almac protein ligation technology webinar presentation 12 09-2012
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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)
• 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
Engineered protein therapeutics
Protein ligation technology
• 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
+
Synthetic molecule recombinant protein
Labelled, semi-synthetic protein
Almac ligation technology
• Express protein of interest fused to an intein domain
• Cleave intein fusion proteins with aqueous hydrazine
• Facile method for the production of recombinant protein C-terminal hydrazides
• Enable chemoselective modification through hydrazone bond forming ligationreactions with aldehydes and ketones
Intercept intein mediated protein splicing
Intein
Intein
N-S acyl shift
NH2NH2
Cotton G. Ligation Method WO200403391
Almac ligation technology
• Express POI fused to an intein domain
• Cleave intein fusion proteins with aqueous dioxyamine reagent
• Facile method for the production of recombinant protein C-terminal aminoxyprotein
• Enable chemoselective modification through oxime bond forming ligationreactions with aldehydes and ketones
Intercept intein mediated protein splicing
Intein
Intein
N-S acyl shift
Cotton G. Ligation Method WO200403391
Versatile technology for site-specific protein modification
Protein-hydrazide
Intein
R
O
FlR
O
PEGR
O
Peptide
Fl PEG Peptide
Protein labelling Polymer modification Peptide ligation
Fluorescent labelling of proteins
Grb2 SH2 – exemplar protein
• Grb2 - 217 amino acid adapter protein linking cell surface growth receptors & Ras signalling pathway – drug target
• SH2 domain mediates binding to autophosphorylated growth factor receptors & tyrosine phosphorylated Shc proteins.
SH3 SH3SH2
Grb2 GNRPP P
RasGDP
RasGTP
regulation of gene expression
Growth factor
Receptor
120 mM NH2NH2, PBS pH 7.4
Recombinant Grb2-SH2 hydrazide
399.0 819.2 1239.4 1659.6 2079.8 2500.0Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100Mass; 12053.0 DaExp; 12051.9.0 Da
ESMS of recombinant Grb2-SH2 C-terminal hydrazideEnzyme digest LC/MS confirms hydrazide at C-terminus
Facile method for the production of C-terminal hydrazide proteins
Time (h)
0 6 25 50 74 144
SH2-Int-CBD
Int-CBD
SH2-hydrazide
Reaction time 4h 24h0h 48h
Grb2-SH2-hydrazide
Green fluorescent
Grb2-SH2-hydrazide
Acetate buffer, pH 4.6
(0.3 mM)
Fl
Site-specifically fluoresceinlabelled Grb2-SH2 domain
Site-specific labelling of Grb2-SH2
• Formation of stabilised alpha-oxo hydrazone bonds
Fl
C-terminal labelled protein fully active in ligand binding assays
0 1 2 3 4
0
10
20
30
40
50
60
70
80
90
One site ligand binding isothermKd = 0.83 ±0.15 µµµµMR2 = 0.985
[TAMRA-phosphopeptide substrate] µM
% C
hang
e in
Em
520
nm
Site-specific protein PEGylation
Site-specific protein PEGylation
IFNα-2b therapeutics
1CDLPQTH SLGSRRTLML LAQMRRISLF S CLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE 165
Receptor binding
Position of PEG in isomers
C-terminus
• 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)
• In vitro anti-viral activity 28% of non-PEGylated form
C-terminal PEGylation applicable to IFNαααα2b
E.Coli expression Oragami(DE3)
Lyse cells – soluble protein
Chitin column
Production of IFN αααα2b – intein fusion CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEG
GyrA CBD
Am
placI
ori
-M13 ori+
pTXB1
1. Mwt markers
2. Purified IFN α2b-intein-CBD
3. IFNα2b hydrazide
27
9766
43
35
20
158
14
56
6
116
212
1 2 3
FOLDED
IFNalpha2b-intein fusion
120 mM NH2NH2, PBS pH 7.40.05% Zwittergent
RP-HPLC
IFNalpha2b-hydrazide
1382.1866
1488.4114
1612.3366
1934.5895
Electrospray MS IFNalpha2b hydrazide(expected mass 19,340 Da)
Intensity
m / z
19,336 Da1758.7379
Generation of folded IFN αααα2b hydrazide
IFNalpha2b-PEG
Site-specific PEGylation of folded IFN αααα2b
O
O
PEG
Pyruvoyl-mPEG (10K)
PEGylation reaction:
10-20 equivs. of PEG reagent
Acidic conditions (0.1% TFA)
25°C (16-20hrs)
Typically 65-75% yield
IFNa2bPEG(10K)
IFNa2b hydrazide
(high mwt PEG contaminant)
27
9766
4335
20
156
6
56
3
212
14
IFNalpha2b hydrazide
NH2NH
O
PEG
Purification of IFN αααα2b-PEG
mAU300
250
200
150
100
50
0
0 5 10 15 20 25 30 ml
mAU2220181614121086420
0 5 10 15 20 25 ml
cont PEG
IFNalpha2bPEG
IFNalpha2b hydrazide control
FT Frs FT Frs
Coomassie PEG stain
Coomassie
Anion exchange (pH7.4, 0.05% Zwittergent)
Gel filtration (PBS pH7.4, 0.05% Zwittergent)
Ion exchange to separate
unreacted pyruvoyl PEG
and IFNα2b-PEG.
