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Published on: March 6, 2018
Influence of structural variations on biological activity of anti-PSMA scFv and immunotoxins targeting prostate
Patrick Bühler1, David Wetterauer, Dorothee Gierschner
1Department of Urology, University Hospital Freiburg, Breisacher Strasse 117, 79106 Freiburg, Germany.
Background:
In this study, different variants of the anti-PSMA single-chain antibody fragment (scFv) D7 were cloned, varying in linker type, position of hexahistidine tags and VH-VL orientation. From these scFv, Pseudomonas exotoxin A-based immunotoxins were constructed and their biological activities against prostate cancer cells were compared.
Materials And Methods:
Binding of the constructs to PSMA-expressing prostate cancer cells was determined by flow cytometry. ADP-ribosyltransferase activity was analysed and cytotoxicity was measured with WST-1 assays.
Results:
Different linker types did not influence the characteristics of the scFv or immunotoxins. The addition of an N-terminal hexahistidine-tag, however, resulted in decreased expression, binding and cytotoxicity. scFv in VH-VL orientation showed the highest expression and binding, whereas immunotoxins in VL-VH orientation exhibited the best binding and cytotoxicity.
Conclusion:
The present study showed how structure influences the characteristics of scFv and immunotoxins. It is therefore suggested that for each individual construct the optimal structure has to be determined separately.
Insights
Optimizing anti-PSMA antibody fragment (scFv) structure is key for effective prostate cancer immunotoxins. VL-VH orientation and avoiding N-terminal tags enhance binding and cell-killing activity.
Area of Science:
- Biotechnology
- Oncology
- Immunotherapy
Background:
- Investigated variants of the anti-prostate-specific membrane antigen (PSMA) single-chain antibody fragment (scFv) D7.
- Constructed Pseudomonas exotoxin A-based immunotoxins using modified scFv D7 variants.
- Compared biological activities of immunotoxins against prostate cancer cells.
Purpose of the Study:
- To determine how structural modifications of anti-PSMA scFv D7 influence immunotoxin characteristics.
- To identify optimal structural features for enhanced binding and cytotoxicity of PSMA-targeted immunotoxins.
Main Methods:
- Flow cytometry to assess binding of constructs to PSMA-expressing prostate cancer cells.
- Analysis of ADP-ribosyltransferase activity.
- WST-1 assays to measure cytotoxicity.
Main Results:
- Linker type did not significantly impact scFv or immunotoxin characteristics.
- N-terminal hexahistidine tags reduced expression, binding, and cytotoxicity.
- VH-VL orientation scFv showed highest expression/binding; VL-VH orientation immunotoxins demonstrated superior binding and cytotoxicity.
Conclusions:
- The study demonstrates a clear structure-activity relationship for anti-PSMA scFvs and their derived immunotoxins.
- Optimal structural configurations, including orientation and tag placement, are crucial for maximizing therapeutic potential.
- Individual optimization of construct structure is recommended for each specific immunotoxin design.
