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ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione)

Palermo

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Fondazione RiMED

Palermo – Italy

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ExperimentalLaboratory

GMP Facility

•  Cell Production Laboratory •  QC Laboratoy

Preclinical Laboratory (large animals)

q  Regenerative Medicine q  Immunobiology q  Molecular Medicine q  Biomedical Technologies

Advanced therapies

Department of Laboratory Medicine and Advanced Biotechnologies

Laboratory of Clinical Pathology, Microbiology and Virology

Adoptive Immunotherapies Transplantation of fetal hepatocytes Transplantation of pancreatic islets

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ü  Immunotherapeutic approaches (CTLs, NK, dendritic cells) for treating viral infections and virus-related tumors

ü  Development of regenerative therapies for treating chronic liver diseases

ü  Characterization of stem/progenitors cells in fetal and adult tissues/organs

ü  Characterization of soluble factors produced by stem cells

Competence

GMP Facility - ISMETT

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Unit of Regenerative Medicine and Biomedical Technologies


Organization and Personnel

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Laboratory of experimental research

n.12 Researchers, n.2 Technicians (supported by RiMED Foundation)

GMP Facility (QP, QA, QC, Validation Manager, Production Head)

ISMETT Labs

Laboratory of molecular biology Laboratory of microbiology Laboratory of immunology

Cytofluorimetry – Mass Spectrometry

Pre-Clinical Research Lab

Animal Facility

n. 1 Veterinarian n. 1 Bioengineer

Technical setting

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§  Facility for experimental cell biology (primary cell cultures, cell immortalization, phenotypic & genotypic analysis , 3D cultures, etc.)

§  Facility for cell production (infected/non-infected cells)

§  Facility for molecular biology (NA amplification, gene expression, sequencing, gene vector production)

§  Facility for experimental and clinical immunology (lymphoid cells purification, activation and characterization; immunopheno yping; cell sorting)

§  Facility for microbiology/virology (virus production and analysis; bacterial/yeast cultures for genetic engineering; control of microbial contamination)

§  Mass spectrometry (proteomics, metabolomics)

§  Biobanking (lymphoid cells for therapy, stem/progenitor cells, primary cell cultures, clinical samples etc.)

§  Clinical setting for phase I/III studies (cohorts of patients affected by end-stage diseases and solid organ transplants, highly-specializedsurgical and interventional procedures)

Services currently offered Ø  Development of adoptive immunotherapies:

Ø  Ag-specific T Cell therapy – Innate immunity–mediated therapy

Ø  Production of stem cells in GMP Facility

Ø  Mass spectrometry (qualitative/quantitative) analysis of stem cells secreted factors

Ø  Biobanking of primary cultures of adult/fetal human tissues and pathologic samples of patients affected by end-stage organ diseases and serious viral/microbial infections

Ø  QA assistance for GMP- compliant Standard Operative Procedures

Ø  Clinical trials (phase I/II studies)

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Ex vivo production and in vivo infusion of autologous (or heterologous) cytotoxic/helper T lymphocyte clones specific to

EBV CMV

Adoptive cell immunotherapy for prevention and treatment of post-transplantation herpesvirus infections

Prevention and treatment of PTLD

Treatment of CMV infections caused by drug-resistant viral

variants

Trapianti – Infettivologia - Immunoterapia

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ITA 005 colon - treatment with anti-EBV-CTLs before treatment after treatment

CD20+ B cells CD20+ B cells

EBER EBER

In vitro production of EBV-specific T cells

Immunoterapia adottiva

CD20+ B cells

EBER

60 Gy irradiation

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Immunoterapia adottiva per il trattamento di patologie indotte da herpesvirus in pazienti immunocompromessi

§  Autorizzazione dell’AIFA per il trattamento delle patologie EBV e CMV-correlate nei pazienti trapiantati QP/QA: QC: Controllo impianti:

§  Uso di CTL eterologhe anti-EBV e anti-CMV §  Biomarkers diagnostici di trasformazione EBV- indotta su linfociti B

