DICHIARAZIONE Relatore: SARAH TETTAMANTI

Transcription

1 DICHIARAZIONE Relatore: SARAH TETTAMANTI Come da nuova regolamentazione della Commissione Nazionale per la Formazione Con>nua del Ministero della Salute, è richiesta la trasparenza delle fon> di finanziamento e dei rappor> con soggee portatori di interessi commerciali in campo sanitario. Posizione di dipendente in aziende con interessi commerciali in campo sanitario (NIENTE DA DICHIARARE) Consulenza ad aziende con interessi commerciali in campo sanitario (NIENTE DA DICHIARARE) Fondi per la ricerca da aziende con interessi commerciali in campo sanitario (NIENTE DA DICHIARARE) Partecipazione ad Advisory Board (NIENTE DA DICHIARARE) Titolarietà di brevee in compartecipazione ad aziende con interessi commerciali in campo sanitario (NIENTE DA DICHIARARE) Partecipazioni azionarie in aziende con interessi commerciali in campo sanitario (NIENTE DA DICHIARARE) Altro

2 A A ML ML Azione in vitro e in vivo dei recenori chimerici an>genici contro an>geni CD33/CD123 nella leucemia mieloide acuta primaria XIII CONGRESSO NAZIONALE SIES SOCIETA ITALIANA DI EMATOLOGIA SPERIMENTALE Sarah Te4aman7 Laboratorio di Terapia Molecolare Prof. ENore Biagi Centro di Ricerca Ma>lde TeNaman> Rimini, 1517 ONobre 2014

3 AML ACUTE MYELOID LEUKAEMIA s>ll associated with a very poor prognosis high rates of relapse 5070% within the first 3 years a_er CR Evidence for the existence of leukaemia stem cells (LSC) in AML (Bonnet and Dick, 1997; Hope et al., 2004) AMLLSCs are quiescent in vivo and reside at the endosteal osteoblas>c niche in the bone marrow (Ishikawa et al., 2007) AMLLSCs may be central to posttreatment relapse and chemoresistance Huntly and Gilliland, Nature Reviews Cancer, 2005 Targeted elimina7on of AML bulk and LSC

4 Redirec7ng T cell ac7vity with Chimeric An7gen Receptors (CARs) ANTIGEN BINDING PROPERTY OF ANTIBODIES TCRTRIGGERING DOMAIN: Tcells ac7va7on Cytokines secre7on Cytotoxicity Homing ADVANTAGES OF CARs HLA independent an>gen recogni>on Ac>ve in both CD4+ and CD8+ T cells Target an>gens include proteins, carbohydrates and glycolipids Immunological memory BeNer biodistribu>on compared to mabs Cartellieri et al., 2010

5 CARs in the clinic Background An7gen # of trials Tumor CD19 19 Hematological malignancies CD30 2 Hematological malignancies Kappa light chain 1 Hematological malignancies GD2 1 Neuroblastoma CEA 1 Adenocarcinoma PSMA 2 Prostate cancer Folate Receptor a 1 Ovarian cancer IL13Rα2 1 Gliobastoma HER2/neu 3 Osteosarcoma, lung cancer, other HER + tumors EGFRvIII 1 Glioma Mesothelin 1 Mesothelioma Adapted from Maher J, ISRN Oncol 2012, Jena, Bipulendu, et al. Current hematologic malignancy reports, 2014 < SAFETY IMPROVEMENTS OF CAR TRANSDUCED CELLS Op>mal effector T cell popula>on for immunotherapy Choice of the target an>gen Gene transfer technology Suicide gene strategy

6 Cytokine Induced Killer (CIK) cells CIK cells Immune effector cells expanded from in vitro cultured PBMC, enriched in CD3 + CD56 + CD1d- unrestricted NKTT cells, which arise from CD3 + CD56 CIK cell progenitors PBMC IFNγ OKT3 IL2. IL2 300 U/mL every 3 dd CIK Day 0 Day +1 Day +4 Day +21 CIK cells are characterized by a non MHCrestricted cytotoxicity against a variety of tumoral targets negligible alloreac7vity and minimal GVHD intrinsic capability of reaching leukemiainfiltrated 7ssues (Linn Journal of Biomed and Biotech 2010, Sangiolo Journal of Cancer 2011) The NKG bind CIK c expr ligan ligan restr

