Maria Teresa Lupo Stanghellini, MD San Raffaele Scientific Institute. Milano, Italy.

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1 : in-vivo HSV-TK suicide machinery is effective in GvHD control and provide a long-term immune-suppressive treatment-free survival Maria Teresa Lupo Stanghellini, MD San Raffaele Scientific Institute. Milano, Italy

2 Haplo-HSCT: a difficult GvHD setting The holy grail of haplo-hsct is to mitigate host-versusgraft and graft-versus-host response while preserving immune responses to infection and the patient s malignancy. Fuchs Hematology 2012

3 Chronic GvHD is NOT beneficial The presence of chronic GVHD is associated with significantly higher treatment-related mortality and worse overall survival across all diseases studied Boyiadzidis et al 2014

4 Haplo-HSCT and T-cell depletion Trends in alternative donor transplantation. EBMT 2013 activity survey Haplo-SCT without T-cell depletion (TCD) is increasingly used, but follow-up is still short to evaluate the impact of chronic GvHD in such a mismatched setting

5 HSV-TK approach: a 20 years history TK cells therapy in haploidentical-hsct Donor haematopoietic stem cells Hospital TK cells Protection from infections Haemat. stem cells Day 0 Day 21+ TK cells Protection from leukaemia relapse Haploidentical donor MolMed GMP facility Genetic engineering of donor T cells No immunesuppression needed (prompt abrogation of GvHD by administration of ganciclovir) T cells Donor T cells HSV-TK Sources: adapted from Bonini et al., Science 1997; Bonini et al., Nat. Med. 2003; Recchia et al., PNAS 2006; Ciceri et al, Blood 2007; Ciceri, Bonini et al., The Lancet Oncology 2009; Oliveira, Curr Opin Hematol 2012 MolMed S.p.A. TK008_IPR/21_Start-up - CONFIDENTIAL 5

6 TK-cells and GvHD kinetics: series analysis 57 adult patients (pts) after haplo-sct for hematologic malignancies years Phase II TK007 trial (Ciceri, Bonini et al, Lancet Oncol 2009) Phase III TK008 trial (NCT ) Graft: peripheral CD34+ selected cells No post-transplant immune-suppression after transplantation as GvHD prophylaxis

7 Study design Phase I-II TK007 NCT Ciceri, Bonini et al, Lancet Oncol 2009 Phase III TK008 NCT Haplo-HSCT* plus TK cells Haplo-HSCT* plus TK cells R (3:1) Haplo-HSCT** *T-depleted (T cells, 1x10 4 /Kg) *T-depleted (T cells, 1x10 4 /Kg) **T-depleted (T cells, 1x10 4 /Kg) or ** unmanipulated BMT/PB + HD CTX Dose of TK cells (1-x10 7 /Kg - 1x10 7 /Kg) Up to 4 monthly doses up to IR (CD3+ cell count >/= 100/mcl) Starting 21 to 49 days after HSCT in absence of IR and/or GvHD Dose of TK cells (1x10 7 /Kg) Up to 4 monthly doses up to IR (CD3+ cell count >/= 100/mcl) Starting 21 to 49 days after HSCT in absence of IR and/or GvHD

8 Patient population n 57 Median age 53y (range 17-66) Male 26 Diagnosis Acute Leukemia Myelodysplasia Chronic Myeloid Leukemia Hodgkin Disease Non Hodgkin Lymphoma 50 (AML 43) Disease status at transplant Source of Stem Cell HLA match type Female Donor in Male Host 12 Graft Manipulation Median Follow-Up Complete Remission 40 (25 CR1) Persistence of Disease 17 Peripheral Blood HLA-mismatched relative with >/= 2 HLA Ag mismatch peripheral CD34+ cells in vivo T and B cell depletion ATG and Rituximab 79 months (range months)

9 Conditioning Regimen Treosulfan 14 g/m 2 X X X Fludarabine 30 mg/m 2 X X X X X ATG Fresenius 10 mg/kg X X X Rituximab 500 mg X T-cell depleted Haplo-PBSC X TBI 200 cgy X X Donor Lymphocytes Infusion X* *TK-cell based strategy

10 TK-cells therapy Patients treated 35/57 N^ Infusion (median) 2 (1-4) CD3 / Kg x10 7 1,1x10 7 /Kg CD3 dose (total cells x10^6) 931x10 6 Immune-reconstitution: patients median time from SCT median time from last TK-cells infusion 25/35 pts 84 days (18-182) 27 days (13-42) n 35

