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

1. PNAS ∣ August 24, 2010 ∣ vol. 107 ∣ no. 34 ∣ 15039–15044 Structural basis for high-affinity HER2 receptor binding by an engineered protein Charles Eigenbrot, Mark Ultsch, Anatoly Dubnovitsky, Lars Abrahmsén, and TorleifHärd Date : 2010.10.11 Speaker : HAN WON SEOK

2. Structural basis for high-affinity HER2 receptor binding by an engineered protein Abstract The human epidermal growth factor receptor 2 (HER2) is specifically overexpressedin 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 affibodymolecule 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.

3. Cancer target Structural basis for high-affinity HER2 receptor binding by an engineered protein staphylococcal protein A, “affibody” HER2 binding affibody, “ZHER2”

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

5. 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 ZHER2-HER2 complex structure

6. 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 ZHER2-HER2 complex structure Staphylococcus aureus (포도상구균 )Resisting Opsonization via Protein A Doc Kaiser's Microbiology Home Page The Fc portion of the antibody IgG, the portion that would normally binds to Fc receptors on phagocytes, 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.

7. 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 ZHER2-HER2 complex structure

8. 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 ZHER2-HER2 complex structure KD = 22pM Nature447, 741-744 (7 June 2007) KD = 0.1nM

9. 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 ZHER2-HER2 complex structure

10. 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 ZHER2-HER2 complex structure Expert Opin. Biol. Ther. (2007) 7(4):555-568

11. 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 ZHER2-HER2 complex structure Journal of Biotechnology 140 (2009) 254–269

12. 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 ZHER2-HER2 complex structure Journal of Biotechnology 140 (2009) 254–269

13. 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 ZHER2-HER2 complex structure In the previous study, Not compete with other antibodies Not have biological effects

14. 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 ZHER2-HER2 complex structure

15. 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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17. Structural basis for high-affinity HER2 receptor binding by an engineered protein References Thank you