Conjugate the smart way

API Services &Chemical Developmentwww.almacgroup.com

> Site specific engineering and labelling

of proteins

> Wide range of labels and modifications

> Proprietary protein ligation technology

> Homogeneous conjugated products

> Low equivalents of label

> Retained biological activity

> High yielding reactions

Site Specific Protein Modification

Delivering improved peptide and protein therapeutics

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Biotherapeutics Market The biotherapeutics’ industry has boomed in recent years with an estimated valuation of US$145 billion in 2014. Increasingly protein engineering technologies are being used to develop protein therapeutics with improved performance and to enable novel therapeutic approaches to be realised using protein-based drugs. Protein engineering has a critical role to play in many aspects of drug development including protein PEGylation, antibody-drug conjugates, bi-specific therapeutics and molecular imaging agents. Consequently, to tackle the current and future challenges, and provide solutions to the drug discovery and healthcare industries, there is a continued requirement for improved protein engineering technologies.

Current shortcomings Established protein labelling technologies generally result in the non-selective and non-specific modification of the target proteins. This yields heterogeneous populations often with compromised biological activity, and large batch to batch variations in their production. Alternative approaches rely on insertion of additional amino acid residues into the sequence, which again can potentially compromise biological activity and may lead to unwarranted issues with protein stability and immunogenicity. Moreover, high label-to-protein stoichiometries may be required to give the required yields of the modified protein, leading to an increased cost of goods. Therefore there is a requirement for robust, high yielding technologies for the site-specific labelling and modification of proteins.

The Almac Advantage Almac’s proprietary protein engineering technology enables proteins and peptides to be site-specifically modified in a highly- selective, high yielding fashion. With only one modification site, at the C-terminus, the resulting protein preparations are homogeneous. In addition, the highly efficient conjugation reactions performed under mild aqueous conditions translate into lower costs and make this technology compatible with native proteins.

Proven technology From the conjugation of large PEG molecules for half-life extension through to the site specific attachment of small molecule probes and labels, the applicability of our technology has been demonstrated on a wide variety of proteins of therapeutic interest.

As examples: > Grb2-SH2> INFa2b > INFb1b> Evasin-3> FKBPL> Range of single domain antibodies > Chemokines

Providing Solutions

Overview

> This thioester intermediate is chemically cleaved under aqueous buffered conditions using hydrazine or dioxyamine to liberate the corresponding C-terminal hydrazide and aminoxy derivatives of the target protein.

> The C- te rm ina l d e r i va t i v e s chemoselectively react with ketone or aldehyde containing moieties (benzaldehyde PEG shown here) resulting in site specific C-terminal modification of the recombinant protein via stabilized hydrazone or oxime linkage.

Key advantages of our technology > Targeted, site specific ligation at the C-terminus – allows total control over the ligation process> Retained biological activity – has the potential to improve upon the native protein i.e. by extending half life while maintaining the activity> Robust, high yielding conjugation technology> Mono conjugated species – COGs savings and lower dosing potential> Conjugate equivalents ratio – low ratio equates to savings on COGs> Compatible with disulphide-bond containing proteins> Applicable to a wide range of labels and modifications> Single modification site means homogeneous products produced

High efficiency - High yield

The recombinant protein of interest is expressed as an N- terminal intein fusion protein. The properties of the intein domain are such that it induces an N to S acyl shift at this protein-intein junction to form the thioester intermediate.