11 TK008 experimental arm: fast and wide immune reconstitution (IR) cells / ul NGFR+ NGFR- Percentage of CD4 + cells Gated on CD4+ Percentage of CD8 + cells 120 T NA 100 T CM T EM 80 T EM RA Gated on CD8+ T NA T CM T EM T EM RA 0 Baseline n=6 at Immune Reconstitution n=6 at 6 months n=6 at 1 year n=4 0 IR 6m 1y time after HSCT (days) 0 IR 6m 1y time after HSCT (days) Spectratyping Complexity Score IR 6m 1Y CTRL Percentage of CD4 + cells Gated on CD4+LNGFR IR 6m 1y time after HSCT (days) Percentage of CD8 + cells Gated on CD8+LNGFR+ 120 T NA T100 CM T EM 80 T EM RA IR 6m 1y time after HSCT (days) T NA T CM T EM T EM RA MolMed S.p.A. Takara Bio MolMed meeting, 19 July 2013 CONFIDENTIAL 11

12 TK-cells: GvHD n 25 GvHD 13/25 Acute GvHD N^ of patients Onset median time days post SCT (20-162) 42 days post 1 st TK-cell infusion (3-118) Grade I Grade II Grade III Grade IV Chronic GvHD N^ of patients Onset time Feature 2 pts 8 pts 1 pts 1 pts days post SCT 146 days post 1 st TK-cell infusion severe, classic, de novo c-gvhd, with sclerodermatous lichenoid skin and mouth features plus moderate dry-eye symptoms

13 TK-cells and GvHD TK-cells infiltrate GvHD affected organs

14 GvHD : ganciclovir treatment TK+ cells/mcl TK- cells/mcl 400 * 600 p p ns pre post 0 pre post 11/13 pts GvHD treatment with ganciclovir iv or VGC per os full control of clinical manifestations of GvHD in a median of 14 d significant reduction in numbers of circulating TK-cells no reduction of CD3+ TK-negative lymphocytes long-term IR In 5 pts additional concomitant treatment with low-dose steroid (prednisone <0.5mg/kg per day for a median of 2 weeks)

15 GvHD: kinetics of ganciclovir treatment Time to GvHD resolution (n=13). Percent of patients GvHD free median 14 days No cases of quiescent or progressive c-gvhd was observed after a median follow-up of 679 days (r 139/4035).

16 TK-cells: Long-Term-Follow-Up According to international guideline for long-term follow-up all pts underwent regular screening and clinical evaluation. Majhail NS et al, BBMT. 2012; 18: No major infections occurred in the late phase No grade IV adverse events regarding ocular, oral, respiratory, hepatic, renal, genitourinary, muscle-skeletal or neurological systems. Noteworthy 1 pt developed 2 nd cancer (non-melanoma skin cancer ), 3 pts hypothyroidism, 5 pts metabolic syndrome. No evidence of psychosocial symptoms was reported.

17 12-years of TK-cells in Haplo-HSCT: conclusion TK-cells are effective in providing IR while controlling GvHD TK-cells provide a long-term immunosuppressive therapy free survival in absence of GvHD GCV/VGCV treatment of GVHD do not impair a long-term IR Phase III TK008: 1-y DFS and OS (all pts) In vivo persistence of gene modified TK cells Bonini et al, ASH 2014 ABS 2535 Oliveira et al, ASH 2014 ABS 4793

18 Acknowledgement Bone Marrow Transplantation Unit Fabio Ciceri Massimo Bernardi Consuelo Corti Jacopo Peccatori Andrea Assanelli Magda Marcatti Sarah Marktel Francesca Lunghi Elena Guggiari Matteo Carrabba Luca Vago Raffaella Greco HLA Tissue Typing Immunohematology and Transfusion Medicine Laura Bellio Milena Coppola Katharina Fleischhauer Salvatore Gattillo Lucia Malabarba Simona Malato Benedetta Mazzi Paola Ronchi Michela Tassara Elisabetta Zino MolMed Claudio Bordignon Antonio Lambiase Monica Salomoni Scialini Colombi Division of Regenerative Medicine, Stem Cell and Gene Therapy - Experimental Hematology Unit Chiara Bonini Giacomo Oliveira Attilio Bondanza Sara Mastaglio Nicoletta Cieri Zulma Magnani Veronica Valtolina Rosaria Carbone

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