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