Use of Ex Vivo Graft Manipulation and Posttransplant Cyclophosphamide Result in Low GvHD Rates and Acceptable Engraftment after RIC Regimens in Pediatric Mismatched SCT
Authors: Lang Peter , Döring Michaela , Lang Anne-Marie , Schlegel Patrick , Seitz Christian M. , Fischer Robin , Holzer Ursula , Brecht Ines , Handgretinger Rupert 
Published: November 2019

Introduction:
There are currently two strategies to prevent Graft-versus-Host Diseases (GvHD) mainly applied in haploidentical transplantation. One is ex-vivo T-cell depletion of TcRa/b T-cells and the other is the T-replete approach, in which the donor T-cells remain in the graft and are tolerized in vivo by post-transplant cyclophosphamide (pCy). The ex-vivo depletion strategy does not require post-transplant immune suppression for GvHD prevention, whereas T-replete transplants require intensive immune suppression. A major obstacle for engraftment is the persistence of patients' T-cells despite intensive and myeloablative condition regimens, thus probably leading to rejection of the graft. We hypothesized that both methods could be combined in a setting of Reduced Conditioning setting (RIC). The ex-vivo T-cell depletion would allow to omit post-transplant immunosuppression and the pCy given at day +3 and +4 could induce in-vivo tolerance of the residual patients' T-cells not eliminated by RIC. Therefore, we applied this strategy in patients who were not eligible based on their poor clinical condition and who were considered to endure only a very reduced conditioning regimen.
Results:
We report on a cohort of 6 pediatric patients who were not eligible for myeloablative condition regimens due to preexisting organ dysfunctions (lungs, gut or liver) but were in urgent need of an SCT from matched unrelated (n=2) or haploidentical family donors (n=4). Diagnoses were: immune deficiencies (n =4; CARMIL 2, STAT 1, ICF 2, 1 not classified), relapsed metastatic ependymoma, refractory Burkitt´s lymphoma. All patients received a non-myeloablative conditioning regimen (ATG (Thymoglobin) 2mg/kg d-9 to d-7, fludarabine 30mg/m² d-6 to d-2, TBI 4Gy d-1, cyclophosphamide 50mg/kg d+3, d+4; adapted from Aversa, Reisner et al. Blood Adv. 2017). One patient additionally received thiotepa 2x5mg/kg on d-2. The CliniMACS® device was used for TCRab/CD19 depletion of peripheral stem cells; a median number of 14x10E6 CD34+ cells/kg bw with 6.4x10E3/kg bw residual TCRa/b T-cells was infused without any further posttransplant immune suppression. Four patients received a single add back of CD45 RA depleted donor T-cells at d+7. Dosages of 1x10E5/kg, 1x10E6/kg or 5x10E6/kg were administered. Two patients received an additional T-cell depleted stem cell boost after application of pCy Engraftment occurred in 4/6 patients; 2 patients rejected their haploidentical grafts and showed complete autologous reconstitution. Median time to reach ANC>500 was 19 days (range 15-23). Four patients had no signs of GvHD; 1 patient had grade I; the patient who had received the highest dose of CD45RA depleted DLI developed grade III but could be treated successfully. No cGvHD occurred. Immune recovery was rapid. Median numbers of CD3+ T-cells, CD3/CD4+ T-cells, CD19+ B cells and CD56+ NK cells at d30 and d100 were 120/µl, 9/µl, 0/µl, 140/µl and 205/µl, 60/µl, 67/µl and 206/µl, respectively.
3 patients are alive and well with a median follow up of 824 days (43-1100). Last observed donor chimerisms were 95-100%. Causes of death in 3 other patients were: MAS/sepsis (STAT 1 deficiency, d 264) and progression in both patients with malignancies (d282 and d73). The patient with relapsed ependymoma showed a transient tumor regression for 3 months posttransplant whereas the patient with refractory Burkitt´s lymphoma had only a short response for 4 weeks.
Conclusions:
The combination of TCRa/b depletion and pCy allowed to use a very reduced conditioning regimen which could be administered in pediatric patients even with preexisting significant organ dysfunctions without severe side effects. GvHD could be effectively prevented (except in one patient who received a high number of DLI) together with an acceptable engraftment rate provided by post cy. Thus, this method might offer the possibility to establish a donor-derived hematopoiesis without using pharmacological myeloablation and with minimal toxicity and might be the basis for future strategies to further reduce the conditioning regimen, especially for patients with non-malignant diseases.

Disclosures
No relevant conflicts of interest to declare.