NEXT GENERATION OFBIOPHARMACEUTICALS

TRANSPORTING THERAPEUTIC PROTEINS, PEPTIDES AND OLIGONUCLEOTIDESAND UNDERSTANDING TRANSPORT PATHWAYS ACROSS BIOLOGICAL

BARRIERS AND CELLULAR MEMBRANES

www.compact-research.org

Contents

COMPACT Key figures ........................................................................................................................................... 3

Mission & Goals ........................................................................................................................................................ 5

Scope of therapeutic modalities for drug delivery system cargos .................................................... 6

Scientific and technical impact .......................................................................................................................... 7

Strategy ....................................................................................................................................................................... 8

Work package 1 and 2: Nanocarrier design, generation andphysico-chemical characterization ................................................................................................................... 9

Workpackage 3: Cellular uptake and trafficking ..................................................................................... 11

Workpackage 4: Oral delivery of peptides ................................................................................................. 12

Workpackage 5: Blood-brain barrier ............................................................................................................ 13

Workpackage 6: Pulmonary delivery ............................................................................................................ 14

Workpackage 7: Skin delivery .......................................................................................................................... 15

Workpackage 8: Consortium management.............................................................................................. 16

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Compact key figures

Project Start Date: 01.11.2012.Duration: 60 Month

○ IMI funding 10 184 909 €

○ EFPIA in kind 18 217 735 €

○ Acad./biotech in kind contribution 3 237 056 €

Total Cost 31 639 700 €

Managing Entity:

Utrecht University Enrico Mastrobattista

Project Coordinator:

SanofiEkkehard Leberer

Deputy Coordinator:

GSKSteve Hood

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cryo TEMLipidoid 5/PLGA NPs

zoomed

cryo TEMLipidoid 5/PLGA NPs

HA-IgGmicroneedle

tips

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Mission

Goals

Overcoming biological barriers

Extension of target space through

Improve next generation biologics by

Crossing cellular membranes

Improve understanding of cellular uptake and trafficking mechanisms of biologics

Provide drug like properties to these drug delivery systems with the potential to move into the clinic.

Develop nanocarrier-based drug delivery systems to deliver biologics

Across epithelial/endothelial barriers such as oIn testine,

brain

lungskin

Across cellular membranes into target cells

Expanding the scope of indications with unmet medical need by making novel targets accessible to biologics.

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Scope of therapeuticmodalities for drugdelivery system cargos

Scaffolds

Antibodies,Antibody

fragments

ASOssiRNAsmiRNAs

Peptides

Size > 1 kDa

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Scientific and technical impact

Better drug delivery systems

COMPACT should provide critical design parameters for drug delivery systems in relation to route of administration with the ability to manufacture drug delivery dystems at suitable scale. This will come with standardized methodologies to characterize drug delivery systems.

Better assay platform for drug delivery system evaluation

Pre-clinical in vitro and in vivo models for drug delivery system-mediated drug delivery to accelerate the translation of nanomedicines into the clinic.

Expanded knowledge

COMPACT will provide a better understanding of the active transport of drug delivery systems across biological barriers and to the cellular target and shall identify novel routes of cellular uptake.

Educational impactTrain and develop talented young researchers through a multidisciplinary cooperative approach in connection with pharma industry. Disseminate knowledge and know how.

Economic impactSpeed up the development of better and safer medicines for patients. Supports collaborative research projects and builds networks of industrial and academic experts in order to boost pharmaceutical innovation in Europe. Enhance the performance on the biopharmaceutical sector and improve the competitiveness of Europe.

Rationally designeddrug deliverysystems

Standardization of models and methodology

Training of young researchers

Development of novel more patient friendly therapies/ threatments

Strategy

Public Private interdisciplinary work package (WP) teams have established state-of-the-art technology platforms for nanocarrier-based drug delivery systems generation, characterization and optimization:

○ Nanotechnology platform to generate drug delivery systems

○ In vitro technology platform

○ In vivo technology platform for biodistribution

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WP1Drug delivery systemsfor proteins/peptides

WP3Cellular uptake & trafficking

• In vitro cytotoxicity and immunogenicity• Characterization of uptake• Monitoring trafficking• Endosomal escape & subcellular targeting• Transcytosis

• In vitro and in vivo evaluation of barrier function and DOS transport• In vivo efficacy studies in animal models of disease

Chemical modifications - Bioconjugates - Nanocarriers

WP8 Consortium management

WP5Brain delivery

WP6Lung delivery

WP7Skin delivery

WP2Drug delivery systems

for oligonucleotide

WP4Oral uptake

Work package 1 and 2: Nanocarrier design, generation and physico-chemical characterizationWP1 and WP2 cover the design, generation and physico-chemical characterization of nanocarriers, the encapsulation or complexation of proteins/peptides and oligonucleotides, respectively, and the characterization of their release.

