1. Site-specific protein labelling: Almac’s unique
conjugation technology enabling protein
conjugation for a wide range of medical
applications
Almac Webinar 12th September 2012 16.00 (UK time)

2. Engineered protein therapeutics
• Targeted drug delivery (ADC)
ADCETRIS(Seattle Genetics) – Hodgkin Lymphoma
Conjugate of anti-CD30 Ab and vcMMAE
Casi G & Neri D., J. Con. Release, 2012, 161, 422
• Pharmacokinetic enhancement through PEGylation
Cimzia (UCB) – Crohn’s disease
PEGylated anti-TNF antibody fragment
Melmed GY., et al, Nature Rev. Drug. Discovery., 2008, 7, 641
• Molecular imaging agents
Cu64 labelled diabody for PET imaging of solid tumours
Increased tumour uptake through engineering
Lin L., et al, Bioconjugate Chem., 2011, 22, 709

3. Protein ligation technology
+
Synthetic molecule recombinant protein
Labelled, semi-synthetic protein
• Chemoselective ligation of recombinant and synthetically derived moieties under
aqueous conditions
• Enables the site-specific incorporation of synthetic moieties into recombinant
proteins for different applications
• Proprietary protein ligation technologies developed by Almac
• Platform technology for site-specific conjugation of synthetic molecules to the C-
terminus of recombinant proteins

4. Almac ligation technology
Intercept intein mediated protein splicing
• Express protein of interest fused to an
intein domain
• Cleave intein fusion proteins with aqueous
Intein hydrazine
• Facile method for the production of
N-S acyl shift recombinant protein C-terminal hydrazides
• Enable chemoselective modification
through hydrazone bond forming ligation
Intein
reactions with aldehydes and ketones
NH2NH2
Cotton G. Ligation Method WO200403391

5. Almac ligation technology
Intercept intein mediated protein splicing
• Express POI fused to an intein domain
• Cleave intein fusion proteins with aqueous
dioxyamine reagent
Intein
• Facile method for the production of
recombinant protein C-terminal aminoxy
N-S acyl shift protein
• Enable chemoselective modification
through oxime bond forming ligation
Intein
reactions with aldehydes and ketones
Cotton G. Ligation Method WO200403391

6. Versatile technology for site-specific protein
modification
Intein
Protein-hydrazide
O O
R Fl O R Peptide
R PEG
Fl PEG Peptide
Protein labelling Polymer modification Peptide ligation

7. Fluorescent labelling of
proteins

8. Grb2 SH2 – exemplar protein
• Grb2 - 217 amino acid adapter protein linking cell surface
growth receptors & Ras signalling pathway – drug target
Growth factor
Receptor
P P
Grb2 GNRP
Ras Ras regulation of
GDP GTP gene expression
SH3 SH2 SH3
• SH2 domain mediates binding to autophosphorylated growth
factor receptors & tyrosine phosphorylated Shc proteins.

9. Recombinant Grb2-SH2 hydrazide
120 mM NH2NH2,
PBS pH 7.4
Time (h)
0 6 25 50 74 144
100
90 Mass; 12053.0 Da
80 Exp; 12051.9.0 Da
70
60
50
SH2-Int-CBD 40
30
Int-CBD 20
10
0
399.0 819.2 1239.4 1659.6 2079.8 2500.0
Mass (m/z)
SH2-hydrazide
ESMS of recombinant Grb2-SH2 C-terminal hydrazide
Enzyme digest LC/MS confirms hydrazide at C-terminus
Facile method for the production of C-terminal hydrazide proteins

10. Site-specific labelling of Grb2-SH2
• Formation of stabilised alpha-oxo hydrazone bonds
Fl (0.3 mM)
Fl
Acetate buffer, pH 4.6
Grb2-SH2-hydrazide Site-specifically fluorescein
labelled Grb2-SH2 domain
90
80
% Change in Em520 nm
70
60
50
Green 40
fluorescent 30
20
One site ligand binding isotherm
10 Kd = 0.83 ±0.15 µM
Reaction time 0h 4h 24h 48h 0
R2 = 0.985
0 1 2 3 4
Grb2-SH2-hydrazide [TAMRA-phosphopeptide substrate] µM
C-terminal labelled protein fully active in ligand binding assays

