Designing an SPR biointerface for transmembrane proteins

Heather FergusonMatthew Linman, Dr. Quan “Jason” Cheng

BRITE Research Presentation

August 20, 2009

Outline

Background informationSPR and EGFR

Experimental approach

Results

Conclusions

Future work

Background - SPRMetals with free electrons

Gold, silver

P-polarized light at resonance angle excites electrons

Angle at which photons couple with plasmons

Plasmons are collective vibrations of electron gas

Cooper, M.A. Nat. Rev. Drug Discovery 2002, 1, 517

Characteristic Sensorgram

(2) Association

(3) Equilibrium

(4) Dissociation

Experimental Setup for SPR

Membrane ProteinsTarget of 60% of drugs Epidermal Growth Factor (EGFR)

Overexpressed in epithelial cancers

Lung, ovary, breast, colon

3 domainsIntracellular tyrosine kinaselipophilic transmembraneExtracellular ligand binding

AntibodiesPolyclonal: anti-EGFR TKmAb: Erbitux® (cetuximab)

Provided by: Eureka Therapeutics Inc.

Huang S., Invest New Drugs 1999, 259-269

Methods – Protein Concentration Assay

Purify cellsExtract proteins

Bio-Rad Protein Assay kit

Colorimetric, similar to ELISA

Folin reagent + alkaline copper tartrate

UV/vis spectroscopy to measure absorbance

Use standards to make calibration curve (1.6 – 0.2 mg/ml)

Determined [EGFR] 4.06 mg/ml

Methods - SAM

Johanna Stettner, Institute of Solid State Physics, Graz University of Technology

SH(CH2)10 COOH

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC)

N-hydroxysulfosuccinimide (Sulfo-NHS)

•Stable bond between sulfur and gold

•Short hydrocarbon chain

•Change functional groups

Covalent Coupling mAb to SAM

mAb binding significantly stronger to EGFR cells than the control

62.0

62.2

62.4

62.6

62.8

63.0

63.2

63.4

63.6 Red = Control CHO CellsBlack = EGFR overexpressed in cells

Signal = 360 mdeg

Signal = 190 mdeg

5.5 x 106 cells (CHO or EGFR overexpressed CHO)

0.25 mg/mL Erbitux

EDC/NHS

Min

. An

gle

(d

eg.)

Time (min.)

SAM-Based Design on Polyclonal Ab

EGFR cells = 84.3 mdegControl = 62.9 mdegControl signal still large

0 25 50 75 100 125 15062.0

62.2

62.4

62.6

62.8

63.0

63.2

63.4

63.6

63.8

Rinse

Control cells and EGFR cells

Control cells and EGFR cells

Anti-EGFR TK (20 g/ml)

Rinse

EDC/NHS4:1

Min

. An

gle

(d

eg.)

Time (min.)

EGFR-expressing Control

EGFR in Tethered Bilayer Membrane

0 25 50 75 100 125 150 175

62.0

62.2

62.4

62.6

62.8

63.0

63.2

63.4

63.6

63.8

Rinsed

PEG-amineblocking agent

Anti-EGFR TK

Rinsed

EDC/NHS 4:1

Rinsed

PC vesicles and cellsMin

An

gle

(d

eg.)

Time (min.)

EGFR cells Control

Small signal increase after injection of Anti-EGFR (2 µg/ml)

Co-injected PC vesicles along with cells

EGFR cells = 413 mdeg

Control cells = 352 mdeg

Tethered membrane provides space

Biologically relevant

Interface Design II: Biotinylation

Biotin-Avidin bondsVery strong (Ka = 1015 M-1 )

orientation specific

Biotinylate TK antibodySulfo-NHS-LC-Biotin kit

BiotinNHS

Effect of Biotinylation

0 20 40 60 80 10063.20

63.25

63.30

63.35

63.40

63.45

Rinse

NeutrAvidin (0.5 mg/ml)

Rinse

Biotin-BSA

Min

. An

gle

(d

eg.)

Time (min.)

0 20 40 60 80 100

63.20

63.25

63.30

63.35

Rinse

NeutrAvidin (0.5 mg/ml)Rinse

BSA (0.5 mg/ml)Min

. An

gle

(d

eg.)

Time (min.)

Biotin BSA signal 3X greater than control

Complete Surface System

Biotin BSA, NeutrAvidin, biotin anti-EGFR, PC vesicles + cells (EGFR and control)

Ideally, the signal should be greater from the EGFR cells, and Erbitux should have a greater signal

0 25 50 75 100 125 150 175 200

62.4

62.6

62.8

63.0

63.2

63.4

63.6 Control Response

Rinse

PC vesicles and control cells

switched buffers from PBS to HEPES

Rinse

biotinylated Anti-EGFR20 g/ml

NeutrAvidin0.25 mg/ml

Biotin BSA0.25 mg/ml

Min

. An

gle

(d

eg.)

Time (min.)

-25 0 25 50 75 100 125 150 175 200 225 250 275 300

62.2

62.4

62.6

62.8

63.0

63.2

63.4EGFR

switched buffersPBS to HEPES

Rinse

Erbitux0.25 mg/ml

Rinse

PC vesicles + EGFR overexpressing cells

biotin Anti-EGFR20 g/ml

NeutrAvidin0.25 mg/ml

biotin-BSA0.25 mg/ml

Min

. An

gle

(d

eg.)

Time (min.)

Conclusions

Biotinylation procedure is effective.

Erbitux shows preferential binding to cells overexpressing EGFR compared to control cells.

Current method of combining EGFR and PC vesicles can be improved.

Lack of signal between EGFR cells in lipid membrane and Erbitux may indicate improper orientation within the membrane

Both SAMs and biotinylated surfaces show promise

Next StepsDetermine ideal membrane interface design for effective and functional EGFR immobilization for protein binding.

Try different lipid mixtures to more closely mimic natural membrane

Create an interface based on the calcinated chip (glassified layer on gold) for direct immobilization of the EGFR in a membrane.

Use mAb Erbitux once ideal interface design is determined

Apply best interface design to a microarray format for high-throughput screening with SPR imaging.

Acknowledgements

Matt Linman and Dr. Cheng

National Science Foundation

Jun Wang and BRITE REU program

ReferencesHopkins, A. L.; Groom, C. R. Nat.Rev. Drug Discovery 2002, 1, 727–730.

Hubbard, S. R. Cancer Cell 2005, 7, 287-288.

Kim, Edward S., et al. Epidermal growth factor receptor biology. Current Opinion in Oncology 2001, 13, 506-513.

Li, Shiqing; et al. Cancer Cell 2005, 7, 301-311.

Liedberg, B., I. Lundstrom, and E. Stenberg. 1993. Principles of biosensing with an extended coupling matrix and surface plasmon resonance. Sensors and Actuators B 11: 63-72.

Linman, M. J.; Culver S.P.; Cheng Q. Langmuir 2009, 25, 3075-3082.

Macher, Bruce A., Yen, Ten-Yang. Proteins at membrane surfaces – a review of approaches. Mol. Biosyst. 2007, 3, 705-713.