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An antibody-calmodulin fusion protein reveals a functional dependence between macromolecular isoelectric point and
Samu Melkko1, Cornelia Halin, Laura Borsi
1Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland.
Purpose:
Human monoclonal antibodies are promising agents for the development of improved anticancer therapeutics, because, unlike low-molecular-weight chemotherapeutic agents, they can selectively localize to solid tumors. In particular, the scFv(L19) antibody fragment, specific for the EDB domain of fibronectin, a marker of angiogenesis, has demonstrated an impressive tumor targeting performance in a variety of tumor-bearing animals and in patients with cancer. The purpose of this study was to develop a tumor pretargeting strategy, based on a novel anti-EDB fusion protein.
Methods And Materials:
We have fused the scFv(L19) to calmodulin, a small acidic protein for which specific binding peptides with a dissociation constant in the picomolar range are available. The resulting fusion protein has been expressed in mammalian cells and purified to homogeneity, before being characterized by quantitative biodistribution analysis in mice bearing the F9 murine teratocarcinoma.
Results:
Surprisingly, we have found that the fusion of scFv(L19) to calmodulin completely abrogated the tumor targeting ability of the antibody in vivo, although both scFv(L19) and calmodulin moieties within the fusion protein retained unaltered binding affinities toward their respective ligand. Furthermore, a systematic analysis of 13 derivatives of scFv(L19) recently produced in our laboratories showed that the 10 derivatives that retain the tumor targeting ability of the parental antibody have isoelectric points (pI) between 5.0 and 9.0, whereas scFv(L19)-calmodulin (pI = 4.49) and two other derivatives of scFv(L19) with pI >9.0 were unable to target tumors in vivo.
Conclusions:
Because the EDB domain of fibronectin is a component of the modified extracellular matrix, predominantly located at the abluminal side of tumor blood vessels, our data suggest that extreme pI values of antibody-based pharmaceuticals may inhibit protein extravasation, perhaps by virtue of electrostatic interactions with endothelial cells and/or components of the extracellular matrix.
Insights
Fusion of the scFv(L19) antibody fragment to calmodulin unexpectedly eliminated tumor targeting. This suggests that extreme isoelectric points (pI) of antibody-based drugs may hinder their ability to reach tumors, potentially impacting cancer therapy development.
Area of Science:
- Oncology
- Biotechnology
- Immunology
Background:
- Human monoclonal antibodies offer targeted anticancer therapeutics due to their selective localization in solid tumors.
- The scFv(L19) antibody fragment targets the EDB domain of fibronectin, a marker of angiogenesis, showing promise in preclinical and clinical studies.
- Developing tumor pretargeting strategies is crucial for enhancing the efficacy of antibody-based cancer therapies.
Purpose of the Study:
- To develop a novel tumor pretargeting strategy using an anti-EDB fusion protein.
- To investigate the impact of fusing scFv(L19) to calmodulin on tumor targeting capabilities.
Main Methods:
- The scFv(L19) antibody fragment was fused to calmodulin, a protein with high-affinity binding peptides.
- The resulting fusion protein was expressed in mammalian cells and purified.
- Quantitative biodistribution analysis was performed in mice bearing F9 murine teratocarcinoma.
Main Results:
- Fusion of scFv(L19) to calmodulin completely abolished in vivo tumor targeting, despite unaltered binding affinities of individual moieties.
- Analysis of 13 scFv(L19) derivatives revealed that those with isoelectric points (pI) between 5.0 and 9.0 retained tumor targeting.
- scFv(L19)-calmodulin (pI = 4.49) and other derivatives with extreme pI values (>9.0) failed to target tumors.
Conclusions:
- Extreme isoelectric points (pI) of antibody-based pharmaceuticals may inhibit extravasation into tumors.
- This inhibition could be due to electrostatic interactions with endothelial cells or extracellular matrix components.
- The findings suggest that pI should be considered when designing antibody-based anticancer agents for effective tumor targeting.