Fifth Annual Ri.MED Scientific Symposium Palermo 24.10.2011 PRIMING THE THERAPEUTIC PIPELINE: NEW STRATEGIES FOR DRUG DISCOVERY Immunotherapeutic strategies in the management of transplantation-related viral infections Pier Giulio Conaldi Unit of Regenerative Medicine and Biomedical Technologies Laboratory of Clinical Pathology, Microbiology and Virology ISMETT
ISMETT- RiMED Foundation Unit of Regenerative Medicine and Biomedical Technologies Pre-clinical Experimental Laboratory Laboratory GMP Facility Preclinical animal Immunobiology models Molecular Medicine Experimental surgery Regenerative Medicine Cell Therapy Adoptive immunotherapy Pancreatic islet transplantation Fetal hepatocyte transplantation 2
Infections in solid-organ transplant recipients (J. A. Fishman, N. Eng. J. Med., 357:2601-14, 2007) 3
Adoptive immunotherapy for prevention and treatment of post-transplantation herpesvirus infections Ex vivo production and in vivo infusion of autologous (or heterologous) cytotoxic/helper T lymphocyte clones specific to EBV Prevention and treatment of PTLD CMV Treatment of CMV infections caused by drug-resistant viral variants 4
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EBV normally infects naive B cells in Waldeyer’s ring, which differentiate into memory B cells, exit the cell cycle and are therefore not pathogenic. Normally, bystander B-cell blasts would be destroyed by CTLs, but if the CTL response is suppressed, the blasts can lead to PTLD 6
Post-transplant lymphoproliferative disease (PTLD) Spectrum of disorders ranging from hyperplasia to invasive malignant lymphoma PTLD incidence is up to 10% of all solid organ transplant recipients (higher incidence in children since PTLD is mainly related to post-transplant EBV primary infection) In all age groups it is the most common single cause of cancer related mortality after solid organ transplantation (overall mortality is approximately 50%) Over 90% of PTLD patients are EBV-positive and the tumor cells generally express all the latent viral genes (latency III) PTLD may involve the lymph nodes or extranodal tissue at any site (kidney, bowel, lung, mediastinum, tonsil, bone marrow and CNS), including the organ allograft 7
EBV-associated malignancies 8
In vitro production of EBV-specific T cells 60 Gy irradiation 9
Immunophenotype of EBV-specific “ CTLs ” 80 Characterization of 70 EBV-specific “ CTLs ” expanded 60 in vitro with serial stimulations by % of positive cells 50 autologous EBV-immortalized LCLs 40 30 20 10 Cytotoxic activity (Cr 51 release assay) 0 CD4 CD8 NK 80 70 Cytotoxic activity (Cr 51 release assay) 60 % of specific lysis 90 50 80 40 70 30 60 20 % of specific lysis 10 50 control group SOT 0 40 auto allo auto allo auto allo auto allo auto allo auto allo 30 40:1 20:1 10:1 5:1 2,5:1 1,25:1 20 Autologous LCLs Allogeneic LCLs 10 High specific reactivity : >40% killing of autologous 0 target cells at 20:1 effector/target ratio 40:1 20:1 10:1 5:1 2,5:1 1,25:1 Transplant patients Healthy controls 10
COD age Transplantation Diagnosis N. infusions CTL dose ITA001 33 kidney High level EBV viral load ITA002 36 Kidney High level EBV viral load ITA003 24 Lung PRE-PTLD (lung) 6 1.5 x 10 6 cells/kg ITA005 5 Liver Polymorphic PTLD (tonsils, bowel) ITA006 9 Liver Polymorphic PTLD (bowel) ITA007 4 Liver Early lesions (tonsils) 3 1.5 x 10 6 cells/kg ITA008 4 Liver Early lesions (gut) ITA009 5 Liver Polymorphic PTLD (tonsils) 2.0 x 10 6 cells/kg 3 ITA010 15 Liver Polymorphic PTLD (tonsils, bowel) ITA011 6 Liver Polymorphic PTLD (tonsils) ITA012 7 Liver Early lesions (liver) ITA014 7 Liver High level EBV viral load ITA016 7 Liver Polymorphic PTLD (tonsils, bowel) ITA017 5 Liver Early lesions (tonsils, bowel) ITA017 5 Liver Early lesions (tonsils, bowel) ITA018 6 Liver Early lesions (tonsils) ITA019 21 Liver Early lesions (tonsils) ITA020 4 Liver High level EBV viral load ITA021 9 Liver Early lesions (tonsils, bowel) ITA022 7 Liver Early lesions (tonsils, bowel) ITA023 9 Liver Early lesions (tonsils, bowel) ITA024 7 Liver Early lesions (tonsils, bowel) ITA025 20 Liver High level EBV viral load ITA026 6 Liver Polymorphic PTLD (bowel) ITA028 9 Liver Early lesions (tonsils, bowel) EBV-DNA EBV-DNA Follow up PTLD histology (copies x10 5 PBMC) (copies/ml blood) (wks) From Polymorphic to From 23 to From 408 to ITA005 32 Early Lesion undetectable undetectable From Polymorphic to From 141 to 20 From 6700 to 1700 ITA010 Early Lesion 21 11