Gel filtration to remove
unreacted IFNα2b.
Activity of C-terminal PEGylated IFN αααα2b
0
50
100
150
200
250
300
350
400
450
IFNalpha2bhydrazidelyophile
IFNalpha2b PEGlyophile
IFNalpha2bstandard
ViraferonPEG
Ant
ivira
l Act
ivity
MIU
/mg
IFN
alph
a2 measured
reported
Site-specifically C-terminal PEGylated IFN αααα2b
IFNα2bPEG(10K)
IFNa2b hydrazide
212
156
966656
35
27
20
14
6
3.5
43
Cytopathic effect inhibition assay using human A549 cells &
EMCV. Referenced against ViraferonPEG (PEG-Intron)
n=8
Thom J. et al., Bioconjugate Chem., 2011, 22, 1017
• 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
• Currently no PEGylated versions
of IFNβ-1b approved
H2N-2SYNLLGFL QRSSNFQSQK LLWQLNGRLE Y CLKDRMNFD IPEEIKQLQQ FQKEDAALTI YEMLQNIFAI FRQDSSSTGW 80NETIVENLLA NVYHQINHLK TVLEEKLEKE DFTRGKLMSS LHLKRYYGRI LHYLKAKEYS H CAWTIVRVE ILRNFYFINR LTGYLRN 166-CO2H
IFNββββ-1b therapeutics
0.0
10.0
20.0
30.0
40.0
50.0
IFNbeta1b hydrazidelyophile
IFNbeta1b PEGlyophile
IFNbeta1b standard
Ant
ivira
l Act
ivity
MIU
/mg
IFN
beta
measured
reported
Anti-viral activity (CPE inhibition assay using A549 cells & EMCV)
n=8
10K mPEG
Thom J. et al., Bioconjugate Chem., 2011, 22, 1017
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)
Attractive target for PEGylation• Increase in vivo half life • Reduced immunogenicity and proteolysis• Better targeting to tumour tissues (Enhanced
Permeability and Retention effect)
Single-domain antibody fragments
Wesolowski J., et al, Med Microbiol. Immunol., 2009, 198, 157Camel Ig
VHH domain(nanobody)
N
C
Classic Ig
1-chitin beads before cleavage2-chitin beads after cleavage3-eluted cleaved protein
1 2 3 M
27kDa
14.3kDa
sdAb hydrazide
sdAb-intein fusion
200 mM NH2NH2, PBS pH 6.9
o/n 24°°°°C
sdAb-hydrazide
Generation of sdAb hydrazide
mPEG (20K)
PEG 20kDa sdAb
0 24 48 72 Time [h]
66
56
43
35
27
20
14
97
sdAb-hydrazide
10 mM aniline, pH 5.5
Ligation of 20 KDa PEG to sdAb with ~90 % yield
n
n
Site-specific PEGylation of anti-EGFR sdAb
time (hours)
PE
Gyl
ated
sdA
b [%
]
0 5 10 15 200
20
40
60
80
100
1:11:5
24
Activity of C-terminal PEGylated anti-EGFR sdAb
66
4335
272014
56
1. PEGylated EGFR sdAb with
reduced hydrazone bond
2. PEGylated EGFR sdAb
3. EGFR sdAb-hydrazide
4. Mwt markers
1 2 3 4
PEGylated EGFR sdAb inhibits binding of radiolabelled [ 125I] EGF to EGFR
20K mPEG
PEG EGFR sdAb
C-terminal PEGylation of sdAbs maintains full activity of protein
-12 -10 -8 -6 -4
0
20
40
60
80
100EGFR sdAb hydrazide, IC
PEG 20KDa EGFR sdAb,IC
Log of EGFR sdAb concentration [M]
Bin
ding
of [
125 I]
EG
F [%
]EGFR sdAb hydrazide, IC50=15 nM
PEG 20KDa EGFR sdAb,IC50=18 nM
PK of C-terminal PEGylated anti-EGFR sdAb
20K mPEG
PEG EGFR sdAbSdAb-PEG1
iv - dose 10 µµµµg per mouse
70 fold increase in in vivo half-lifeChemistry applicable for in vivo applications
0 5 10 150
1000
2000
