Nat ional I nst itute For Cellular B iotechnology

Research Overview

NICB – Dublin City University

• Government-designated National Centre of Expertise in Basic

and Applied Molecular Cell Biotechnology since 1987

• New 3,200m2 building (opened 2006)

Main Areas of Research

• Molecular basis for biopharmaceutical production by animal

cells

• Tissue Engineering/Stem Cell Therapy – ocular diseases,

diabetes

• Cells as research tools and models for disease research

• Cancer – drug resistance, invasion and biomarkers

Cells as products

Stem cel l therapy/ Tissue engineering

Tissue Engineering/

Stem Cell Therapy

• Twin focus – basic research and clinical application

• Corneal stem cell transplant – entering clinical phase

• Pancreatic islet transplant – currently technology

transfer stage (Partner: University of Oxford)

• The Corneal surface consists of an

epithelial layer that, in the healthy eye,

is constantly renewed.

• Located at the limbus a narrow ring of

stem cells surrounding the cornea.

Stem Cell Treatments For

Eye Injur ies 1

Stem Cell Treatments For

Eye Injur ies 2

• Patients with limbal deficiencies are unable to maintain a stable

cornea. e.g. Chemical and thermal burns, Stevens-Johnson syndrome

• Cornea transplantation in these patients typically fail due to

conjunctival invasion, vascularization, and persistent epithelial defect.

• The NICB in collaboration with its partners, proposes to introduce a

stem cell therapy for these patients.

• Isolate and culture stem cells from the limbal ring.

These cultured cells are then transplanted on to the

surface of the injured eye, repopulating the

damaged cornea

• Research to refine techniques, find better

differentiation markers and investigate biology of

the process, e.g. using microarrays and proteomics

Stem Cell Treatments For Eye

Injur ies - 3

Diabetes Research

at the NICB

5-10% of these have severe

recurring hypoglycemia

23,000 adults in I re land

suffer f rom Type 1 Diabetes

(T1DM)

Significant sustained decrease in

severe hypoglycaemic events and

improved quality of life.

Islet

transplantation

Islets from the Donor’s pancreas are

infused into the recipient’s hepatic

portal vein and produce insulin

Islet

Transplantation Islet Isolation &

Purification

Donated

Pancreas

DRIVE – Horizon 2020

funded project

Diabetes

Reversing

Implants with enhanced

Viability and long-term

Efficacy

Mitochondrial

Function

Oroboros oxygraph

Measures oxygen consumption

in cells and tissues in real time

Cells as factories

Manufacture of recombinant

proteins

Research Re la ted to B iopharmaceut ica l

Product ion

Our goal is to work closely with biopharmaceutical companies to improve basic

understanding of the molecular basis for recombinant protein production by

mammalian cells (including CHO and hybridomas) and

• to translate this understanding into improved biopharmaceutical

production processes

• Special focus on miRNA

Why do we need improved b iopharmaceut ica l

product ion processes?

• Increasing number of products

• Current high unit cost

• Reduced production costs will contribute to

(a) Access for patients to treatments they need

(b) Long-term health of the biopharmaceutical industry

Invited talks

Cells as research tools

& as models for

disease research

Cell culture

Access to l iving human material

- pr imar y cul tures

- ce l l l ines

Cel l cul ture systems may not a lways

complete ly ref lect complex mult i -ce l l type

and spat ia l organisat ion in the body

C o r e t e c h n o l o g i e s :

M o l e c u l a r p r o f i l i n g o f c e l l u l a r r e s p o n s e

e . g . t o d r u g s ; t o t e m p e r a t u r e c h a n g e : t o o x i d a t i v e s t r e s s

• mRNA (Affymetrix)

• miRNA (ABI)

• Proteins

• Importance of bioinformatics analysis

Proteomics

• Proteomics Infrastructure at the NICB

• 2D DIGE (Difference Gel Electrophoresis)

• Mass Spectrometry for Protein

Identification/Characterisation, Including

phosphoproteomics

• Quantitative LC-MS approaches (i.e. gel-free)

Mass Spectrometr y Suite

• LTQ Orbitrap XL (Thermo Fisher Scientific)

High mass accuracy

Interfaced with Dionex Ultimate 3000

1D and 2D LC capabilities

• LTQ XL with ETD

Electron Transfer Dissociation (ETD)

PTM analysis (e.g. phosphorylation)

• MALDI TOF-TOF 4800 (Applied Biosystems)

High throughput MALDI MS/MS

LC-MALDI

Using proteomic profi l ing to identify

new cancer markers in blood

Cancer Biomarkers

for What?