§  Linee guida di diagnostica molecolare, immunologica e immunoistochimica per trattamenti terapeutici virus-specifici in fase sintomatica e pre-emptive (Gruppo di Lavoro su Infezioni in Trapianto: AMCLI-SIV)

§  Partecipazione di ISMETT (socio fondatore) a EATRIS

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Workshop: L’Infrastruttura di Ricerca EATRIS e il Nodo Italiano: Stato di avanzamento e prospettive

26-27 Febbraio 2013 - Istituto Superiore di Sanità

The EATRIS consortium of academic institutes provides cutting edge infrastructure and expertise along the entire translational value chain. With top quality facilities for basic research, manufacture, non-clinical and clinical development, our EATRIS institutes offer a truly multidisciplinary environment. Prfoducts: Advanced Therapy Medicinal Products – Biomarkers - Imaging and Tracing - Small Molecules - Vaccines

Isolation of Buffy coat from liver perfusate

Leukapheresis

Hepatic Lymph nodes PBMC

from HCV+ patients and healthy donors

B cell

B cell

HCV-specific Neutralizing Antibodies

B cell

B cell B cell

EBV- Immortalized HCV-specific B cells

IV infusion activated NK IL2/IFNα/IL12

Low viremic Recipient (anhepatic phase)

Adapted from: Ohira M et al JCI 2009,119:11 3226-3235

Depletion of CD3+ cells Isolation of CD56+ NK cells

CliniMACS

Adoptive immunotherapy plan against HCV recurrence after liver tranplantation

Liver perfusate (whole hepatic blood) processing

Infusion in Liver Recipient

Transfer in 500ml conical Centrifugation

In vitro NK cells Activation/expansion

3 X ~3Lt Reservoirs

Volume reduction 7-10 Lt → 0.5-1.5 Lt

buffy coat Isolation

Apheresis COM.Tec

Transfer in double blood bag

Purification of CD3-CD56+ NK cells

CliniMACS Clinical grade

MagMACS research grade

GMP-conforming

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Isolamento Isole di Langerhans

Programma di trapianto insule pancreatiche - trapianto eterologo, autotrapianto –

Insule pancreatiche – diabete

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Nano Engineering for Cross Tolerance: New approaches for bioengineered, vascularized, chimeric islet transplantation in non-immunosuppressed hosts (NEXT)

Progetto presentato per FP7 HEALTH.2013.1.3-2: Innovative approaches to address adverse immune reactions to biomedical devices, implants and transplant tissues - Coordinator dr. Severino – Explora srl

Studio di piccole molecole cito-protettive con duplice applicabilità nella demenza di Alzheimer e nel trattamento del diabete mediante trapianto di insule pancreatiche

Progetto finanziato PON 02- 00697 Potenziamento laboratori pubblico-privati In collaborazione con CNR IBB di Catania, RiMED, Myrmex SpA (ccordinatore CNR –IBB)l

Transplantion of human fetal liver cells in patients with end-stage liver cirrhosis

Fetal liver cell suspension

Collagenase digestion

Infusion into patient splenic artery Human fetal

liver

Phase I-II Control Study of Human Fetal Liver Cell Transplantation for Treatment of Chronic Liver Disease (Submitted manuscript)

Efficient human fetal liver cell isolation protocol based on vascular perfusion for liver cell-based therapy and case report on cell transplantation

(Liver Transpl. 18:226-237, 2012)

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Human fetal hepatocytes cultured in high density demonstrate mature hepatic functionality

* 0 50

100 150 200 250

Con

c (n

g/m

l)/1.

8 x

10 6 c

ells

Albumin secretion

Fetal Adult Liver MSCs

0 2 4 6 8 10 12 14 16 18

Con

c (m

g/dL

)/1.8

x 1

0 6 cel

ls

Urea production


0 50000

100000 150000 200000 250000 300000

RL

U/1

.8 x

10 6 cell

s

CYP3A4 activity


G6Pase activity Glycogen storage ICG uptake

Primary culture of fetal hepatocytes

High density culture Low density culture Flat cell culture

1:3 split

0 50

100 150 200 250

Con

c (n

g/m

l)/1.