7 CIK cells in the clinic 2007 Haematologica 2007; 92: IRCC CIK registry Good FEASIBILITY and SAFETY & Encouraging EFFICACY Results 2013

8 AML targeted therapy with CARs: An7CD33.CAR and An7CD123.CAR + CIK cells < SAFETY IMPROVEMENTS OF CAR TRANSDUCED CELLS Op>mal effector T cell popula>on for immunotherapy Choice of the target an>gen Gene transfer technology Suicide gene strategy CD33 CIK cells + CAR Gemtuzumab Ozogamicin AntiCD33 antibody (113 clone ) Low EFFICACY High TOXICITY in vivo Calicheamicin Efficient killing of leukemic cells in vitro by an>cd33.car + CIK cells BUT robust level of toxicity against HSPCs, highly expressing the CD33 molecule on all myeloid precursors

9 AML targeted therapy with CARs: An7CD33.CAR and An7CD123.CAR + CIK cells CD123 BETTER DISCRIMINATION BETWEEN HSPCs and AML cells % of (Lin ) CD34+ CD38 cells MFI (Taussig et al. Blood 2005) Overexpressed on AML blasts, CD34+ LP and AMLLSCs VS normal HSPCs High CD123 expression in AML pa>ents is associated with a poor prognosis IL3 induces AML prolifera>on in vitro (Jordan et al. Leukemia 2000; Testa et al. Blood 2002; Graf et al. Eur. J Hematol. 2004; Florian et al. Leuk Lymphoma 2006; Van Rhenen et al. Leukemia 2007)

10 An7CD123.CAR genera7on 1 7G3 VL and VH Angel Lopez, CSL Limited, Australia VL VH 2 Overlapping PCR scfvcd123 LS VL Linker VH 3 An>CD123.CARζ An>CD123 scfv NcoI VL VH BamHI LTR SFGCD123.CAR LTR CD28TM domain zchain RETROVIRAL VECTOR

11 An7CD123.CAR vs An7CD33.CAR in vitro TeNaman> et al., BJH 2013 CB Lin cells LESS TOXIC PROFILE vs HSPCs Primary AML SAME KILLING EFFICIENCY vs primary AML samples!

12 An7CD123.CAR + CIK cells exert minimal ly7c ac7vity against CD123 low- expressing HUVEC and monocytes

13 An7CD123.CAR vs An7CD33.CAR in vivo AML MNCs Vehicle or 10x10 6 CIK injec7on 3 weeks NSG BM puncture days Sacrificed (day +37) Dr. Irene Pizzitola Dr. Dominique Bonnet (UCL, London, UK)

14 LOW ENGRAFTMENT AML Experimental plan AML MNCs 3 weeks Vehicle or 10x10 6 CIK injec7on BM puncture days Sacrificed (day +37) LOW ENGRAFTMENT Mouse CD45 Human CD45

15 LOW ENGRAFTMENT AML Results AML sample 1 % of hcd45 + CD33 + cells Vehicle CIK NT CIK an7cd33car CIK an7cd123 CAR AML sample 2 % of hcd45 + CD33 + cells Vehicle CIK NT CIK an7cd33car CIK an7cd123 CAR

16 HIGH ENGRAFTMENT AML Experimental plan AML MNCs 5 weeks Vehicle or 10x10 6 CIK injection BM puncture days Sacrificed (day +37) HIGH ENGRAFTMENT Mouse CD45 Human CD45

17 HIGH ENGRAFTMENT AML Results AML MNCs 5 weeks Vehicle or 10x10 6 CIK injection BM puncture days Sacrificed (day +37) Sorted AML MNCs 4 weeks Vehicle or 10x10 6 CIK injection BM puncture days Sacrificed (day +37) % of hcd45 + CD33 + cells % of hcd45 + CD33 + cells Vehicle CIK NT CIK an7cd33car CIK an7cd123 CAR Vehicle CIK an7cd33car CIK an7cd123 CAR NO RESISTENCE MECHANISM!! NO RESISTANCE MECHANISM!!

