Transplantation of TcRαβ/CD19 Depleted Stem Cells From Haploidentical Donors: Robust Engraftment and Rapid Immune Reconstitution In Children with High Risk Leukemia
Authors: Handgretinger Rupert , Lang Peter , Feuchtinger Tobias F. , Schumm Michael , Teltschik Heiko-Manuel , Schulz Ansgar S , Huppert Volker , Moretta Lorenzo , Moretta Alesandro , Bernardo Maria Ester , Caniglia Maurizio , Bertaina Alice , Zinno Francesco , Locatelli Franco 
Published: October 2019

Abstract
Abstract 1005
In haploidentical transplantation with mobilized peripheral blood stem cells (PBSC's), in vitro T-cell depletion of the graft is an effective method to prevent or completely avoid Graft-versus-Host Disease (GvHD). In order to increase the T-cell depletion efficacy of PBSC's while maintaining the anti-tumor and anti-infectious properties of the graft, we have investigated a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, Natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of posttransplant EBV-associated lymphoproliferative disease. The αβ+ T-cells and CD19+ B cells were then removed using the CliniMACS system. So far, 23 patients have been treated in two centers, namely Tübingen and Rome. Graft manipulation resulted in a consistent efficiency of αβ+ T-lymphocyte removal at the two centers. The overall depletion of αβ+ T-cells was 4.5 log (range 3.8–5.0) and 4.3 log (range 3.7–5.0) in Tübingen and Rome, respectively, with a median number of transplanted αβ+ T-cells of 14 × 103/kg. The recovery of CD34+ stem cells was 72% and 89% in the two centers, and the median number of infused CD34+ stem cells was 11.9 ×106/kg (range 7.5 –30 × 106/kg) and 13.3 ×106/kg (range 8.3 –19.8 × 106/kg), respectively. Patients were given 107 × 106/kg (range 35 –186 ×106/kg) and 123 × 106/kg (range 51–202 ×106/kg) CD56+ NK cells in Tübingen and Rome, respectively. The median number of infused γδ+ T-lymphocytes was 11.9 ×106/kg (range 7.5–30.2 × 106/kg) and 10.3 ×106/kg (range 6.5–25.1 × 106/kg) respectively.
The 10 patients transplanted in Tübingen had advanced/refractory leukemias (ALL, n=5; AML, n=5; active disease, n=6; 2nd transplantation, n=2; CR2, n=2). For this poor-prognosis patients, a reduced-intensity conditioning regimen (melphalan, thiotepa, fludarabin or clofarabin and OKT-3 or ATG) was used. No further post-transplant GvHD prophylaxis was given. All 10 patients engrafted. The median time to reach neutrophil (PMN) and platelet (PLT) recovery was 9 (range 8– 12) and 15 days (range 6 –28) respectively. All patients reached complete donor chimerism and showed a very rapid immune reconstitution with 350 (range 21–824) CD3+ T cells/μl, 66 (12–177) CD3+4+/μl and 599 (227–1390) CD56+ NK cells/μl at day +28 posttransplant. Three patients had no signs of acute GvHD, 5 patients had GvHD grade I and 2 patients had skin GvHD grade II. Only 1 patient experienced a transient grade 3 GvHD of the skin which required only topical treatment. No patient experienced chronic GvHD. Three patients relapsed after transplantation, 7 patients are in remission for 5 months (range 3 –12). There was no transplant-related death sofar.
The second cohort given transplantation in Rome comprised 13 patients with ALL (9), AML (3) and NHL (1). All children but 1 had relapsed/refractory disease. In particular, 11 patients were transplanted in CR and 2 with active disease. Conditioning regimen was myeloablative and consisted of fractionated TBI, Thiotepa, fludarabine and ATG (8 mg/Kg). No further post-transplant GvHD prophylaxis was given. All patients engrafted, the median time to reach PMN and PLT recovery being 11 (range 7–13) and 12 (range 10–16) days, respectively. Only 2 patients had skin grade I acute GvHD. No patient experienced chronic GvHD. With a median follow-up of 4 months (range 1–9) 10 patients are alive and disease-free; 2 patients relapsed (1 died) and 1 had fatal lung aspergillosis. In both cohorts, γδ+ T cells started to expand faster than αβ+ T cells in the early post-transplant period, whereas at day +100, αβ+ T-cells were predominant. In addition to a rapid reconstitution of αβ+ T-lymphocytes, Vbeta spectratyping revealed a broad T-cell receptor repertoire early after transplantation.
Altogether, these data indicate that transplantation of TcR αβ+/CD19 depleted cells from a haploidentical donor results in sustained engraftment, rapid immune reconstitution and low incidence of both acute and chronic GvHD. The anti-leukemic efficacy of this approach in comparison to other methods of T-cell depletion needs to be evaluated with a longer patient follow-up.

Disclosures:
No relevant conflicts of interest to declare.