Rupert Handgretinger Children s University Hospital, Tübingen, Germany Is in vitro T-cell depletion necessary for Haploidentical TransplantationTitle of Presentation Disclosure of Interest: Nothing to Disclose
How to avoid GvHD?
In vitro T-cell depletion is the most effective way to reliably avoid GvHD independent of the HLA disparity Schöttken et al. Five donors-one recipient: modeling a mosaic of granulocytes, natural Killer and T-cells from cord blood and third-party donors. Nature Practice Clinical Oncology 2008; 5: 291
CD34 selection: BM +G-PBSC s: E-rosetting + CD34 selection (CellPro) Aversa F et al.: Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. New England J. Medicine 1998;339:1186-1193. Delayed immune reconstitution, high incidence of infections
Handgretinger et al.; Megadose transplantation of purified peripheral blood CD34+ Progenitor cells from HLA-mismatched parental donors in children. Bone Marrow Transplant. 2001; 27: 778-83. GvHD 0-I II III-IV 91% 7% 2%
Reconstitution of CD3+ T-cells after haploidentical with CD34+ positively selected stem cells Cumulative Incidence 1 0 0 % of p a tie n ts w ith C D 3 > 0.1 x1 0 /L 9 5 0 0 > 20x1 0 6 /kg < 20x10 6 /kg CD34+ 0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0 0. 3 days after t r ansp lantation 0.2 lethal viral infections (ADV, CMV, HSV) 0.1 lethal viral infections(onlyadv) 0.0 0.0 0.2 0.4 0.6 0.8 1.0 years
The important role of NK alloreactivity Ruggeri et al., BLOOD 2007; 110: 433-440.
Probability of relapse The role of NK alloreactivity in pediatric acute Lymphoblastic Leukemia W.Leung et al., Journal of Immunology 172, 644-650, 2004. 1.00 0.75 0.50 (NK nonalloreactive) High Risk P=0.01 0.25 0.00 Low Risk (NK alloreactive) 0 1000 2000 3000 Days after transplantation Lowe EJ et al. T-cell alloreactivity dominates natural killer alloreactivity in minimally T-cell depleted HLA-nonidentical pediatric bone marrow transplantation. Br. J Hematol. 2003; 123: 323
T-cell depletion: positive selection versus negative depletion CD34+ positive selection CD3/19 depletion Gordon et al.: A large-scale method for T cell Depletion: towards graft engineering of mobilized peripheral Blood stem cells. Bone Marrow Transplant 2002; 30:69-74. Barfield et al.: A one-step large-scale Method for T-and B-cell depletion of Mobilized PBSC for allogeneic transplantation. Cytotherapy 2004; 6:1-6. NK cells Monocytes/myeloid cells Comitted precursors Dendritic cells Determination of residual T-and B-cells: Schumm et al. Cytotherapy 2006; 8: 465-472.
percent 100 80 60 CD3/19 depletion Incidence of GvHD (grade 1-3) (Children) 1; 36.7% 2; 23.5% 3, 4.4% 35.3% without GvHD 40 20 0 0 50 100 150 200 days from transplantation
Percent death 100 Comparison of TRM: Positive selection vs. CD3/19 depletion (Children) 80 60 CD3/19 depletion CD34+ selection 40 20 p<0.05 Day 100 TRM: 0 0 0.0 0.5 1.0 1.5 2.0 years from transplantation
CD3/19 depletion in adults Federman B et al. Haploidentical allogeneic hematopoietic cell transplantation in adults using CD3/CD19 depletion and reduced intensity conditioning: a phase II study. Haematologica 2012; 97: 1523
Federmann B et al. Immune reconstitution after haploidentical hematopoietic cell transplantation: impact of reduced intensity conditioning and CD3/CD19 depleted grafts. Leukemia 2011; 25: 121
The potential role of gamma/delta T-cells Godder et al., Long term disease-free suzrvival in acute leukemic patients Recovering with increased g/d T cells after partially mismatched related Donor bone marrow transplantation. BMT 2007; 39,751-757. Vantourout P. Six-of-the best: unique contributions of γδ T cells to immunology. Nat Reviews 2013; 13: 88 Otto M... Handgretinger R. Human g/d T cells From G-CSF mobilized donors retain Strong tumoricidal activity and produce immunostimulatory cytokines after clinical scale isolation. J Immunotherapy 2005: 28: 73