11. Site-specific protein
PEGylation

12. Site-specific protein PEGylation

13. IFNα-2b therapeutics
1CDLPQTH SLGSRRTLML LAQMRRISLF SCLKDRHDFG FPQEEFGNQF QKAETIPVLH
EMIQQIFNLF STKDSSAAWD ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL
AVRKYFQRIT LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE165
• IFNα2b is 165 amino acid protein – 2 disulphide bonds
• PEG-Intron® (Schering Plough)
• IFNα2b non-selectively PEGylated with 12 KDa linear PEG
• 14 PEG positional isomers
• Half-life in humans is 40 hrs (cf Intron® A, T1/2 ~4-7h)
C-terminus
• In vitro anti-viral activity 28% of non-PEGylated form
C-terminal PEGylation applicable to IFNα2b
Receptor binding
Position of PEG in isomers

14. α
Production of IFNα2b – intein fusion
CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLH
EMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDS
ILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG
Gyr
A
CB
D
Amp -
lacI
pTXB1
M1
3o
ri+
ori
E.Coli expression Oragami(DE3)
Lyse cells – soluble protein
Chitin column

15. α
Generation of folded IFNα2b hydrazide
120 mM NH2NH2,
PBS pH 7.4
0.05% Zwittergent
RP-HPLC
IFNalpha2b-intein fusion IFNalpha2b-hydrazide
212
158 Electrospray MS IFNalpha2b hydrazide
116 FOLDED
97 (expected mass 19,340 Da)
66
56 Intensity
1758.7379
43
19,336 Da
35
1612.3366
27
1488.4114
1934.5895
20
1. Mwt markers
14 1382.1866
2. Purified IFNα2b-intein-CBD
6
3. IFNα2b hydrazide m/z
1 2 3

16. α
Site-specific PEGylation of folded IFNα2b
O
Pyruvoyl-mPEG (10K)
O
NH NH2 PEG
PEG
O
IFNalpha2b hydrazide IFNalpha2b-PEG
212
156 PEGylation reaction:
97
66
56 (high mwt PEG contaminant)
10-20 equivs. of PEG reagent
43
35 IFNa2bPEG(10K) Acidic conditions (0.1% TFA)
27
20 25° (16-20hrs)
C
IFNa2b hydrazide
14
6
Typically 65-75% yield
3

17. α
Purification of IFNα2b-PEG
Anion exchange (pH7.4, 0.05% Zwittergent)
mAU
300
Coomassie PEG stain
250
Ion exchange to separate
200 unreacted pyruvoyl PEG
150 and IFNα2b-PEG.
100
50
FT Frs FT Frs
0
0 5 10 15 20 25 30 ml
Gel filtration (PBS pH7.4, 0.05% Zwittergent)
mAU IFNalpha2bPEG Coomassie
22 Gel filtration to remove
20
18
IFNalpha2b
unreacted IFNα2b.
16
14 hydrazide control
12
10
8
6 cont PEG
4
2
0
0 5 10 15 20 25 ml

18. α
Activity of C-terminal PEGylated IFNα2b
α
Site-specifically C-terminal PEGylated IFNα2b
n=8
450
Antiviral Activity MIU/mg IFNalpha2
400 measured
reported
212 350
156 300
96
66 250
56
43 200
35 IFNα2bPEG(10K)
150
27
100
20
14 IFNa2b hydrazide 50
6 0
3.5 IFNalpha2b IFNalpha2b PEG IFNalpha2b ViraferonPEG
hydrazide lyophile standard
lyophile
Cytopathic effect inhibition assay using human A549 cells &
Thom J. et al., Bioconjugate Chem., 2011, 22, 1017
EMCV. Referenced against ViraferonPEG (PEG-Intron)

19. β
IFNβ-1b therapeutics
H2N-2SYNLLGFL QRSSNFQSQK LLWQLNGRLE YCLKDRMNFD IPEEIKQLQQ FQKEDAALTI
YEMLQNIFAI FRQDSSSTGW 80NETIVENLLA NVYHQINHLK TVLEEKLEKE DFTRGKLMSS
LHLKRYYGRI LHYLKAKEYS HCAWTIVRVE ILRNFYFINR LTGYLRN166-CO2H
• IFNβ−1b is 165 amino acid protein – 1 disulphide bond
• Betaseron® / Betaferon® (Bayer / Schering) - Treatment of MS
• Rapid clearance from blood stream → frequent administration required
• Neutralizing Abs form in 45% of patients
• Physical instability Anti-viral activity
(CPE inhibition assay using A549 cells & EMCV)
• Currently no PEGylated versions
50.0 n=8
of IFNβ-1b approved
Antiviral Activity MIU/mg IFNbeta
measured
reported
40.0
30.0
20.0
10K mPEG 10.0
0.0
IFNbeta1b hydrazide IFNbeta1b PEG IFNbeta1b standard
lyophile lyophile
Thom J. et al., Bioconjugate Chem., 2011, 22, 1017

