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Structural basis for high-affinity HER2 receptor binding by an engineered protein Date : 2010.10.11 Speaker : HAN WON SEOK Charles Eigenbrot, Mark Ultsch, Anatoly Dubnovitsky, Lars Abrahmsn, and Torleif Hrd PNAS August 24, 2010 vol. 107 no. 34 1503915044

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Structural basis for high-affinity HER2 receptor binding by an engineered protein Abstract The human epidermal growth factor receptor 2 (HER2) is specifically overexpressed in tumors of several cancers, including an aggressive form of breast cancer. It is therefore a target for both cancer diagnostics and therapy. The 58 amino acid residue ZHER2 affibody molecule was previously engineered as a high-affinity binder of HER2. Here we determined the structure of ZHER2 in solution and the crystal structure of ZHER2 in complex with the HER2 extracellular domain. ZHER2 binds to a conformational epitope on HER2 that is distant from those recognized by the therapeutic antibodies trastuzumab and pertuzumab. Its small size and lack of interference may provide ZHER2 with advantages for diagnostic use or even for delivery of therapeutic agents to HER2-expressing tumors when trastuzumab or pertuzumab are already employed. Biophysical characterization shows that ZHER2 is thermodynamically stable in the folded state yet undergoing conformational interconversion on a submillisecond time scale. The data suggest that it is the HER2-binding conformation that is formed transiently prior to binding. Still, binding is very strong with a dissociation constant KD=22pM, and perfect conformational homogeneity is therefore not necessarily required in engineered binding proteins. A comparison of the original Z domain scaffold to free and bound ZHER2 structures reveals how high-affinity binding has evolved during selection and affinity maturation and suggests how a compromise between binding surface optimization and stability and dynamics of the unbound state has been reached.

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Structural basis for high-affinity HER2 receptor binding by an engineered protein Cancer target staphylococcal protein A, affibody HER2 binding affibody, Z HER2

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Structural basis for high-affinity HER2 receptor binding by an engineered protein

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1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure

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Structural basis for high-affinity HER2 receptor binding by an engineered protein 1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure Staphylococcus aureus ( )Resisting Opsonization via Protein A The Fc portion of the antibody IgG, the portion that would normally binds to Fc receptors on phagocyt es, instead binds to protein A on Staphylococcus aureus. In this way the bacterium becomes coated with a protective coat of antibodies that do not allow for opsonization. Doc Kaiser's Microbiology Home Page

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Structural basis for high-affinity HER2 receptor binding by an engineered protein 1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure

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Structural basis for high-affinity HER2 receptor binding by an engineered protein 1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure Nature 447, 741-744 (7 June 2007) K D = 0.1nM K D = 22pM

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Structural basis for high-affinity HER2 receptor binding by an engineered protein 1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure

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Structural basis for high-affinity HER2 receptor binding by an engineered protein Expert Opin. Biol. Ther. (2007) 7(4):555-568 1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure

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Structural basis for high-affinity HER2 receptor binding by an engineered protein 1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure Journal of Biotechnology 140 (2009) 254269

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Structural basis for high-affinity HER2 receptor binding by an engineered protein 1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure Journal of Biotechnology 140 (2009) 254269

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Structural basis for high-affinity HER2 receptor binding by an engineered protein 1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure In the previous study, Not compete with other antibodies Not have biological effects

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Structural basis for high-affinity HER2 receptor binding by an engineered protein 1. Why using Affibody, instead of Ig-antibody for target 2. How engineering high-affinity affibody for target 3. What knowing from Z HER2 -HER2 complex structure

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Structural basis for high-affinity HER2 receptor binding by an engineered protein Conclusion 1. Zher2 does not compete with commercial antibodies 2. Zher2 does not have biological effects 3. Zher2 allows for molecular imaging of HER2 without interfering with ongoing therapy using commercial antibodies 4. Zher2 may be used as a carrier of other therapeutic agents to HER2 target

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Thank you

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Structural basis for high-affinity HER2 receptor binding by an engineered protein References