COMPACT is developing a broad range of drug delivery systems to allow best use of synergies between the different technology platforms:

Lipid-based carriers • Liposomal formats: Functionlzed; CPP modified; hydrogel-filled;• coiled-coil fusogenic• Protamine-lipid complexes• Solid lipid nanospheres• Self-nanoemulsifying drug delivery systems (SNEDDS)• Exosomes

Polymers • PLGA microspheres• PLGA-based nanoparticles• Chitosan-PLGA• Polyamidoamines

Peptidic carriers • Cell penetrating peptides (CPPs)• Designed ankyrin repeat proteins (DARPins)• Bacterial toxins

Microneedles • Dissolving microneedles with nanocarriers• Hollow microneedles with nanocarriers

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“Macromolecular biopharmaceuticals (biologics)

such as therapeutic antibodies, peptides and

oligonucleotides are among the most promising

drugs that are currently being developed in

pharmaceutical research. They can act with

high accuracy on their therapeutic targets

and hence are suited for individual therapy as

envisioned in “personalized medicine”. However,

their therapeutic scope is currently limited by

their poor delivery across biological barriers

and cellular membranes. For example, all

therapeutic antibodies that are currently on

the market are directed against extracellular

targets. Therefore, finding ways to extend

the target space by bringing biologics to their

target locations in the body while preserving

their effectiveness is a primary goal pursued

by all the pharmaceutical companies which are

involved in research, but also of universities

and other research institutions, irrespective of

whether they engage in drug development or

fundamental research.The intake of biologics

in the form of tablets is a goal which remains

unattained. The COMPACT consortium offers an

excellent possibility to join forces to address this

major challenge in the development of innovative

biologics by combining academic fundamental

research and applied drug development in

the pharmaceutical industry. The goal of this

consortium is to generate the basis for the

development of the next generation biologics.”

Ekkehard Leberer coordinator of the project

from Sanofi

Work package 3:Cellular uptake and traffickingThe goal of WP3 is to gain a better understanding of transport pathways into cells and across cell membranes, and how to maximize the capacity to utilize these processes with the drug delivery systems developed in WP1 and WP2. This knowledge is used to design improved formulations for exploiting trafficking pathways leading to enhanced cargo delivery into cells or more efficient delivery across the barriers via transcytosis. The analysis is performed in representative cell models of the gut, blood-brain barrier, lung barrier and skin allowing mechanistic analysis of:

For each of these specific barriers, the WP3 isaddressing following objectives:

• Initial interactions at the cell surface and the mechanism of

uptake into defined endocytic pathways;

• Characterizing traffic through the endolysosomal network for

○ Determination of subcellular fate; ○ Endosomal escape; and ○ Transcytosis.

• Assessment of cytotoxicity and cellular immunogenicity;

• Evaluation of the data from low and high output screening

State-of-the-art imaging technologies are beingemployed to measure cellular uptake and trafficking:

• High throughput and high content cellular imaging by

○ Confocal laser microscopy; ○ Coherent anti-stokes Raman scattering (CARS) microscopy; ○ Four wave mixing imaging (FWM) multiphoton microscopy;

• Single particle tracking by live cell imaging using

○ Raster image correlation spectroscopy (RICS); ○ Fluorescence recovery after photobleaching (FRAP); ○ Fluorescence correlation spectroscopy (FCS).

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Particle analysis

Cell b

inding

Uptake and traff icking

Hig

h co

ntent a

nalysis

poor absorptioni.e. low bioavailability

drug inactivation

stomachpH 1.2

proteolyticdegradation in

GI tract

intestinepH 7.4

Work package 4: Oral delivery of peptides

For biopharmaceuticals, oral delivery is challenging due to degradation in the gastro-intestinal (GI) tract and poor permeability into and across the mucosal barrier. However, by combining detailed knowledge on GI physiology with advanced drug delivery technologies, this challenge may be met.

The goal of WP4 is to gain an improved understanding of the require-ments for enabling delivery of therapeutic macromolecules into and through the intestinal barrier. Therefore, WP4 is developing robust and predictive in vitro and in vivo models and characterization methods as predictive tools for oral delivery of peptides and proteins. The use of these models provide a generic understanding of the critical proper-ties of drug delivery systems and mechanisms for successful (trans)mucosal delivery of a therapeutic peptide or protein.

The objectives of the WP4 are to

• Establish representative models for assessment of the stability of the drug delivery systems and the peptide cargo during transit of the GI tract;

• Explore the interaction of the payload and drug delivery systems with mucus present in the GI tract;

• Mechanistically assess the delivery of the active peptide/protein across relevant mucosal models of the GI tract in vitro; and

• Establish and explore relevant in vivo models for studies of dosing and correlation with in vitro models.

challenges in oral delivery

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The brain prevents the passage of drugs from the blood stream into the brain parenchyma by a vascular barrier system referred to us as the blood-brain barrier. One of the most im-portant factors for successful CNS drug development is therefore the ability to effectively deliver drugs across this barrier, which is especially difficult for biopharmaceuticals.

