Pharmacokinetics (PK) Study Of Antithymocyte Globulins Fresenius (ATG-F) Prior To Allogeneic Stem Cell Transplantation: Implications For Timing Of Graft and Early Adoptive Immunotherapy Infusions
Authors: Mastaglio Sara , Forcina Alessandra , Seitz Isabell , Martinius Holger , Peccatori Jacopo , Bernardi Massimo , Clerici Daniela , Corti Consuelo , Crotta Alessandro , Stanghellini Maria Teresa Lupo , Messina Carlo , Bonini Chiara , Ciceri Fabio 
Published: October 2019

Background
Antithymocyte globulins Fresenius (ATG-F) are purified, concentrated preparations of polyclonal immunoglobulin G from hyperimmune serum of rabbits immunized with human thymus activated lymphocytes. These immunoglobulins induce immunosuppression through T-cell depletion and immune modulation. The polyclonal nature of ATG-F is responsible for its effects on the immune system: T-cell depletion in blood and peripheral lymphoid tissues through complement-dependent lysis and T-cell activation and apoptosis; modulation of molecules involved in leukocyte-endothelium interactions; induction of apoptosis in B-cell lineages; interference with dendritic cells. ATG-F administered before allogeneic hematopoietic stem cell transplantation (allo-HSCT) reduces the risks of graft rejection and graft-versus-host disease (GvHD), but the slow clearance of the xenoserum might delay immune reconstitution, increase the risk of disease relapse and impair the activity of a donor lymphocyte infusion (DLI) performed early after allo-HSCT.

Methods
We studied 24 patients with hematologic malignancies, who underwent allo-HSCT from familiar or unrelated donors, after a conditioning regimen based on myeloablative treosulfan and fludarabine. As graft rejection and GvHD prophylaxis, 17 patients received ATG Fresenius at the dose of 10 mg/kg over 16 hours at day -4,-3 and -2 before HSCT; 5 patients received in vivo T-cell depletion at the dose of 20 mg/kg at day -4,-3 and -2 before HSCT; 2 more patients received ATG Fresenius at the dose of 10 mg/kg much earlier before allo-HSCT (day -14, -13 and -12) since the treatment protocol included a donor DLI at day +3 after transplantation. We collected serum samples at different timepoints, from the first ATG dose to at least 3 weeks after HSCT. We used a flow cytometry-based assay to detect the concentration of free T-cell specific rabbit IgG (SRIgG) which corresponds to the serum biological activity against human T-lymphocytes, as opposed to the levels of unspecific rabbit IgG (RIgG) by ELISA, which lack anti-T-cell function.

Results
In our cohorts of patients we observed a concentration peak at 64 hours after the first ATG administration, corresponding to the end of the last immunoglobulin dose administration. Interestingly, patients who received the 20 mg/kg dose of serum reached four times higher levels of SRIgG, suggesting a non-linear correlation between the administered dose and the measured plasmatic peak concentrations. Moreover, the terminal elimination half life of SRIgG is significantly shorter than the one of RIgG: 14 vs 67 days (data not shown), indicating that, for the dose of 10 mg/kg, 10 days after HSCT, SRIgG titre has already reached sub-therapeutic levels.

Conclusions
Previous results indicated that in vivo specific activity of rabbit ATG Fresenius disappears from circulation around day +7 after their last administration (at the dose of 10 mg/kg, day -4, -3, 2). Based on our current data, we can suggest that residual specific anti-lymphocyte activity is dependent upon i. timing and ii. dosage of ATG administration, although this correlation might not be linear. According to these results, current policies of a fixed schedule for DLI infusion should be revisited and possibly adapted to each patient, based on specific conditioning protocols.

Disclosures:
Seitz: Fresenius Biotech GmbH: Employment. Martinius:Fresenius Biotech GmbH: Employment.