Is in vitro T-cell depletion necessary for Haploidentical TransplantationTitle of Presentation. Disclosure of Interest: Nothing to Disclose

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1 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

2 How to avoid GvHD?

3 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

4 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: Delayed immune reconstitution, high incidence of infections

5 Handgretinger et al.; Megadose transplantation of purified peripheral blood CD34+ Progenitor cells from HLA-mismatched parental donors in children. Bone Marrow Transplant. 2001; 27: GvHD 0-I II III-IV 91% 7% 2%

6 Reconstitution of CD3+ T-cells after haploidentical with CD34+ positively selected stem cells Cumulative Incidence % of p a tie n ts w ith C D 3 > 0.1 x1 0 /L > 20x1 0 6 /kg < 20x10 6 /kg CD days after t r ansp lantation 0.2 lethal viral infections (ADV, CMV, HSV) 0.1 lethal viral infections(onlyadv) years

7 The important role of NK alloreactivity Ruggeri et al., BLOOD 2007; 110:

8 Probability of relapse The role of NK alloreactivity in pediatric acute Lymphoblastic Leukemia W.Leung et al., Journal of Immunology 172, , (NK nonalloreactive) High Risk P= Low Risk (NK alloreactive) 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

9 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: 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:

10 percent CD3/19 depletion Incidence of GvHD (grade 1-3) (Children) 1; 36.7% 2; 23.5% 3, 4.4% 35.3% without GvHD days from transplantation

11 Percent death 100 Comparison of TRM: Positive selection vs. CD3/19 depletion (Children) CD3/19 depletion CD34+ selection p<0.05 Day 100 TRM: years from transplantation

12 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

13 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

14 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, 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

15 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: Biotin-anti-αβ (BMA031) + anti-biotin mab magnet Graft magnet Waste CD34+ and CD34- progenitors NK cells Dendritic/myeloid cells γδ T-cells

16 log Depletion Efficacy of TcRαβ T-cell depletion 5.5 Efficacy of T cell depletion n=139 n= 125 n= CD34 pos CD3 neg TCRab CD3+ T cells TcRab+ T cells Schumm M et al., Cytotherapy 15; : 2013

17 0.00 CUMULATIVE INCIDENCE (95% CI) 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% DAYS AFTER HSCT

18 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

19 Cell/μl Posttransplant T lymphocyte recovery 1800 CD3 CD4 CD CD3 300 CD4 0 CD Days since transplant HSCT Program University of Parma

20 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 αβ CD3+ CD8+ CD4 CD8 CD19 NK alpha/beta gamma/delta CD4+ Ɣδ CD3+ CD days HSCT Program University of Parma

21 Comparison of CD3+ recovery at day +30 (children) p< CD3/19 n = 45 TcRab n = 19

22 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

23 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 Inhibititory signal KIR HLA Fc receptor + CD 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.

24 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* nd haplo SCT > 2 year

25 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 Haplo Trp. ca. 200x10 6 /kg NK anti GD2 mab (CH14.18/CHO) 8 hour infusion day mg/m 2 20mg/m 2 20mg/m cycles Evaluation after cycles 3,6 Interleukin 2 s.c. 1 Mio U day 6,8,10 day

26 absolute cell count/µl Bispecific T-cell engaging antibodies (BiTE) induce posttransplant donor-derived T- cell proliferation w/o causing GvHD 2500 start end abscd3 abscd19 abscd4 abscd8 abscd16/ Blinatumomab after Tx

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28 Recovery and log Depletion after TcRab Depletion with the fully automated Prodigy Recovery CD % CD % TcRgd+ 51% Log Depletion TcRab % CD % CD3 1.63% n = 1

29 Fill out the gaps: Tumor-specific T-cells? Prep.regimen Vaccination????? T-cell depleted stem cells No GvHD prophylaxis, no GvHD

30 University Children s Hospital Tübingen: Peter Lang Tobias Feuchtinger Michael Schumm Heiko-Manuel Teltschik Matthias Pfeiffer Martin Ebinger Patrick Schlegel Karin Schillbach MiltenyiBiotec