Negative depletion strategy of αβ+ T-cells Chaleff S. et al.: A large scale method for the selective Depletion of αβ+ T-lymphocytes from PBSC for allogeneic Transplantation. Cytotherapy 2007; 9: 746-754. Biotin-anti-αβ (BMA031) + anti-biotin mab magnet Graft magnet Waste CD34+ and CD34- progenitors NK cells Dendritic/myeloid cells γδ T-cells
log Depletion Efficacy of TcRαβ T-cell depletion 5.5 Efficacy of T cell depletion n=139 n= 125 n=102 5.0 4.5 4.0 3.5 3.0 2.5 CD34 pos CD3 neg TCRab CD3+ T cells TcRab+ T cells Schumm M et al., Cytotherapy 15; 1253-8: 2013
0.00 CUMULATIVE INCIDENCE (95% CI) 0.25 0.50 0.75 1.00 Cumulative incidence of grade I-II skin-only acute GvHD in children Unpublished data kindly provided By Franco Locatellia and Alicia Bertaina 50 patients with hematological malignancies Chronic limited (skin): 2 agvhd I-II = 26% 0 10 20 30 40 50 60 70 80 90 100 DAYS AFTER HSCT
TcRαβ/CD19 depletion in adults No. of Patients 14 Primary Engraftment Overall Engraftment Median (range) days to: Neutrophils 0.5x10 9 /L Platelets 25 x 10 9 /L Acute GvHD grade 0- I grade II grade III-IV Chronic GvHD moderate/severe TRM: 2/14 Data kindly provided by Franco Aversa 13 (93%) 14 (100%) 11 (9-15) 10 (5-13) 12 (skin) 1 (skin) 1 (skin,gut)(αβ>10x 5 /kg) 0 HSCT Program University of Parma
Cell/μl Posttransplant T lymphocyte recovery 1800 CD3 CD4 CD8 1500 1200 600 CD3 300 CD4 0 CD8 15 30 45 60 75 90 105 120 135 Days since transplant HSCT Program University of Parma
n/μl 3000 Posttransplant immunological recovery 47 y-old man CML-BT Relapse after MUD Refractory to TK inhibitors CR, 4 months after Haplo 2500 NK cells 2000 1500 1000 αβ CD3+ CD8+ CD4 CD8 CD19 NK alpha/beta gamma/delta 500 0 CD4+ Ɣδ CD3+ CD19+ 15 45 70 days HSCT Program University of Parma
Comparison of CD3+ recovery at day +30 (children) p<0.0001 CD3/19 n = 45 TcRab n = 19
Haploidentical Tx as platform for further immunotherapy Anti-CD19 (ongoing) Prep.regimen Anti-GD2 (ongoing study) Bispecific antibodies CAR T-or NK cells (CD19, GD2) NK cells/cik cells Virus-specific T-cells TcRαβ-depleted stem cells No GvHD prophylaxis, no GvHD
n o a n t i b o d y a n t i - C D 1 9 a n t i - H L A % specific lysis ADCC overrides KIR mediated inhibition: CD16 is the stongest activatory signal for NK cells 100 80 Inhibititory signal KIR HLA - 60 40 Fc receptor + CD19 20 0 Activatory signal Lang P. et al.: Chimeric CD19 antibody mediates cytotoxic activity against leukemic Blasts with effector cells from pediatric patients who received T-cell depleted allografts. BLOOD 15: 3982, 2004.
Second HaploTx in NR and post-transplant treatment with anti-cd19 antibody 4G7 (Prof.G.Jung, Tübingen) (20 mg as a 3 hour infusion weekly or every other week) 1 x 10e-1 PCR-MRD FACS-MRD 1 x 10e-5 1 x 10e-6 negative* 0 +14 +32 +53 +67 +91 +113 +161 2nd haplo SCT > 2 year
MIBG Phase II feasibility study using ch14.18/cho antibody and subcutaneous Interleukin 2 after haploidentical stem cell transplantation in children with relapsed neuroblastoma Eudra CT 2009-015936-14 Haplo Trp. ca. 200x10 6 /kg NK anti GD2 mab (CH14.18/CHO) 8 hour infusion day 1-5 20mg/m 2 20mg/m 2 20mg/m 2 6-9 cycles Evaluation after cycles 3,6 Interleukin 2 s.c. 1 Mio U day 6,8,10 day 0 30 60 90 120
absolute cell count/µl Bispecific T-cell engaging antibodies (BiTE) induce posttransplant donor-derived T- cell proliferation w/o causing GvHD 2500 start end 2000 1500 1000 abscd3 abscd19 abscd4 abscd8 abscd16/56 500 0 Blinatumomab after Tx
Recovery and log Depletion after TcRab Depletion with the fully automated Prodigy Recovery CD34+ 80 % CD56+ 74 % TcRgd+ 51% Log Depletion TcRab+ 5.05 % CD19 4.98 % CD3 1.63% n = 1
Fill out the gaps: Tumor-specific T-cells? Prep.regimen Vaccination????? T-cell depleted stem cells No GvHD prophylaxis, no GvHD
University Children s Hospital Tübingen: Peter Lang Tobias Feuchtinger Michael Schumm Heiko-Manuel Teltschik Matthias Pfeiffer Martin Ebinger Patrick Schlegel Karin Schillbach MiltenyiBiotec