5 x

10 6 c

ells

Down-regulation albumin secretion in flat cell cultures

Fetal hep. Flat cells

Liver MSCs

*

Cryopreservation up to 1 year storage is well tolerated as did not significantly affect viability (A), and functions (B, C) of fetal hepatocytes

60

65

70

75

80

85

90

FRE SH CRYO

Pe

rce

nta

ge

of

via

bili

ty

Fresh Cryo

10

30

50

70

90

110

130

150

170

190

F R ESH C R Y O

Con

c. (n

g/m

l)/1.

8 x

106 c

ells

Fresh

Cryo

A BC

Cryo

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Medicina rigenerativa: cellule staminali dermiche

Ø  Multipotent fetal dermal cells are easy to isolate, with a high yield

Ø  Cultured fetal dermal stem cells possess a mesenchymal phenotype

Ø  Cells with a regenerative potential can be successful isolated from a

small fetal skin biopsy and maintained in culture for long periods without

losing their potential, thus generating large quantities for clinical

applications.

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In vitro Expansion and Characterization of Skin Precursor Cells Isolated from Human Fetal Dermis

CD71

CD105

dermal papilla dermal

papilla

A B C

CD105

CD90 CD73 CD105

Fetal Adult SSEA4 SSEA4

90% 10.5%

Pluripotency marker SSEA-4 in fetal and adult dermal cells

Approx. 90% positive for the classical mesenchymal markers CD90, CD73 and CD105

Localized in close proximity of skin dermal papillae

Low immunogenicity. (A) Fetal dermal cells co-cultured with allogenic PBMCs do not evoke T cell proliferation. (B) Positive control MLR

A B A

0 5 10 15 20 25 30

P1 P3

P5

P7 P9

P11

P13

P15

P17

P19

P21

P23

P25

P27

P29

Passage number

Num

ber o

f day

s to

the

next

pa

ssag

e

Adult Fetal

Day 0

C B

Day 6

Retention of a stable phenotype after 12 subcultivations

Expansion potential of fetal vs. adult dermal cells

(A) In vitro angiogenic potential. (B, C) Epithelial-like potential DMSO-induced

A B

AD P10 Fet P25

Higher retention of differentiation potential in fetal than adult cells

0

20

40

60

80

100

120

CD

90

CD

105

CD

73

HLA

cla

ss I

CD

44

CD

166

CD

71

Vim

entin

CD

29

Nes

tin

CD

49b

CD

49e

CD

106

CD

117

CD

45

CD

34

CD

14

HLA

DR

% p

ositi

ve c

ells

P3P12

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Cel

l num

ber

(10 6 )

(C) Non enzymatic isolation technique of “cell outgrowth”. (D) cell yield after isolation: fetal cells can be isolated with a 3-fold higher efficiency than adult cells

Adult

Fetal

Tissue Stem/Progenitor Cells & Microenvironment Modulation

TISSUE OF ORIGIN: ≠ types of tissues ; ≠ age (e.g. fetal and adult tissues)

DEVELOPMENT & OPTIMIZATION of CULTURE SYSTEMS:

2D and 3D ; use of scaffolds ; growth factors + ; hypoxia Mesenchymal Stem Cells “S

tem

ness

deg

ree”

Tu

ning

& E

valu

atio

n

Models of assessment of therapeutic and regenerative potentials in vivo

SILAC

Microvescicles

cell-cell contacts

Secreted Factors: Qualitative & Quantitative Proteomics Secret-OMICS

miRNA

Light AA Heavy AA Conditioned Media Processing

Quantitative identification of full differential secretome

LC-MS/MS

Systems Biology

Bioinformatics Data Functional Analysis

Integration of Transcriptomics Data

Secreted Factors Mix & Systems Biology

In Gel Digestion

Protein identification

Protein quantification

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Technology