This document discusses site-specific protein modification using Almac's proprietary protein ligation technology. The technology enables proteins to be modified at their C-terminus in a highly selective and high-yielding manner, resulting in homogeneous conjugated protein products with retained biological activity. This overcomes limitations of existing nonspecific protein modification methods. The technology has been demonstrated on various therapeutic proteins and can attach a wide range of labels and molecules like PEG for half-life extension.

1. Site Specific Protein Modification
Conjugate the smart way
> 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
Delivering improved peptide and protein therapeutics
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Chemical Development
www.almacgroup.com

2. Overview
Biotherapeutics Market Current shortcomings
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The biotherapeutics’ industry has boomed in recent Established protein labelling technologies generally result
years with an estimated valuation of US$145 billion in the non-selective and non-specific modification of the
in 2014. Increasingly protein engineering technologies target proteins. This yields heterogeneous populations
are being used to develop protein therapeutics with often with compromised biological activity, and large
improved performance and to enable novel therapeutic batch to batch variations in their production. Alternative
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approaches to be realised using protein-based drugs. approaches rely on insertion of additional amino acid
residues into the sequence, which again can
Protein engineering has a critical role to play in many potentially compromise biological activity and may lead to
aspects of drug development including protein unwarranted issues with protein stability and immunogenicity.
PEGylation, antibody-drug conjugates, bi-specific
therapeutics and molecular imaging agents. Moreover, high label-to-protein stoichiometries may be
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Consequently, to tackle the current and future required to give the required yields of the modified protein,
challenges, and provide solutions to the drug leading to an increased cost of goods. Therefore there is
discovery and healthcare industries, there is a continued a requirement for robust, high yielding technologies for
requirement for improved protein engineering technologies. the site-specific labelling and modification of proteins.
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Providing Solutions
The Almac Advantage
Almac’s proprietary protein engineering
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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.
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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.
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As examples:
> Grb2-SH2
> INFa2b
> INFb1b
> Evasin-3
> FKBPL
> Range of single domain antibodies
> Chemokines

3. Almac’s proprietary ligation technology
Commercial Services
> 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 > This thioester intermediate is
protein-intein junction to form the thioester intermediate. chemically cleaved under aqueous buffered
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conditions using hydrazine or dioxyamine to
liberate the corresponding C-terminal
hydrazide and aminoxy derivatives of the
target protein.
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> The C-terminal derivatives
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
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linkage.
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Key advantages of our technology
> Targeted, site specific ligation at the C-terminus –
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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
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proteins
> Applicable to a wide range of labels and
modifications
> Single modification site means homogeneous
products produced
High efficiency - High yield

4. Site-Specific protein ligation applications
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Protein PEGylation
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Site-specifically
PEGylated protein
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 Protein labelling
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Aqueous buffer
Site-specifically fluorescein
labelled protein
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Imaging applications
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Rapid, high yielding protein fluorination
using 2 equivalents of label
Fluorinated protein
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 Targeted cytotoxics
Protein-drug conjugate (e.g. ADCs)
Aqueous buffer

5. Bi-Specific proteins
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Bi-functionalised linker
Aqueous buffer
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Protein 1
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Protein 2
Bi-specific molecule
Peptide conjugation
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c-myc peptide
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Aqueous buffer Site-specifically peptide
conjugated protein
Technology compatible with prokaryotic and eukaryotic expression
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Expression in Echerichia coli Expression in Pichia pastoris
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> Cytoplasmatic expression of Evasin 3- intein in > Secreted expression of EGFR sdAb-intein
bacteria from yeast
> Intein cleavage followed by site specific C-terminal > Intein cleavage followed by site specific
PEGylation of target protein C-terminal PEGylation of target protein
> Technology also compatible with periplasmic
expression of the intein-fusion proteins

6. PEGylation of antibody fragments: anti-EGFR single domain antibody
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Antibody fragments Attractive target for PEGylation
> Nanobodies are the smallest available intact antigen > Increase in vivo half life
binding domains > Reduced immunogenicity and proteolysis
> 120-130 amino acids > Better targeting to tumour tissues
> Contain 1 or 2 conserved disulphide bridges (Enhanced permeability and retention effect)
> Rapid clearance from blood (half life ~10 mins)
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Labelling is high yielding and cost-effective
PEGylation reaction is fast and high yielding
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PEGylated sdAb [%]
Aqueous buffer
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PEGylated protein maintains full activity
Binding of [125I] EGF [%]
> Ligation reaction is very efficient, >80%
yield even with a 1:1 protein to PEG ratio
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> Full activity of single domain anti
bodies is maintained after C-terminal
PEGylation
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PK properties are improved by Almac PEGylation technology
> Mild reaction conditions
Concentration [ng/ml]
> Without modification, the anti-EGFR
single domain antibody has a half-life
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of just 3.7 minutes in mice
> PEGylation increases the half-life by
approximately 70 fold, to 4.3 hours

7. PEGylation of protein therapeutics: INFa2b & INFb1b
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INFa2b INFb1b
> 165 aa residues – 2 disulphide bridges > 165 aa residues – 1 disulphide bridge
> PEG-Intron® (Schering Plough) has been > No PEGylated INFb1b approved
approved to treatment of Hepatitis C: > Rapid clearance from blood stream – frequent
> non-selective PEGylation (12 kDa linear) administration required
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> 14 PEG positional isomers > Neutralizing Abs form in 45% of patients
> in vitro antiviral activity is 28% of non-modified > Physical instability
form
INFa2b
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> Site-specific PEGylation of
INFa2b
Antiviral Activity MIU/mg IFNalpha2
> Homogeneous population,
C-terminal conjugation of
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a single 10 kDa PEG
> INFa2b-PEG has significantly
improved activity over the
approved non-specifically
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PEGylated INFa2b therapeutic
Cytopathic effect inhibition assay using human A549 (Viraferon PEG)
cells & EMCV. Referenced against WHO calibrated IFNa
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INFb1b
Anti-viral activity
(CPE inhibition assay using A549 cells & EMCV) > Site-specific PEGylation of
INFb1b
Antiviral Activity MIU/mg IFNbeta
> Homogeneous population,
C-terminal conjugation of
a single 10 kDa PEG
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> INFb1b-PEG has similar
activity to non-PEGylated
INFb1b

8. www.almacgroup.com
Commercial Services
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SUMMARY
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> 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
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> High yielding process with low ligand or label
equivalents, under aqueous conditions
> Platform technology for the engineering and labelling of
single domain antibodies
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> Generic robust technology for the site-specific attachment of
small molecules, large polymers and peptides onto a variety
of proteins
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Biomarker Discovery & Development
Almac Contact:
Sciences Robert Grundy, PhD
Almac House Director of Commercial Development
20 Seagoe Industrial Estate, and Licensing
Craigavon, BT63 5QD, T: +44 (0) 78 2732 2608
United Kingdom. E: robert.grundy@almacgroup.com
T: +44 (0) 28 3833 2200
E: sciences@almacgroup.com
www.almacgroup.com