>

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Almac’s proprietary ligation technology

Site-Specific protein ligation applications

Protein PEGylation

Protein labelling

Imaging applications

Targeted cytotoxics

Aqueous buffer

Aqueous buffer

Site-specificallyPEGylated protein

Site-specifically fluorescein labelled protein

Protein-drug conjugate (e.g. ADCs)Biom

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Fluorinated protein

Rapid, high yielding protein fluorination using 2 equivalents of label

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Technology compatible with prokaryotic and eukaryotic expression

Bi-Specific proteins

Expression in Echerichia coli Expression in Pichia pastoris

Peptide conjugation

Bi-functionalised linker

Bi-specific molecule

Aqueous buffer

Aqueous buffer

c-myc peptide

Site-specifically peptide conjugated protein

Protein 2

Protein 1

> Cytoplasmatic expression of Evasin 3- intein in bacteria> Intein cleavage followed by site specific C-terminal PEGylation of target protein> Technology also compatible with periplasmic expression of the intein-fusion proteins

> Secreted expression of EGFR sdAb-intein from yeast > Intein cleavage followed by site specific C-terminal PEGylation of target protein

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PEGylation of antibody fragments: anti-EGFR single domain antibody

Antibody fragments > Nanobodies are the smallest available intact antigen binding domains> 120-130 amino acids> Contain 1 or 2 conserved disulphide bridges > Rapid clearance from blood (half life ~10 mins)

Attractive target for PEGylation > Increase in vivo half life> Reduced immunogenicity and proteolysis > Better targeting to tumour tissues (Enhanced permeability and retention effect)

Labelling is high yielding and cost-effectivePEGylation reaction is fast and high yielding

PEGylated protein maintains full activity

> Mild reaction conditions

> Without modification, the anti-EGFR single domain antibody has a half-life of just 3.7 minutes in mice

> PEGylation increases the half-life by approximately 70 fold, to 4.3 hours

PK properties are improved by Almac PEGylation technology

Bin

ding

of [

125 I]

EG

F [%

]C

once

ntra

tion

[ng/

ml]

PE

Gyl

ated

sdA

b [%

]

Aqueous buffer

> Ligation reaction is very efficient, >80% yield even with a 1:1 protein to PEG ratio

> Full activity of single domain anti bodies is maintained after C-terminal PEGylation

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PEGylation of protein therapeutics: INFa2b & INFb1b

INFa2b > 165 aa residues – 2 disulphide bridges > PEG-Intron® (Schering Plough) has been approved to treatment of Hepatitis C: > non-selective PEGylation (12 kDa linear) > 14 PEG positional isomers > in vitro antiviral activity is 28% of non-modified form

INFb1b > 165 aa residues – 1 disulphide bridge > No PEGylated INFb1b approved> Rapid clearance from blood stream – frequent administration required> Neutralizing Abs form in 45% of patients > Physical instability

INFa2b

INFb1b

> Site-specific PEGylation of INFb1b

> Homogeneous population, C-terminal conjugation of a single 10 kDa PEG

> INFb1b-PEG has similar activity to non-PEGylated INFb1b

Cytopathic effect inhibition assay using human A549 cells & EMCV. Referenced against WHO calibrated IFNa

Anti-viral activity(CPE inhibition assay using A549 cells & EMCV)

> Site-specific PEGylation of INFa2b

> Homogeneous population, C-terminal conjugation of a single 10 kDa PEG

> INFa2b-PEG has significantly improved activity over the approved non-specifically PEGylated INFa2b therapeutic (Viraferon PEG)

Ant

ivira

l Act

ivity

MIU

/mg

IFN

alph

a2A

ntiv

iral A

ctiv

ity M

IU/m

g IF

Nbe

ta

AlmacSciencesAlmac House20 Seagoe Industrial Estate, Craigavon, BT63 5QD, United Kingdom.T: +44 (0) 28 3833 2200E: sciences@almacgroup.comwww.almacgroup.com

Contact:Robert Grundy, PhDDirector of Commercial Development and Licensing T: +44 (0) 78 2732 2608E: robert.grundy@almacgroup.com

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SUMMARY

> Versatile technology developed for the site-specific engineering and labelling of recombinant proteins > Compatible with disulphide bond containing proteins > Provides a cost-effective, high yielding method for the site- specific C-terminal modification of proteins > High yielding process with low ligand or label equivalents, under aqueous conditions > Platform technology for the engineering and labelling of single domain antibodies > Generic robust technology for the site-specific attachment of small molecules, large polymers and peptides onto a variety of proteins

www.almacgroup.com