3000
4000
5000
Time [h]C
once
ntra
tion
[ng/
ml]
SdAb t 1/2 ~3.7 minutes
SdAb-PEG1 t 1/2 ~4.3 hours
Periplasmic expression of sdAb-Intein fusion proteins
Production of sdAb – intein fusion through periplasmic expression
E.Coli expression (BL 21 (DE3))
18°C overnight, 0.1 mM IPTG
Lyse cells (osmotic shock)
soluble protein
GyrA CBD
Am
placI
ori
-M13 ori+
pTXB1
PelB sdAb
Ratio Protein:PEG
Time (h) 0 24 48 72 0 24 48 72
66 kDa
14.4 kDa
1:1 1:5
20K mPEG
HN O
O
O
O
H
PEGylatedsdAb
sdAbhydrazide
pH 5.5 at 18 ºC10 mM aniline80 µµµµM sdAb
Site-specific C-terminal PEGylation of sdAbexpressed in periplasm
High yield of C-terminal PEGylated protein (20K mPEG) using 1 equivs.
High yielding with 1:1 protein:PEG
Secreted expression of sdAb-Intein fusion proteins from
yeast followed by site-specific C-terminal PEGylation
A collaboration with VTU
Production of sdAb – intein fusion through secreted yeast expression ( Pichia pastoris)
Secreted expression from Pichia Pastoris
Media containing sdAb-Intein-polyHis(desired fusion protein produced at 1 g / L using s mall scale fermentation)
Immobilisation of protein on IMAC column(>90% recovery of sdAb-intein fusion protein from the media)
polyHissdAb Intein
GyrA
CBD
Am
placI
ori
-M13 ori+
Yeast ExpressionVector
sdAb-intein fusion
(i) Elute from column
(ii) NH2NH2, PBS pH 6.9
o/n 24°CsdAb-hydrazide
Generation and PEGylation of sdAb-hydrazide
Intein polyHis IMAC
20K mPEG
mPEG (20K)
10 mM aniline, pH 5.5
n
EGFR sdAb-PEG(20K)
Coomassie PEGstain
High yielding, site-specific PEGylation technology for proteins generated through secreted expression from yeast
sdAb-intein fusionC-terminal aminoxy sdAb
PEGylation of C-terminal aminoxy sdAb
• Rapid site-specific C-terminal PEGylationusing low number of PEG equivalents
(80% C-terminal PEGylation after 15 mins)
• Overall isolated yield from un-purified intein-fusion protein in Pichia Pastoris
media to pure isolated C-terminal
PEGylated sdAb is > 60%
mPEG (20K)
10 mM aniline, pH 5.5
n
M 1 2 3 M 1 2 3
A) B)
66kDa 66kDa
14.3kDa 14.3kDaEGFR sdAb-aminoxy
PEGylated EGFR sdAb34.6kDa34.6kDa
M 1 2 3M 1 2 3 M 1 2 3
A) B)
66kDa 66kDa
14.3kDa 14.3kDaEGFR sdAb-aminoxy
PEGylated EGFR sdAb34.6kDa34.6kDa
(A) Monitoring of PEGylation reaction at 15 min (lane 1), 6h (lane 2) 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 PEG stain (lane 3).
Engineering bispecificproteins
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
Bispecific protein therapeutics
Bi-specific protein therapeutics with novel defined topologies
• Almac ligation technology is bio-orthogonal to other conjugation chemistries
• Enabling the development of bi-specific protein constructs
LinkerC-terminal aminoxy protein C-terminal thiol protein
Linker
Linker
Green-3rd domain involved in FcRn bindingBlue-Cys34 with free sulfhydryl group in yellow.