20. Single-domain antibody fragments
N
Antibody fragments
• Nanobodies are the smallest available intact
antigen binding fragment
• 120-130 amino acids
• Contain 1 conserved disulphide bridge
• Increased tissue penetration
• Rapid clearance from blood (half life ~10 min)
C
VHH domain
(nanobody)
Attractive target for PEGylation
• Increase in vivo half life
• Reduced immunogenicity and proteolysis
• Better targeting to tumour tissues (Enhanced
Permeability and Retention effect)
Wesolowski J., et al, Med Microbiol. Immunol., 2009, 198, 157
Classic Ig Camel Ig

21. Generation of sdAb hydrazide
200 mM NH2NH2,
PBS pH 6.9
°
o/n 24°C
sdAb-intein fusion sdAb-hydrazide
1 2 3 M
1-chitin beads before cleavage
2-chitin beads after cleavage
3-eluted cleaved protein
27kDa
14.3kDa
sdAb hydrazide

22. Site-specific PEGylation of anti-EGFR sdAb
mPEG (20K)
n
10 mM aniline, pH 5.5
n
97 100
PEGylated sdAb [%]
66 80
56
PEG 20kDa sdAb 60
43
1:1
35 40
1:5
27 20
20
0
0 5 10 15 20 24
14 sdAb-hydrazide
time (hours)
0 24 48 72
Time [h] Ligation of 20 KDa PEG to sdAb with ~90 % yield

23. Activity of C-terminal PEGylated anti-EGFR sdAb
20K mPEG
PEGylated EGFR sdAb inhibits binding of
PEG EGFR sdAb radiolabelled [125I] EGF to EGFR
Binding of [125I] EGF [%]
100
12 34
EGFR sdAb hydrazide, IC
66 80
56 PEG 20KDa EGFR sdAb,IC
43 60
35
40
27
20 20
14
0
1. PEGylated EGFR sdAb with -12 -10 -8 -6 -4
reduced hydrazone bond Log of EGFR sdAb concentration [M]
2. PEGylated EGFR sdAb
EGFR sdAb hydrazide, IC50=15 nM
3. EGFR sdAb-hydrazide
PEG 20KDa EGFR sdAb,IC50=18 nM
4. Mwt markers
C-terminal PEGylation of sdAbs maintains full activity of protein

24. PK of C-terminal PEGylated anti-EGFR sdAb
20K mPEG
5000
Concentration [ng/ml]
iv - dose 10 µg per mouse
4000
PEG EGFR sdAb
SdAb-PEG1 3000
2000
1000
0
0 5 10 15
Time [h]
SdAb t1/2 ~3.7 minutes
SdAb-PEG1 t1/2 ~4.3 hours
70 fold increase in in vivo half-life
Chemistry applicable for in vivo applications

25. Periplasmic expression of
sdAb-Intein fusion proteins

26. Production of sdAb – intein fusion through
periplasmic expression
PelB sdAb
Gyr
A
CB
D
Amp -
lacI
pTXB1
M1
3o
ri+
ori
E.Coli expression (BL 21 (DE3))
18° overnight, 0.1 mM IPTG
C
Lyse cells
(osmotic shock)
soluble protein

27. Site-specific C-terminal PEGylation of sdAb
expressed in periplasm
O 20K mPEG pH 5.5 at 18 ºC
H 10 mM aniline
H
N O 80 µM sdAb
O
O Ratio Protein:PEG
1:1 1:5
Time (h) 0 24 48 72 0 24 48 72
66 kDa PEGylated
sdAb
sdAb
14.4 kDa hydrazide
High yielding with 1:1 protein:PEG
High yield of C-terminal PEGylated protein (20K mPEG) using 1 equivs.