• Early detection

• Monitoring of tumour burden (response to

therapy)

• Detection of relapse

• Predicting response to specific therapy

Advanced Lung

Cancer Haptoglobin ELISA

0

1000

2000

3000

4000

5000

6000

ug

/mL

Control

Squamous

Adenocarcinoma

Small cell carcinoma

Ultimate aims of our

Cancer Research Programmes

Pa i red d iagnos t i c and therapeut i c approaches to

t rea tment of ind iv idua l cancer pa t ients

Toxicity Testing Drug Resistance in Cancer Mechanisms of Cancer Cel l Invasion

CHEMOTHERAPY WORKS,

BUT ONLY SOMETIMES:

WHY?

One reason is drug resistance

(acquired or intrinsic)

Adr iamyc in D is t r ibut ion in Res i s tant Cancer Ce l l s

DLKP-A resistant cancer cells

after exposure to Adriamycin.

Fluorescent view

DLKP sensitive cancer cells after

exposure to Adriamycin

Fluorescent view

Immunodetection of MRP-1

MRP-1 Negative Breast Carcinoma MRP-1 Positive Breast Carcinoma

Cancer invasion & Metastasis

• Cell models, including clonal variants

• In vitro properties and investigation of relevance in human cancer

• Molecular profiling

• New antigens/monoclonal antibodies

Therapeutic Targeting Using ADCs

Ident i f i ca t ion and Inves t iga t ion of nove l

membrane prote in ta rgets w i th h igh cancer

spec i f i c express ion for potent ia l the rapeut i c

ta rget ing us ing Ant ibody Drug Con jugates (ADCs )

Colon Cancer

The 9E1 target antigen is highly

expressed in colon cancer and show

limited expression in normal colon

Pancreatic Cancer

Novel membrane target is highly

expressed in Pancreatic Cancer

Breast Cancer

Novel Membrane Target is highly

expressed in triple negative breast

cancer (TNBC) and shows very

limited expression in normal breast

Development of funct ional ant i - invas ive

MAbs to ident i f y potent ia l drug targets

associated with cancer invas ion/metastas is .

MAb 7B7 target ing the Ku70/80 heterodimer

s igni f icant ly b locks the invas ion of

MiaPaCa-2 pancreat ic cancer ce l ls

Ef fec t of LY6G6F s iRNA on 2D co lony format ion

of Mia Paca-2 ce l l s

Cell number seeded:

100 200 400

Control

Negative

siRNA

LY6G6F

siRNA #2

Uveal Melanoma

Proteomic Analysis of uveal melanoma

to understand metastatic disease

Enucleation & Plaque

Radiotherapy

Treatments for

Primary Tumor

metastasis in

50% of cases

Primarily to the liver with

average survival of

5-8 months

Loss of chromosome 3

Class I v Class 2 gene signature

Loss of BAP1

GNAQ/GNA11 mutation

Current

Biomarkers

A i m s o f t h e S t u d y :

I d e n t i f y d i f f e re n t i a l l y e x p re s s e d p ro t e i n s i n p r i m a r y

u v e a l m e l a n o m a t i s s u e s o f pa t i e n t s w h o d e v e l o p e d

m e t a s t a t i c d i s e a s e v e r s u s t h o s e w h o d i d n o t .

U n d e r s t a n d t h e b i o l o g y o f t h e d i s e a s e

I d e n t i f y n e w t h e r a p e u t i c t a rg e t s

Cancer Biotherapeutics

Background

• NSABP-B31 (Paik et al. 2007) and N9831 (Perez et al.