ID:2BXG.pdbHuman Albumin (Sugio et al., Prot.Eng.,1999, 439-446)
-Cysteine 34 is on the surface of albumin molecule but the sulfhydrylis 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.
Novel Bifunctional linker for site-specific protein-protein conjugation
.
Half life extension – HSA conjugation
Linker
Generation of sdAb-maleimide
C-terminal maleimide protein
Linker
pH 5.5
sdAb-intein fusion C-terminal aminoxy sdAb
Intein CBD
PBS pH 7.0
1 2 M
14.6KDa
27KDa
1-EGFRsdAb-aminoxy2-EGFR sdAb coupled to linker via oxime bond
ESI-MS of sdAb-maleimide
13775.2
130000100020003000400050006000
Intens.
13500 14000 14500 15000m/z
Expected = 13774.0 DaLinker
Linker
Generation of HSA-sdAb (C-terminal to side chain)
+
Ratio of EGFR sdAb-linker to Albumin: 1.5 : 1
EGFR sdAb-linker
Albumin
EGFR sdAb-linker-Albumin conjugate
14.6 KDa
66 KDa
96 KDa
EGFR sdAb-linker
Albumin
EGFR sdAb-linker-Albumin conjugate
14.6 KDa
66 KDa
96 KDa
Ratio of EGFR sdAb-linker to Albumin: 1 : 1.5
0 24h 0 24h
PBS pH 6.0
Linker
Linker
HSA - benzaldehyde
PBS pH 6.0
66774.9
0
2000
4000
6000
8000
Intens.
62000 63000 64000 65000 66000 67000 68000 m/z
66596.266934.9
ESI-MS of human serum albumin coupled to linker
Expected Mass 66788.0 Da
Albumin-linker
Generation of HSA-sdAb (C-terminal to side chain)
+
HN
N
O
O
O
S
O
Ratio of Albumin-linker to EGFR sdAb-aminoxy: 1:5
pH 5.5
Control
EGFR sdAb
Albumin linkeror Albumin (control)
0 1 2 4 24 h 0 1 2 4 24 h
EGFR sdAb-linker-Albumin conjugate
Facile approach for site-specific protein derivatisation
enabling bio-orthogonal protein engineering
sdAb-intein fusion C-terminal thiol sdAb
Generation of C-terminal ‘thiol’ sdAb
Intein Tag
• Direct cleavage of resin bound his-tagged intein-fusion protein with cysteamine under
physiological conditions
• High yielding, chemoselective approach to generating C-terminal thiol proteins
PBS pH 7.0
Quantitative ‘on-resin’ cleavage of the inteinfusion protein to generate highly pure C-terminal thiol sdAb
B0 B E B E
24h 48h
EGFR sdAb_Npu_CBD
EGFR sdAb-cysteamine
sdAb-intein fusion C-terminal alkyne sdAb
Generation of C-terminal alkyne sdAb
Intein Tag
• Direct cleavage of resin bound his-tagged intein-fusion protein with aminoxy-propyne
under physiological conditions.
• High yielding, chemoselective approach to generating C-terminal alkyneproteins
PBS pH 7.0
Quantitative ‘on-resin’ cleavage of the inteinfusion protein to generate highly pure C-terminal alkyne sdAb
R0h R24hE24hR48hE48h
EGFR sdAb-propynyl-hydroxylamine
Bi-specific proteins through ‘Click’ chemistry
+
Application in molecular imaging
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
Protein-based imaging agents
Intein
DOTA
Metal Chelators Radiolabelling
Site-specific labelling with 18F for PET imaging applications.Fast reaction under aqueous buffered conditions enables site-specific protein labelling within minutes.
Site-specific labelling of recombinant protein with metal chelating groups for PET / SPECT imaging applications.
18F
Rapid, high yielding protein fluorination using
2 equivalents of label
aqueous buffer, pH 5.5
Illustrative example - site-specific Fluorination of recombinant proteins
C-terminal modified protein generated using intein cleavage methodology
Rapid, high yielding fluorination using 2 equivalents of fluoro-label to protein
F
F
Site-specific incorporation of Fluorine at the C-terminus of proteins
• 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
Protein Drug Conjugates
• 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
Almac protein ligation technology - Summary
Thank You
Please contact:Robert Grundy PhDDirector of Commercial Development and LicensingTel: + 44 (0) 7827322608robert.grundy@almacgroup.comwww.almacgroup.com
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