28. Secreted expression of sdAb-
Intein fusion proteins from
yeast followed by site-specific
C-terminal PEGylation
A collaboration with VTU

29. Production of sdAb – intein fusion through
secreted yeast expression (Pichia pastoris)
sdAb Intein polyHis
Gyr
A
CB
D
Amp -
Yeast
lacI
Expression
Vector
M1
3o
ri+
ori
Secreted expression from Pichia Pastoris
Media containing sdAb-Intein-polyHis
(desired fusion protein produced at 1 g / L using small scale fermentation)
Immobilisation of protein on IMAC column
(>90% recovery of sdAb-intein fusion protein from the media)

30. Generation and PEGylation of sdAb-hydrazide
(i) Elute from column
Intein polyHis IMAC
(ii) NH2NH2, PBS pH 6.9
o/n 24°
C
sdAb-intein fusion sdAb-hydrazide
EGFR sdAb-PEG(20K)
mPEG (20K)
n
10 mM aniline, pH 5.5
20K mPEG
Coomassie PEG
stain
High yielding, site-specific PEGylation technology for
proteins generated through secreted expression from yeast

31. PEGylation of C-terminal aminoxy sdAb
mPEG (20K)
n
10 mM aniline, pH 5.5
C-terminal aminoxy sdAb sdAb-intein fusion
A) B)
M 1 2 3 M 1 2 3
• Rapid site-specific C-terminal PEGylation
66kDa 66kDa
using low number of PEG equivalents
PEGylated EGFR sdAb
34.6kDa 34.6kDa
(80% C-terminal PEGylation after 15 mins)
14.3kDa 14.3kDa
EGFR sdAb-aminoxy
• Overall isolated yield from un-purified
intein-fusion protein in Pichia Pastoris
(A) Monitoring of PEGylation reaction at 15 min (lane 1), 6h (lane 2) media to pure isolated C-terminal
and 24h (lane 3) on gel stained with Coomassie stain.
(B) EGFR sdAb-aminoxy (lane 1) stained with Coomassie stain and
PEGylated EGFR sdAb stained with Coomasie stain (lane 2) or
PEGylated sdAb is > 60%
PEG stain (lane 3).

32. Engineering bispecific
proteins

33. Resurgence of bi-specific antibodies
• First bispecific antibody (Removab) obtains market approval 2009
• Number of different modes of action now being exploited with bi-
specific approaches
Redirecting cytotoxic cells of the immune system
Binding to two different ligands
Bidentate interactions with one target
Receptor cross-linking
• Many different formats and technology platforms
Amgen (Micromet)
F-Star
Macrogenics
Zymeworks
Current platforms based on genetic fusion of antigen binding domains

34. Bispecific protein therapeutics
• Almac ligation technology is bio-orthogonal to other conjugation chemistries
• Enabling the development of bi-specific protein constructs
Linker
C-terminal aminoxy protein Linker C-terminal thiol protein
Linker
Bi-specific protein therapeutics with novel defined topologies

35. Half life extension – HSA conjugation
-Cysteine 34 is on the surface of albumin molecule but the sulfhydryl
is pointed into the interior. It is located in the 1st domain of albumin.
-Calculated distance of cysteine34 sulfhydryl group to the surface of
albumin is 10-15Å.
ID:2BXG.pdb Novel Bifunctional linker for site-specific protein-protein conjugation
Human Albumin (Sugio et al., Prot.Eng.,1999, 439-446)
.
Green-3rd domain involved in FcRn binding
Blue-Cys34 with free sulfhydryl group in yellow.
Linker

36. Generation of sdAb-maleimide
PBS pH 7.0
Intein CBD
sdAb-intein fusion C-terminal aminoxy sdAb
1 2 M Linker
ESI-MS of sdAb-maleimide
Intens
.
13775.2 Linker
6000
5000
4000 pH 5.5
3000 27KDa
2000
1000
0 13000 13500 14000 14500 15000 14.6KDa
m/z
Linker
Expected = 13774.0 Da 1-EGFRsdAb-aminoxy
2-EGFR sdAb coupled to
linker via oxime bond
C-terminal maleimide protein

37. Generation of HSA-sdAb (C-terminal to side chain)
PBS pH 6.0 Linker
+
Linker
Ratio of EGFR sdAb-linker to Albumin: 1.5 : 1 Ratio of EGFR sdAb-linker to Albumin: 1 : 1.5
EGFR sdAb-linker-Albumin 96 KDa EGFR sdAb-linker-Albumin 96 KDa
conjugate conjugate
66 KDa 66 KDa
Albumin Albumin
EGFR sdAb-linker 14.6 KDa EGFR sdAb-linker 14.6 KDa
0 24h 0 24h