The objectives of WP 5 are to

• Establish in vitro blood-brain barrier models to analyze delivery of model cargos by drug delivery systems;

• Analyze and optimize existing drug delivery systems such as exosomes, liposomes and poly meric nanoparticles;

• Enable blood-brain barrier specificity by using already established ligands such as RVG and the transferrin receptor;

• Improve blood-brain barrier specificity by using novel ligands that are being generated by COMACT against blood-brain barrier specific cell surface proteins identified in a genomics/proteomics approach;

• Demonstrate the in vivo efficiency of the drug delivery systems in disease-relevant pharmacological models, e.g. Alzheimer’s disease.

Work package 5: Blood-brain barrier

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Until recently, pulmonary drug delivery has been primarily considered as an area of aerosol technology, with the aim to maximize the deposition in the lung. Traditional target of aerosol medicines were the lungs itself, especially in state of disease (asthma, COPD, etc.) while more recently also the option for systemic delivery across the healthy air-blood-barrier came in the picture (e.g., insulin).

The goal of WP6 is to use aerosol technology for “air-to-lung” delivery of biopharmaceuticals across cellular and non-cellular pulmonary barriers, e.g. epithelial cells, marcophages, mucus and surfactant.

The objectives of WP6 are to

• Generate and validate 3D in vitro lung barrier models;

• Validate the delivery of model cargos by drug delivery systems in the 3D in vitro lung barrier models and assess their safety;

• Validate the delivery of disease-relevant therapeutic model cargos in in vivo disease models (e.g. lung fibrosis models or chronic allergic pulmonary inflammation models).

Work package 6: Pulmonary delivery

Alveolar sacks

Alveolar duct

Pulmonary vein

Pulmonary artery

Alveoli

Capillary beds

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The goal of WP7 is to identify and evaluate formulations and devices (microneedles) for patient-friendly biopharmaceutical transport both into the skin (dermal) and through the skin (transdermal) to the systemic circulation.

The objectives of WP7 are to

• Optimize of microinjection through hollow microneedles into human skin in vitro;

• Optimize the delivery of model cargos into in vitro skin using hollow micorneedles in combination with novel drug delivery systems;

• Develop biodegradable microneedles for release of therapeutic proteins/peptides, and optimize thes microneedles by testing in in vitro human skin;

• Demonstrate in vivo in rats or mice intradermal and/or transdermal delivery of model cargos by using microneedles

Work package 7: Skin delivery

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Traditional s.c.i.m. needle

Micro needle

Stratum corneum15 - 20 μm

Viable epidermis130 - 180 μm

Dermis2000 μm

Subcutaneousfat tissue

Nerve

Blood vessels

Sweat gland

Hair folliclesebaceous gland

WP8 carries out the activities of the consortium management, organise the governance of the project, provide administrative oversight and support to WPs 1 to 7.

The Sanofi team (Coordinator), composed of experts in the fields of project management, communication, legal aspects and intellectual property has been put in place to constantly support and advise the consortium.

The objectives of WP8 are to:

•Establish and maintain the governance structure of COMPACT;

•Ensure that scientific, technical and managerial decisions are taken for the implementation of the work plans;

•Oversee fund allocation and ensure that the project stays within budget;

•Manage the IP tracking;

•Manage communication and dissemination of results.

The Matrix structure improves the role and responsibilities of Leaders. With this structure WP Leaders are responsible for technologies and assay systems whereas Project Leaders (PLs) responsible for drug delivery system (DDS) project planning and execution.

Work package 8: Consortium management

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WP 1/2PLs

DDS1

TECHNOLOGY PL ATFORMS

PROJECTS

DDS2

DDS3

DDS4

DDS5...

WP 3 WP 4 WP 5 WP 6 WP 7

Consortium partners

EFPIA partners

Academic partners

• Ultrect university Dept. Pharmaceutics

• University of Copenhagen

• Helmholtz Inst. for Pharmaceutical

Research Saarland

• Cardiff University

• Stockholm University

• Norwegian University of Science and

Technology

• University of Vienna

• LMU Munich - Dept. of Pharmacy

• University of Zurich

• University of Ghent

• Ultrecht University Dpt. Infectious Diseases

and Immunology

• University of Helsinki

• Leiden University

• Oxford University

Biotech partners

• Pharmacoidea Ltd. (Hungary) • BioneerPharma (Copenhagen)

For more information please visit www.compact-research.org or please contact us anytime at info@compact-research.org.