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Updated: Oct 5, 2025
In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research
Published on: September 16, 2019
Use of photoimmunoconjugates to characterize ABCB1 in cancer cells
Barry J Liang1, Sabrina Lusvarghi2, Suresh V Ambudkar3
1Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA; and Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
Accurate detection of ATP-binding cassette drug transporter ABCB1 expression is imperative for precise identification of drug-resistant tumors. Existing detection methods fail to provide the necessary molecular details regarding the functional state of the transporter. Photo-immunoconjugates are a unique class of antibody-dye conjugates for molecular diagnosis and therapeutic treatment. However, conjugating hydrophobic photosensitizers to hydrophilic antibodies is quite challenging. Here, we devise a photoimmunoconjugate that combines a clinically approved benzoporphyrin derivative (BPD) photosensitizer and the conformational-sensitive UIC2 monoclonal antibody to target functionally active human ABCB1 (i.e., ABCB1 in the inward-open conformation). We show that PEGylation of UIC2 enhances the BPD conjugation efficiency and reduces the amount of non-covalently conjugated BPD molecules by 17%. Size exclusion chromatography effectively separates the different molecular weight species found in the UIC2-BPD sample. The binding of UIC2-BPD to ABCB1 was demonstrated in lipidic nanodiscs and ABCB1-overexpressing triple negative breast cancer (TNBC) cells. UIC2-BPD was found to retain the conformation sensitivity of UIC2, as the addition of ABCB1 modulators increases the antibody reactivity in vitro. Thus, the inherent fluorescence capability of BPD can be used to label ABCB1-overexpressing TNBC cells using UIC2-BPD. Our findings provide insight into conjugation of hydrophobic photosensitizers to conformation-sensitive antibodies to target proteins expressed on the surface of cancer cells.
Insights
We developed a novel photo-immunoconjugate targeting functionally active ABCB1 transporters in drug-resistant tumors. This conjugate enhances detection and offers insights into cancer cell surface protein targeting.
Area of Science:
- Bioconjugation Chemistry
- Cancer Molecular Diagnostics
- Drug Transporter Research
Background:
- Accurate detection of ATP-binding cassette drug transporter ABCB1 is crucial for identifying drug-resistant tumors.
- Current methods lack molecular detail on transporter functional states.
- Conjugating hydrophobic photosensitizers to hydrophilic antibodies presents a significant challenge.
Purpose of the Study:
- To devise a photo-immunoconjugate targeting functionally active human ABCB1 (inward-open conformation).
- To overcome challenges in conjugating hydrophobic photosensitizers to hydrophilic antibodies.
- To develop a tool for molecular diagnosis and potential therapeutic treatment of ABCB1-overexpressing cancers.
Main Methods:
- Developed a photo-immunoconjugate combining a benzoporphyrin derivative (BPD) photosensitizer with the conformational-sensitive UIC2 monoclonal antibody.
- Utilized PEGylation to enhance BPD conjugation efficiency to UIC2.
- Employed size exclusion chromatography for purification.
- Validated binding in lipidic nanodiscs and ABCB1-overexpressing triple-negative breast cancer (TNBC) cells.
Main Results:
- PEGylation of UIC2 enhanced BPD conjugation efficiency and reduced non-covalently conjugated BPD by 17%.
- UIC2-BPD demonstrated specific binding to ABCB1 in lipidic nanodiscs and TNBC cells.
- The conjugate retained conformation sensitivity, with ABCB1 modulators affecting antibody reactivity.
- Inherent fluorescence of BPD enabled labeling of ABCB1-overexpressing TNBC cells.
Conclusions:
- Successfully conjugated a hydrophobic photosensitizer (BPD) to a conformation-sensitive antibody (UIC2) for targeting functionally active ABCB1.
- The developed UIC2-BPD photo-immunoconjugate can effectively label ABCB1-overexpressing TNBC cells.
- This approach provides valuable insights for conjugating photosensitizers to antibodies for targeting cell surface proteins in cancer.
