| Zusammenfassung: | CD39 and CD73 are the two key ecto-enzymes in the process of converting pro-inflammatory ATP into anti-inflammatory adenosine. The enzymatic activity of both ecto-enzymes modulates the course inflammation associated diseases, including cancer. Compared to conventional antibodies, nanobodies are much smaller, exhibit better tissue penetration and are well suited for targeting the active site of enz... CD39 and CD73 are the two key ecto-enzymes in the process of converting pro-inflammatory ATP into anti-inflammatory adenosine. The enzymatic activity of both ecto-enzymes modulates the course inflammation associated diseases, including cancer. Compared to conventional antibodies, nanobodies are much smaller, exhibit better tissue penetration and are well suited for targeting the active site of enzymes.
In this thesis, nanobodies against mouse and human CD39 and CD73 were successfully generated. After screening on transfected HEK cells, 11 and 9 families of specific binders to CD39 and CD73 were identified, respectively. Among 11 CD39 specific families, four families were specific for mouse CD39, six families were specific to human CD39, and one family was cross-reactive to mouse and human CD39. For CD73, three mouse CD73 and three human CD73 specific families and three cross-reactive families were identified. The specificity to primary mouse and human immune cells was also evaluated. Here, human CD39, mouse and human CD73 specific nanobodies could showed propper bind to their specific target on primary cells, whereas most mouse CD39 specific nanobodies (except for fam3 and fam13) failed to bind to primary mouse cells.
The capacity of selected nanobodies towards modulating the enzymatic activity of their target was further evaluated. For human CD39-specific nanobodies, fam11 and fam15 showed strong inhibition of CD39 enzymatic activity, while fam17 and fam19 only slightly inhibit ATP degradation. For mouse CD39 specific nanobodies, fam2 exhibited CD39-inhibiting capacity, while fam10 and fam16 showed only minor inhibitory effect. The cross-reactive fam3 could slightly inhibit mouse CD39 but had no impact on human CD39 activity.
Among all CD73 specific nanobodies, four mouse CD73 specific and five human CD73 specific nanobodies were evaluated towards their CD73-modulating capacity. Regarding mouse CD73 specific nanobodies, clone 18-6 showed proper inhibition of mouse CD73 activity, whereas clone 20-1 showed moderate inhibitory capacity. All five human CD73 binders showed moderate inhibition of CD73 activity in the AMP degradation assay.
In summary, mouse and human CD39 and CD73 specific nanobodies were successfully generated in this project and promising antagonists have been identified for each target. These newly developed nanobodies could serve as new generation of CD39/CD73 inhibiting drugs to be tested in preclinical mouse models and, after positive results, also in clinical trials e.g. as new anti-tumor drugs. |