18 CORDBLOOD (autologous seeng) Results CB CD34 + Vehicle or 10x10 6 CIK injection 11 weeks BM puncture days Sacrificed (day +37) Vehicle CIK NT CIK an7cd33car CIK an7cd123 CAR

19 CORDBLOOD (autologous seeng) Results <695/40 Blue-A>: CD <530/30 Blue-A>: Lin CD38 <780/60 Red-A>: CD <695/40 Blue-A>: CD34 CD <576/26 Blue-A>: CD CD34 + CD38 + Progenitor cells CD34 + CD Hematopoie>c stem cells enriched compartment 5 <780/60 Blue-A>: CD45RA 10 5 <660/20 Red-A>: CD <780/60 Blue-A>: CD45RA Vehicle CIK NT CIK an7cd33car CIK an7cd123 CAR Vehicle CIK NT CIK an7cd33car CIK an7cd123 CAR

20 CORDBLOOD (autologous seeng) Results CB CD weeks Vehicle or 10x10 6 CIK injection BM puncture days Sacrificed (day +37) MNCs Secondary mice Primary and secondary colony

21 CORDBLOOD (autologous seeng) Results CB CD weeks Vehicle or 10x10 6 CIK injection BM puncture days Sacrificed (day +37) MNCs Secondary mice Primary and secondary colony Vehicle CIK NT CIK anti CD33 CAR CIK anti CD123 CAR Number of positive mice 3/3 3/3 1/3 3/3 %hcd

22 CONCLUSIONS and FUTURE PERSPECTIVES Tcell gene modifica>on by CARs is a promising tool for immunotherapy of AML Targe>ng of CD123 an>gen appears safer for normal HSPCs compartment < < Gene>callymodified cells carry some risks of toxicity SAFETY IMPROVEMENTS OF CAR TRANSDUCED CELLS Op>mal effector T cell popula>on for immunotherapy Choice of the target an>gen Gene transfer technology Suicide gene strategy (i.e. icasp9)

23 Transposons: an easy alterna7ve to viral vectors for gene therapy Sleeping Beauty (SB) transposon (collabora>on with L. Cooper, MD Anderson, Houston, TX, USA) Modified from Singh et al., Immunological reviews, 2014

24 icasp9: the suicide gene of choice SAFETY IMPROVEMENTS OF CAR TRANSDUCED CELLS Op>mal effector T cell popula>on for immunotherapy Choice of the target an>gen Gene transfer technology Suicide gene strategy (i.e. icasp9)

25 ACKNOWLEDGEMENTS! Prof. Andrea Biondi Prof. E4ore Biagi Sarah TeNaman> Virna Marin Irene Pizzitola ElisabeNa Cribioli Francesca Maltese Greta Maria Paola Giordano AEanese Chiara Francesca Magnani Silvia Arcangeli Nice Turazzi Maria Caterina Ro>ro> Dr. Dominique Bonnet (UCL, London, UK) Prof. Alessandro Rambaldi Dr. Mar>no Introna Dr. OrieNa Spinelli Funding:

26

27 An7CD123.CAR + CIK cells are potently cytotoxic against AML targets, like an7cd33.car + CIK cells Short term cytotoxicity assay THP1 Primary AML

28 An7CD123.CAR + CIK cells display limited toxicity against normal HSPCs Colonyforming assay

29 An7CD123.CAR + CIK cells exert minimal ly7c ac7vity against CD123 low- expressing HUVEC and monocytes

30 Cytokines release by an7cd123.car + CIK cells aper s7mula7on with leukemic targets, HUVEC and monocytes