2010)

• Could a strong immune response to trastuzumab in the

adjuvant setting be responsible for the response in HER2

low patients?

• Trastuzumab has two mechanisms of action 1) it inhibits

HER2 signaling and 2) engages the immune system

through ADCC.

Antibody-Dependent Cell-

mediated Cytotoxicity

ADCC

NK cells and peripheral blood

mononuclear cells (PBMCs)

Immune cells from

healthy Volunteers

Immunoblotting and high

content analysis.

HER2 Levels

quantified

Examine the effects of TKIs on

HER2 expression in a HER2-

positive cell line (SKBR3)

Laser scanning

Confocal

Microscopy

Determining the effect of PD-1/PD-L1

inhibition on response to

trastuzumab in preclinical HER2-

expressing breast cancer models.

Assessing the expression of

immunomodulatory proteins in

HER2-targeted therapy-resistant

tumour cells.

Pancreatic Cancer Programme

• St Lukes Radiation Oncology

Network, Rathgar

• University of Buffalo

Collaboration

Generation of primary pancreatic

cancer and stromal cell lines

With pancreatic cancer

cells and how they impact

treatment success

Stromal Cancer

Cell interactions

L e v e l o f c o l o n y f o r m a t i o n i n P a n c - 1 c e l l s p o s t c o - c u l t u r e

w i t h p a n c r e a t i c p a t i e n t - d e r i v e d f i b r o b l a s t s .

Medicinal Inorganic Chemistry

artificial

metallonuceases

• First reported “self-active” oxidative system

capable of inducing single-stranded DNA scission

in the absence of exogenous reductant or oxidant.

• Copper based complex, di-nuclear structure.

• Displays excellent in vitro chemotherapeutic

activity toward cisplatin-resistant ovarian cancer.

• Detection and quantification DNA

oxidation by synthetic artificial

metallonucleases using capillary

electrophoresis.

• Analysis completed using Agilent

Bioanalyzer 2100 located within the

molecular biological laboratory at NICB.

Chemical nuclease detection

using microfluid ics

• Potent non-covalent DNA binding agents where nucleic

acid recognition is achieved through use of the

“phosphate clamp”.

• Phosphate clamp-DNA interactions result in

condensation of superhelical and B-DNA.

• Triplatin-DNA binding inhibits endonuclease activity by

type II restriction enzymes.

• High chemotherapeutic potential for human cancer.

Novel chemotherapeutic

platinum( I I ) complexes

• Detection of apoptosis induced mitochondrial

depolarization through exposure to ROS active

copper developmental therapeutics.

• Flow cytometry and confocal imaging analyses.

• Broad spectrum of activity identified using the

National Cancer Institute (NCI) 60-human cancer

cell panel.

• Unique mechanism compared to marketed

therapeutics.

Copper chemotherapeutic

drug development

• Individual morphine molecules and derivatives

thereof lack nucleic acid recognition properties.

• In the triplet drug form unique properties

emerge with this alkaloid substructure interacting

with dsDNA and condensing superhelical DNA.

Tripodal opio ids as

unique DNA interacting

agents

Phenazine based

biomaterials

• Enhanced high affinity DNA binding

• Distinctive nucleotide binding

specificity.

• High intercalative capacity

• In vitro chemotherapeutic potential

• Metal catalyzed reactive oxygen species (ROS)

in biological systems can cause a wide variety of

pathological conditions including cancer.

• The extent of DNA damage owing to these

radicals can be quantified through 8-oxo-2’-

deoxyguanosine (8-oxo-dG) lesion detection

using both ELISA or LC-MS/MS analysis.

Detection of free radical

DNA damage

Metallodrug

topoisomerase

inhibitors

• Development of intercalating metal complexes to

unwind and relax negatively supercoiled DNA.

• Applications as unique topoisomerase inhibitors.

Collaboration with Industry

• Cells as factories

• Cells as products

• Cells as Research Tools

• Cells for toxicity assay and bioassay

• Making monoclonal antibodies

• Whole genome mRNA and miRNA profiling

• Proteomic profiling

• Pharmacokinetics/drug analysis

Some past & present industr y col laborators

Quest ions?

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