Cell factories for production of biological therapeutics

Thursday, June 9, 2016 Mathias Uhlen Science for Life Laboratory (KTH) Stockholm, Sweden

A silent revolution- from chemical to biological drugs

7 out of 8 most sold drugs in the world are proteins

More than 98% of all drugs today are towards proteins

Insulin

HIV Reverse Transcriptase Collagen Lipase

Map of All Human Proteins

A systematic analysis to build a map of the all human proteins

A network of scientists Sweden (six universi.es)

Seoul site

Hangzhou site

10 research groups in US 20 research groups in Europe

Mumbai site

Tissue Atlas •  •  •  • 

IHC (immunohistochemistry) Single cell resolution Annotated by pathologist >10 million images

CD47 Fallopian tube 6

Tissue-based map of the human proteome

Uhlen et al ”Tissue-based map of the human proteome” Science 347: 394 (January 23, 2015)



Nature, May 29, 2014 7!

The tissue-enriched genes HPA and GTEx GTEx only HPA only

n=2,500

Uhlen et al (2015) Science 347: 394 (HPA) – January 23 GTEX consortium (2015) Science 348: 648 (GTEX) - May 6

Uhlen et al, Mol. Systems Biol, 2016

“take-home” messages Almost half of the genes expressed in all tissues of the human body

Very few proteins unique for one tissue

Many drug targets localized to all tissues

Human Protein Atlas Web Portal

>150,000 visitors per month

Human Protein Atlas Portal 400000

Sessions

300000

200000

100000

0 United States

Germany

UK! China!

Canada

South Korea

Italy

Switzerland

Belgium

Sweden! France! Japan! India!

Users from >170 countries

Israel

Norway

Other!

Cell Atlas •  IF (immunofluorescence) •  Subcellular resolution •  >100,000 images

BUB1B Kintochores

To be launched: December 4, 2016 13

Atlas strategy Atlas

Launch

No of genes

Objectives for end of 2018

Tissue

2014

18,000

Special Gssues, GTEx-Fantom integraGon, AnnotaGon, missing genes, validated anGbodies.

Brain

2015

1,200 IntegraGon with Allen Brain Atlas and Gensat

Cell

2016

14,000

Merge with Cell Line Atlas, cell cycle, organell definiGons

Pathology

2017

18,000

New design with HPA and TCGA data incorporated

Metabolic

2017

18,000

Visualize data from the metabolic atlas (links to Chalmers atlas)

2018

Absolute concentraGon across healthy and 2,000 disease individuals, focus on secreted proteins and some leakage proteins

Plasma

The Human Protein Atlas

•  More than 1000 person years of research (KAW funded) •  Open source knowledge-base for medical research •  More than 150,000 visits every month •  Altogether more than 350 “in-house” peer-review papers •  Five external publications using HPA antibodies every day •  Funding ended in April 2016 (after 12 years)

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Wallenberg Center for Protein Research (2016-2023) 1.  Development of bioproduc9on 2.  Discovery of new biological drugs, including systems biology 3.  Human Protein Atlas

Cell factories at KTH Bacterial (E.coli)

Mammalian (CHO)

Targets

All human proteins

Secreted proteins + ECDs

Constructs

PrESTs (Protein fragments)

Full-length proteins

Cloning

RT-PCR

Gene synthesis

Expression host

E. coli

CHO cells

Protein purification

IMAC (Co2+)

Monoclonal (HPC4)

Through-put

600 genes/month

80 genes/month

Time-line

2003 -2015

2016 -

Proteins produced

45,000 (MS validated)

640 (MS validated)

CHO cell factories – the secretome project

Overall objec.ve:

•  ProducGon of all human secreted proteins and membrane proteins (ECD) •  High-throughput producGon in CHO cells •  Create a resource of reagents for drug discovery and development

Funding (equal contribu.on): •  •  •  • 

Knut and Alice Wallenberg FoundaGon (WCPR) Novo Nordisk FoundaGon (CFB) AstraZeneca (MedImmune) Faculty funding (KTH)

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Collaboration between KTH and AstraZeneca

CHO cell factories – Secretome project

Overall objec.ve:

•  ProducGon of human secreted proteins and membrane proteins (ECD) •  High-throughput producGon in CHO cells •  Create a resource of reagents for drug discovery and development

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Summary - secretome • The first 600 proteins of the secretome have been produced • Systems are established for expression, purification, dispensing, storage and screening of purified proteins • Initial hits have been obtained • A plan is in place to produce and screen all proteins • Secretomics has the potential to unlock areas of biology that have previously been closed • Secretomics will lead to the identification of new drug targets and mechanisms for therapeutic intervention in diseases of high ummet medical need

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Human cell factories for bioproduction Overall objec9ve Develop the next generaGon pladorms for producGon in human cells

Short descrip9on Establish human cell factories (alternaGve to CHO cells) for expression of anGbodies and human biologics by engineering of secreGon and metabolic pathways in human cells

Fresh medium!

Johan Rockberg Magdalena Malm Anna-Luisa Rosa Volk

Cell-free supernatant!

Regulator!



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Next generation antibody therapeutics Overall objec.ve: The overall aim of this program is to develop next generaGon monoclonal anGbodies and scaffold binders to targets of pharmaceuGcal interest Affibody molecule 6 kDa

Antibody 150 kDa

Soluble ligand A Soluble ligand B

Stefan Ståhl John Löhlom Johan Rockberg Anna-Luisa Rosa Volk

Affibody® Technology – Next Generation Protein Therapeutics Biopharmaceuticals

Next generation therapeutics

Traditional pharmaceuticals

Monoclonal Antibodies

Affibody® Molecules

Small Molecules

•  •  •  •  •  • 

High specificity High COGS Size: 150 kDa High affinity Long half-life Injectable only

•  •  •  •  •  • 

High specificity Low COGS Size: 6.5 kDa High affinity Flexible half-life Injectable and oral

•  •  •  •  •  • 

Low specificity Low COGS Size: