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Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers
Anna-Karin Haylock1,2, Johan Nilvebrant3, Anja Mortensen2
1Department of Surgical Sciences, Uppsala University, Uppsala, Sweden.
Aim:
The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients.
Materials And Methods:
Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I.
Results:
Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p.i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor.
Conclusion:
The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging.
Insights
Researchers developed novel single-chain variable fragment (scFv) antibodies targeting CD44v6 for cancer imaging. Radiolabeled antibodies showed excellent tumor targeting and fast clearance, demonstrating potential for molecular imaging in patients.
Area of Science:
- Oncology
- Immunology
- Radiochemistry
Background:
- CD44v6 is a promising target for cancer molecular imaging.
- Development of specific targeting agents is crucial for effective imaging.
Purpose of the Study:
- Generate and characterize scFv antibodies against human CD44v6.
- Evaluate radiolabeled scFv candidates for CD44v6-targeted molecular imaging in vitro and in vivo.
- Assess potential for clinical application in cancer patients.
Main Methods:
- Phage display for scFv isolation and affinity maturation.
- Chain shuffling for enhanced antibody affinity.
- Radiolabeling with 111In, 125I, and 124I.
- In vitro cell binding assays.
- In vivo biodistribution studies in tumor-bearing mice.
- Small animal PET-CT imaging.
Main Results:
- Two affinity-matured scFv clones (CD44v6-scFv-A11 and CD44v6-scFv-H12) showed strong binding.
- Radiolabeled conjugates exhibited specific CD44v6 binding.
- In vivo studies revealed favorable tumor-to-organ ratios and fast clearance.
- PET-CT imaging clearly visualized CD44v6-expressing tumors.
Conclusions:
- CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind CD44v6.
- Radioiodinated scFvs offer high tumor-to-blood ratios and rapid clearance.
- These antibodies are suitable for molecular imaging in oncology.