1. Designing an SPR biointerface for transmembrane proteins Heather Ferguson Matthew Linman, Dr. Quan “Jason” Cheng BRITE Research Presentation August 20, 2009

2. Outline Background information SPR and EGFR Experimental approach Results Conclusions Future work

3. Background - SPR Metals 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

4. Cooper, M.A. Nat. Rev. Drug Discovery 2002, 1, 517 Characteristic Sensorgram (2) Association (3) Equilibrium (4) Dissociation Experimental Setup for SPR

5. Membrane Proteins Target of 60% of drugs Epidermal Growth Factor (EGFR) Overexpressed in epithelial cancers Lung, ovary, breast, colon 3 domains Intracellular tyrosine kinase lipophilic transmembrane Extracellular ligand binding Antibodies Polyclonal: anti-EGFR TK mAb: Erbitux® (cetuximab) Provided by: Eureka Therapeutics Inc. Huang S., Invest New Drugs 1999, 259-269

6. Methods – Protein Concentration Assay Purify cells Extract 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

7. 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

8. Covalent Coupling mAb to SAM mAb binding significantly stronger to EGFR cells than the control

9. SAM-Based Design on Polyclonal Ab EGFR cells = 84.3 mdeg Control = 62.9 mdeg Control signal still large

10. EGFR in Tethered Bilayer Membrane 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

11. Interface Design II: Biotinylation Biotin-Avidin bonds Very strong (Ka = 10 15 M -1 ) orientation specific Biotinylate TK antibody Sulfo-NHS-LC-Biotin kit Biotin NHS

12. Effect of Biotinylation Biotin BSA signal 3X greater than control

13. 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

14. 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

15. Next Steps Determine 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.

16. Acknowledgements Matt Linman and Dr. Cheng National Science Foundation Jun Wang and BRITE REU program

17. References Hopkins, 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.