ITA 005 colon - treatment with anti-EBV-CTLs before treatment after treatment CD20+ B cells CD20+ B cells EBER EBER EBER 12
ITA 010 tonsil - treatment with anti-EBV-CTLs before treatment after treatment CD20+ B cells CD20+ B cells EBER EBER 13
CMV-specific T cell therapy X ray IL-4 +GM-CSF Co-colture with lymphocites PBMC CMV-specific T cells Pulsed with pp65 and IL-2 IE-1 peptide-mix Evaluation for cell therapy purpose: specifity, phenotype and sterility of CMV-specific T cells expanded in vitro CMV pp65 and IE-1: a mix of 15-mers peptides overlapping by 11 amino acids spanning the entire IE-1 and pp65 protein sequences 14 14
Treatment of a SOT recipient with severe and persistent CMV infection with allogenic CMV-specific T cells Male, 16 yrs old, affected by primary sclerosing cholangitis Liver transplantation: D+/R- for anti-CMV Abs Primary CMV infection after transplantation with serious clinical manifestations Severe leucopenia, CMV infecton of bone marrow Ganciclovir therapy (GC) * 9700 copies/ml of CMV-DNA in Severe leucopenia bone marrow GC Pneumonitis GC Cut-off of CMV-DNA level in blood for pre-emptive therapy * 15
In vitro production of CMV-specific Cytotoxic activity (Cr 51 release assay) T cells using the PBMC of the 100 mother of the patient 90 80 % of specific lysis 70 T1 autologous PHA-blasts +pept 60 Immunophenotype T2 autologous PHA-blasts 50 T3 recipient PHA-blasts +pept) 40 T3 recipient PHA-blasts 30 20 10 0 40:1 20:1 10:1 5:1 2,5:1 Effector/target ratio Follow-up after CMV-specific T GC No GC treatment cell infusions infusion of CMV-specific T 1 w after 1° inf.: significant reduction of cells (1x10 6 cells/kg) CMV-DNA level in bone marrow * evaluation of CMV infection in the bone marrow 2 w after 1° inf.: normalization of blood CMV-DNAemia leucocyte number (still now) normal level of blood leucocytes detectable CMV-specific T cells in the peripheral blood (ELISPOT) until 2 > 6000/ m l months after 1° inf. Undetectable level of CMV-DNA emia (still after 1 year) <2000/ m l * No further treatment with ganciclovir 16
Adoptive immunotherapy for the prevention of HCV recurrence after liver transplantation Funded by With a global burden of 180 million infected people HCV is a major public health challenge. Of the infected patients 60-80% develop chronic hepatitis, leading cause of liver cirrhosis and hepatocellular carcinoma. HCV persistence is mainly due to virus capability to evade host immune response. Particularly, in the early phase of the infection HCV attenuates innate immune responses (including NK cells functions), weakening the development of specific T cell responses, and impairs the production of anti-HCV neutralizing Abs. Chronic hepatitis C is the most common (almost 50%) indication of liver transplantation, but viral recurrence of the graft is universal and the damage of the new livers occurs usually and is associated with accelerated progression to cirrhosis, graft loss and death. A prophylactic strategy for prevention of HCV recurrence is lacking and drug-based antiviral therapies have limited efficacy and tolerability in liver transplantation recipients. 17
Innate immune response Adaptive immune response Impaired differentiation & maturation Delayed trafficking to the liver Inhibiti on of Treg Impaired Resistance of Reduced effector infected proliferation functions hepatocytes to the effect of IFN CD4+ DC CD8+ HCV antigen HCV mutations and uptake quasispecies escape from antibody and T Reduced cell responses T cell priming Modulation of DCs function Late and limited NK humoral immune responses Inhibition of NK cells by HCV E2 Poly- & monoclonal B cell expansions 18 18 Adapted from Rehermann B at el. Nat Rev Immunol Mar 2005: Vol 5; 215-229
HCV infection B. Rehermann J. Clin. Invest. 119: 1745, 2009 The incubation phase : viral attenuation of the innate immune responses (hepatocytes, DC, NK cells) 19 P. Georgel et al. Trends Molecular Medicine 16: 277, 2010
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