38. HSA - benzaldehyde
PBS pH 6.0
Intens.
66774.9
8000
Expected Mass 66788.0 Da
Albumin-linker 6000
4000 66934.9
66596.2
2000
0
62000 63000 64000 65000 66000 67000 68000 m/z
ESI-MS of human serum albumin coupled to linker

39. Generation of HSA-sdAb (C-terminal to side chain)
O
O
S H
N
N
O
O
pH 5.5
+
Ratio of Albumin-linker to EGFR sdAb-aminoxy: 1:5
Control
0 1 2 4 24 h 0 1 2 4 24 h
EGFR sdAb-linker-Albumin
conjugate
Albumin linker
or Albumin (control)
EGFR sdAb

40. Facile approach for site-
specific protein derivatisation
enabling bio-orthogonal
protein engineering

41. Generation of C-terminal ‘thiol’ sdAb
PBS pH 7.0
Intein Tag
sdAb-intein fusion C-terminal thiol sdAb
24h 48h
B0 B E B E
• Direct cleavage of resin bound his-tagged
intein-fusion protein with cysteamine under
physiological conditions
EGFR sdAb_Npu_CBD
• High yielding, chemoselective approach
EGFR sdAb-cysteamine
to generating C-terminal thiol proteins
Quantitative ‘on-resin’ cleavage of the intein
fusion protein to generate highly pure C-
terminal thiol sdAb

42. Generation of C-terminal alkyne sdAb
PBS pH 7.0
Intein Tag
sdAb-intein fusion C-terminal alkyne sdAb
R0h R24hE24hR48hE48h
• Direct cleavage of resin bound his-tagged
intein-fusion protein with aminoxy-propyne
under physiological conditions.
EGFR sdAb-propynyl-hydroxylamine
• High yielding, chemoselective approach
to generating C-terminal alkyne
Quantitative ‘on-resin’ cleavage of the intein proteins
fusion protein to generate highly pure C-
terminal alkyne sdAb

43. Bi-specific proteins through ‘Click’ chemistry
+

44. Application in
molecular imaging

45. Imaging applications of protein technologies
• Development of in vivo imaging agents for medical diagnostics
Radiolabelled protein targeted to pathologically important molecule
Development as a diagnostic or prognostic imaging product
• Development of imaging agents for drug development
Biodistribution & Pharmacokinetics
- labelled protein therapeutic
Development of pharmacological tools
- For use in displacement studies
Pharmacodynamic markers
- Imaging biological event relevant to the PD effect of a therapeutic

46. Protein-based imaging agents
Intein
DOTA 18F
Metal Chelators Radiolabelling
Site-specific labelling of recombinant Site-specific labelling with 18F for PET
protein with metal chelating groups imaging applications.
for PET / SPECT imaging Fast reaction under aqueous buffered
applications. conditions enables site-specific protein
labelling within minutes.

47. Illustrative example - site-specific Fluorination of
recombinant proteins
F
aqueous buffer, pH 5.5
F
C-terminal modified protein generated Site-specific incorporation of Fluorine at
using intein cleavage methodology the C-terminus of proteins
Rapid, high yielding protein fluorination using
Rapid, high yielding fluorination
2 equivalents of label
using 2 equivalents of fluoro-label
to protein

48. Protein Drug Conjugates
• Area of significant interest
• Clear clinical and commercial potential
• Technology applicable to the development of ADCs
Homogeneous product
Site specificity
Retained biological function
• Focus of current R&D activities

49. Almac protein ligation technology - Summary
• Versatile technology developed for the site-specific (C-terminal selective)
engineering and labelling of recombinant proteins
• Allows total control over the ligation process
• Retains biological activity – has the potential to improve upon the native protein
i.e. for protein PEGylation by extending half life while maintaining the activity
• Provides a cost-effective, high yielding method for the site-specific C-terminal
labelled proteins
• High yielding process with low equivalents of label under aqueous conditions
• Generic robust technology for the site-specific attachment of small molecules,
large polymers and peptides onto proteins
Compatible with disulphide bond containing proteins
Compatible with cytosolic and periplasmic E.coli expression
Compatible with secreted expression from Pichia pastoris
• Complementary to other bio-orthogonal chemistries – enable bispecific proteins /
multivalent proteins to be constructed and engineered

50. Thank You
Please contact:
Robert Grundy PhD
Director of Commercial Development and Licensing
Tel: + 44 (0) 7827322608
robert.grundy@almacgroup.com
www.almacgroup.com