraftment. There is important inter-patient variability in ATG exposure with standard weight-based dosing, and the optimal exposure range may be context dependent (conditioning intensity, GVHD prophylaxis, ATG schedule). This study evaluated how measured post-transplant ATG exposure (PT-ATG-exp) relates to clinical outcomes, including GVHD and donor chimerism, in a homogeneous cohort receiving reduced intensity conditioning (RIC) and a fixed Thymoglobulin regimen.
This retrospective pharmacokinetic-pharmacodynamic analysis included 68 adult patients undergoing first RIC-alloSCT from 10/10 HLA matched unrelated donors at two Australian centres between August 2018 and February 2024. Conditioning consisted of fludarabine and melphalan, and ATG (Thymoglobulin®) was administered as a total of 4.5 mg/kg across day -3 to -1 (0.5 mg/kg on day -3; 2 mg/kg on days -2 and -1). GVHD prophylaxis included cyclosporin and short-course methotrexate. Standard infection prophylaxis and monitoring for CMV and EBV by PCR twice weekly until day 100 were performed. The study had Human Research Ethics Committee approval from the Royal Melbourne Hospital.
Plasma samples were prospectively collected from all patients at day 0, day 14 and day 30 post-alloSCT. ATG concentrations were measured by flow cytometric methods and reported in arbitrary units per millilitre (AU/mL). The PT-ATG-exp was calculated as area under the curve (AUC) by summing exposure across day 0–14 and day 14–30, expressed as AU per day/mL. The cohort yielded 204 plasma samples in total.
The primary outcome was GVHD-free, relapse-free survival (GRFS), which incorporates freedom from relapse, death, grade III–IV acute GVHD or moderate–severe chronic GVHD. Secondary outcomes included overall survival (OS), acute and chronic GVHD incidence and grade, disease relapse, non-relapse mortality (NRM) and donor-recipient chimerism measured by PCR of short tandem repeat markers on peripheral blood at days 30, 60 and 100. Mixed donor chimerism (MDC) was defined as <95% donor cells in the CD3+ (T cell) or CD3- (myeloid) fractions.
Median PT-ATG-exp for the cohort was 125 AU per day/mL (range 47–420). Median ATG concentrations were 8.4 AU/mL at day 0, 1.1 AU/mL at day 14 and 0.32 AU/mL at day 30. The strongest correlates of PT-ATG-exp were the day 0 ATG concentration (R = 0.925, P < 0.001) and ATG AUC over day 0–14 (R = 0.993, P < 0.001). Graft CD3+ cell dose, recipient body weight and absolute lymphocyte count at first ATG administration did not correlate with PT-ATG-exp in this cohort.
Using proportional hazard modelling of log relative hazard against PT-ATG-exp, the authors identified a range of PT-ATG-exp (80–135 AU per day/mL) associated with inferior 2-year GRFS. Patients within this range had lower estimated 2-year GRFS (25%, 95% CI 11%–56%) versus the remainder of the cohort (54%, 95% CI 40%–73%, P = 0.02; HR 2.01). The inferior GRFS in this PT-ATG-exp band was attributable to a higher incidence of grade III–IV acute GVHD (33% vs 12%, P = 0.034; HR 2.95).
When PT-ATG-exp was examined by quartiles (Q1: 46–94; Q2: 95–124; Q3: 125–184; Q4: 185–420 AU/day/mL), the highest quartile (Q4) had the lowest incidence of grade III–IV acute GVHD (0% at 180 days). There was no significant association between quartiles and estimated 2-year GRFS, overall survival, relapse or NRM in the analyses shown. For chronic GVHD, the reported 2-year incidence was greater in Q4 compared to Q3, but moderate–severe chronic GVHD did not differ significantly. In high-risk CMV recipient groups, CMV reactivation was greater in Q4 compared to Q3.
Donor chimerism data were available for 66 patients at day 30, 60 patients at day 60 and 58 patients at day 100. Median CD3+ donor chimerism declined in Q4 compared to other quartiles: at day 60 Q4 median CD3+ chimerism was 85% versus 98% in Q2 (P = 0.03); at day 100 Q4 median CD3+ chimerism was 85% versus 97–99% in Q1–Q3 (P values 0.005–0.001). Mixed CD3+ donor chimerism at day 100 was most frequent in Q4 (93%). CD3- (myeloid) donor chimerism remained near 100% early but was lower in Q4 at day 100 compared to Q2. These findings indicate that higher PT-ATG-exp protects against severe acute GVHD but is associated with impaired T-cell donor engraftment.
Overall cohort outcomes included a 2-year OS of 70% and a 2-year relapse incidence of 12%. Acute GVHD occurred in 34% of patients with a majority being grade III–IV. CMV reactivation at 6 months occurred in 54% and EBV reactivation in 71%, with EBV requiring therapy in 34% of patients. High PT-ATG-exp was associated with increased CMV reactivation in predefined high-risk recipient groups; other infection associations were not consistently reported.
This analysis demonstrates marked heterogeneity in PT-ATG-exp despite standardized weight-based dosing and identifies day 0 ATG level as the central determinant of exposure. A mid-range PT-ATG-exp (80–135 AU/day/mL) was paradoxically associated with worse GRFS driven by severe acute GVHD, while the highest exposures reduced severe acute GVHD but impaired donor chimerism, notably CD3+ reconstitution. These observations highlight a trade-off between GVHD prevention and immune engraftment linked to ATG exposure and support the need for population- and protocol-specific pharmacokinetic-pharmacodynamic studies to define optimal ATG targets.
Limitations include the retrospective design and a single standardized regimen population; mechanistic explanations for the associations and whether adaptive management based on measured ATG would alter outcomes were not reported in the source. Long-term follow-up beyond median 668 days and detailed management of mixed chimerism were not provided.
Post-transplant ATG exposure varies widely after standardized RIC and Thymoglobulin dosing. Measured PT-ATG-exp correlates strongly with day 0 ATG concentration and predicts both acute GVHD risk and the degree of donor T-cell engraftment. These findings support further investigation of individualized ATG exposure monitoring to balance GVHD prevention with preservation of donor immune reconstitution. The source did not report interventional strategies based on ATG measurements or longer-term outcomes beyond the reported follow-up.