1. Selection and characterization of nanobodies for the early detection of ovarian cancer biomarkers
Lan-Huong Tran, PhD Student - Hasselt University
4th NanoBio Surfaces and Interfaces in Health Care and Science 2019
Workshop, May 17th 2019, Potsdam, Germany

2. Ovarian cancer Epithelial ovarian tumors 90-95%, postmenopausal
Increases with age
Stromal tumors
Benign or malignant, age 0-20
Non-aggressive in the adult form
Germ cell tumors
Benign or malignant, age 10-30
3% of OC
Diagnosed in the early stages
Tend to grow rapidly

4. Biomarkers Problems Aims of this project Cancer Antigen 125 (CA125)
Human Epididymis protein 4 (HE4)
Secretory leukocyte protease inhibitor (SLPI)
Progranulin (PGRN)
Problems
Specificity & sensitivity: still poor at early stage (50% for stage I )
Aims of this project
Attempt to develop more efficient biosensor detection strategies based on bioconjugation between Nbs and the biomarkers HE4, SLPI, PGRN
To modifify nanobodies (Nbs) targeting biomarker by site-specific functionalization using Expressed protein ligation (EPL)

5. Expressed protein ligation - EPL

6. Expressed protein ligation - EPL
Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) Click Chemistry

7. Materials and Methods Human Epididymis protein 4 (HE4) : 13 strains
Available strains of Nbs from Vrije Universiteit Brussel (VUB): 35 strains in total
Human Epididymis protein 4 (HE4) : 13 strains
Secretory leukocyte protease inhibitor (SLPI) : 10 strains
Progranulin (PGRN) : 12 strains
All strains: Nb-His

8. An overview of experiment
Glycerol stock
Pre-culture 37oC
Transformation
Pre-culture O/N, 37oC
Plasmid extraction
+IPTG
Binding affinity
test
Expression O/N, 37oC
SDS-PAGE on gel
2nd purification, SEC
1st purification, IMAC

9. Kinetics of Nbs – Binding affinity tests
Indirect Elisa
Surface plasmon resonance (SPR)
KD = koff / kon
Epitope mapping
koff
kon

10. Results - Elisa A B C

11. Results – SPR

14. Coupling of alkynated Nbs to PEG-N3 using click chemistry reaction
15 kDa
25 kDa
M: Marker
1: HE4-LEY-CA
2: HE4-LEY
3: SLPI-LEY-CA
4: SLPI-LEY
5: PGRN-LEY-CA
6: PGRN-LEY

15. Summary Best strains of Nbs from HE4, SLPI and PGRN were chosen
The selected nanobodies were successfully site-specifically alkynated at their C-terminus using the Expressed protein ligation (EPL) technique and were coupled to an azidified PEG counterpart using “click”-chemistry.

16. Thank you for your attention!

17. Posibilities

18. Epitope mapping