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EphB4-targeted imaging with antibody h131, h131-F(ab')2 and h131-Fab
Dan Li1, Shuanglong Liu, Ren Liu
1Department of Radiology, The Third Affiliated Hospital of Sun Yat-sen University , Guangzhou, 510630, China.
Abstract:
Accumulating evidence suggests that overexpression of the tyrosine kinase receptor EphB4, a mediator of vascular development, is a novel target for tumor diagnosis, prognosis and therapy. Noninvasive imaging of EphB4 expression could therefore be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. In this study, we systematically investigated the use of anti-EphB4 antibody h131 (150 kDa) and its fragments (h131-F(ab')2, 110 kDa; h131-Fab, 50 kDa) for near-infrared fluorescence (NIRF) imaging of EphB4 expression in vivo. h131-F(ab')2 and h131-Fab were produced through pepsin and papain digestion of h131 respectively, whose purity was confirmed by FPLC and SDS-PAGE. After conjugation with Cy5.5, in vivo characteristics of h131, h131-F(ab')2 and h131-Fab were evaluated in EphB4-positive HT29 tumor model. Although h131-Cy5.5 demonstrated highest tumor uptake among these probes, its optimal tumor uptake level was obtained at 2 days post injection (p.i.). For h131-Fab-Cy5.5, maximum tumor uptake was achieved at 4 h p.i. However, no significant difference was observed between h131-Fab-Cy5.5 and hIgG-Fab-Cy5.5, indicating the tumor accumulation was mainly caused by passive targeting. In contrast, h131-F(ab')2-Cy5.5 demonstrated prominent tumor uptake at 6 h p.i. The target specificity was confirmed by hIgG-F(ab')2-Cy5.5 control and immunofluorescent staining. Collectively, h131-F(ab')2 exhibited prominent and specific tumor uptake at early time points, which suggests it is a promising agent for EphB4-targeted imaging.
Insights
The anti-EphB4 antibody fragment h131-F(ab")2 shows promising results for near-infrared fluorescence imaging of EphB4 expression in tumors. It demonstrates specific and prominent tumor uptake early after injection, making it suitable for early disease detection.
Area of Science:
- Biomedical Imaging
- Molecular Biology
- Oncology
Background:
- EphB4 receptor tyrosine kinase is crucial for vascular development and is overexpressed in tumors.
- Noninvasive imaging of EphB4 expression is valuable for monitoring tumor progression and treatment efficacy.
- Targeting EphB4 offers potential for cancer diagnosis, prognosis, and therapy.
Purpose of the Study:
- To evaluate anti-EphB4 antibody fragments for near-infrared fluorescence (NIRF) imaging of EphB4 expression in vivo.
- To compare the in vivo characteristics of the full antibody (h131) and its fragments (h131-F(ab")2, h131-Fab) for EphB4-targeted imaging.
- To determine the optimal fragment for early and specific tumor imaging.
Main Methods:
- Production of h131-F(ab")2 and h131-Fab fragments via pepsin and papain digestion of the anti-EphB4 antibody h131.
- Confirmation of fragment purity using FPLC and SDS-PAGE.
- Conjugation of fragments with Cy5.5 and evaluation in an EphB4-positive HT29 tumor model using NIRF imaging.
- Assessment of tumor uptake at various time points post-injection and comparison with control antibodies (hIgG).
- Confirmation of target specificity through immunofluorescent staining.
Main Results:
- The full antibody h131-Cy5.5 showed highest tumor uptake but required 2 days post-injection.
- The h131-Fab-Cy5.5 fragment achieved maximum uptake at 4 hours but lacked specificity, similar to control IgG.
- The h131-F(ab")2-Cy5.5 fragment demonstrated prominent and specific tumor uptake at 6 hours post-injection.
- Specificity was confirmed by control antibody imaging and immunofluorescence.
Conclusions:
- The anti-EphB4 antibody fragment h131-F(ab")2 is a promising agent for targeted EphB4 imaging.
- Its ability to achieve specific tumor uptake at early time points is advantageous for noninvasive monitoring.
- This fragment could facilitate early diagnosis and assessment of anti-EphB4 therapy efficacy.
