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 Torleif Härd
Hello
I am 한원석, and work in omics center, KIST.
Today, I will talk about cancer target binding protein.
Date :
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 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.

3. Cancer target
Structural basis for high-affinity HER2 receptor binding by an engineered protein
staphylococcal protein A, “affibody”
HER2 binding affibody, “ZHER2”
HER2 is famous breast cancer target.
And in this title, engineered protein is staphylococcal protein A. another name “affibody”
Especially, HER2 binding protein A, ZHER2,
In this paper, Engineered protein means Zher2.
ZHER2 is three helix bundle, shape is like Z.
In the previous study, ZHER2 was engineered as a high-affinity binder of HER2
Goal of this paper is determination of X-ray crystal structure of HER2-ZHER2 complex.

4. Structural basis for high-affinity HER2 receptor binding by an engineered protein
This complex structure is HER2 and Zher2 complex structure suggested in this paper.
Rendered structure is HER2, and orange three helix bundle is Zher2.
Figure B descript binding site between these two proteins.
And cyan and blue are commercial antibodies for HER2.
I will explain more detail about ZHER2, and this complex structure.

5. 1. Why using Affibody, instead of Ig-antibody for target
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
I will discuss three topics.
First, background of affibody, and engineering of high-affinity binding affibody and
ZHER2-HER2 complex structure analysis.
First, talk about affibody.

6. 1. Why using Affibody, instead of Ig-antibody for target
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
Affibody is immune protein like ig-anitbody.
I explain why affibody is immune protein.
Origin of Affibody is staphylococcal protein A, which is surface protein of staphylococcus aureus.
Circle is staphylococcus aureus, orange is protein A.
Green is antibody.
Fab domain bind antigen. and
Fc domain normally binds to Fc receptor of phagocytes,
But Fc domain binds to protein A under staphyloccocus infection,
As shown in figure, antibody coat is generated,
In this way, staphylococcus evade host immune defenses system .
Protein A and antibody interaction was studied.
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. 1. Why using Affibody, instead of Ig-antibody for target
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
This figure show Fc domain- protein A complex structure.
Protein A bind CH2-CH3 domain of Fc domain.
And this site is Fc receptor binding site.
Therefore, staphylococcal protein A inhibit Fc-FC receptor interaction,
staphylococcal protein A is very useful, because of its structural stability.

8. 1. Why using Affibody, instead of Ig-antibody for target
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
Tree helix bundle is very robust fold, a few mutations do not disrupt this fold.
Therefore, We can easily engineer protein A having some interest properties.
As a example, we can generate affibody with high affinity for target protein.
Zher2 have high-affinity for HER2 and
This binding affinity between two proteins is higher than other commercial antibodies.
This is reason, why use affibody.
In this paper, some experimental data show structural stability of Zher2.
Nature 447, (7 June 2007)
KD = 0.1nM

9. 1. Why using Affibody, instead of Ig-antibody for target
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
These figures show biophysical characters of Zher2
Figure A is CD spectrum, at twenty degree,
This graph implies that a major fraction is helix at twenty degree.
Figure B present thermo-stability of Zher2. melting temperature is about 67degree.
-Melting temperature of Wild type is 75degree
Figure C show folding stability of Zher2.
Zher2 is stabilized by a free energy 3.1 kcal/mol.
-wild type free energy is about 7 kcal/mol.
Zher2 is less stable than wild type,
but Zher2 is still stable. At room temperature, have high helicity,
And have high thermo- and fold stability.
above mentioned staphylococcal protein A is three helix bundle.

10. 2. How engineering high-affinity affibody for target
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
surface of First and second helix is binding site for target protein.
Function of third helix is related structural stability.
Affibody is mutated at 13 positions.
This figure display 13 mutation position on the structure and
On the sequence.
We can Build large library and select target binding mutants
Expert Opin. Biol. Ther. (2007) 7(4):

11. 2. How engineering high-affinity affibody for target
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
This is screening method, using phage display.
Maybe black box is GIII, gray box affibody mutant, these expressed affibody mutant
This parge library is exposed to target protein,
select target binding affibody mutants and identify.
Using phage display, several affibodies were published.
Journal of Biotechnology 140 (2009) 254–269

12. 2. How engineering high-affinity affibody for target
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
Affibody bind to various target proteins.
This table show possible target proteins of affibody.
Affinity is KD value.
Commercial antibodies have nM KD values.
EGFR binding affibody has 5nM KD value
And TNF-alpha binding affibody has 95pM.
This table show that We can make affibody with high-affinity for various target proteins
Above mentioned, The goal of this paper is determination of complex structure, HER2 and Zher2
Journal of Biotechnology 140 (2009) 254–269

13. 3. What knowing from ZHER2-HER2 complex structure
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
This complex structure can explain previous experimental result of affibody.
In the previous study, Zher2 bind to her2 with higher-affinity and
Zher2 did not competes with commercial antibodys.
And Zher2 did not show biological effects.
In the complex structure, Zher2 binding site is distant from other antibodies binding site.
We understand easily, why zher2 does not compete with other antibodies.
Zher2 has high-affinity for HER2
But Zher2 did not affect HER2 function.
HER2 is epidermal growth factor receptor family.
EGFR family carry out conformational change, home- hetero dimerization.
This dimer form is active form.

14. 3. What knowing from ZHER2-HER2 complex structure
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
Commercial antibodies bind to dimerization site, and inhibit dimerization of her2
Zher2 bind here. Remote from dimerization site.
Zher2 does not inhibit dimerization.
This site is ligand binding site. Zher2 binding site is also distant from ligand binding site.
Zher2 does not inhibit ligand binding.
Maybe Zher2 binding site is not effective functional site.
In this paper, author proposed Zher2 application based on these results.

15. Conclusion 1. Zher2 does not compete with commercial antibodies
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
Author proposed two applications of Zher2
First, Zher2 may be used as a imaging tracer of HER2
without interfering with antibody therapy
And Another using is as a carrier of other therapeutic agents to HER2.
In this case, expect synergy effect with commercial antibodies.

16. Thank you

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