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Published on: November 13, 2012
Development of a novel anti-CEACAM5 VHH for SPECT imaging and potential cancer therapy applications
Ying Cong1, Rianne Biemans1, Natasja G Lieuwes1
1The M-Lab, Department of Precision Medicine, GROW- Research Institute for Oncology and Reproduction, Maastricht University, UNS50/23, Maastricht, PO Box 616, 6229 ER, Maastricht, The Netherlands.
Purpose:
In this study, we investigated the utility of a novel developed anti-CEACAM5 VHH for cancer diagnosis and its potential of being a targeting-moiety of VHH-drug conjugates for cancer therapy.
Methods:
Anti-CEACAM5 VHH (6B11) affinity and specific cellular binding was confirmed by ELISA, FACS and immunofluorescence in cancer cell lines with varying CEACAM5 expression levels. Intracellular penetration ability within tumor spheroids was tested with Oregon Green 488 labeled 6B11 (OG488-6B11). Biodistribution and binding specificity of 99mTc-radiolabeled 6B11 was tested in A549 CEACAM5 overexpressing (A549-CEA5-OV) and knockout (A549-CEA5-KO) tumor-bearing mice upon SPECT/CT imaging, γ-counting and autoradiography. The therapeutic efficacy of 6B11 and 6F8 (anti-CEACAM5 VHH with lower binding affinity) was tested by viability, wound healing and adhesion assays. To verify the potential of VHHs as a warhead for VHH-drug conjugation, an internalization assay with OG488 labeled VHH was performed.
Result:
6B11 demonstrated high binding affinity (EC50 0.5nM) and cellular binding. OG488-6B11 penetrated tumor spheroids completely at 24 h, while a conventional antibody was only visible at the spheroid periphery. SPECT imaging indicated higher uptake (p < 0.05) in A549-CEA5-OV tumors, resulting in increased tumor-to-blood ratios especially at 4 (2.0016 ± 1.1893, p = 0.035) and 24 (2.9371 ± 2.0683, p = 0.003) hpi compared to A549-CEA5-KO tumors at 4 (0.5640 ± 0.3576) and 24 (0.8051 ± 0.4351) hpi. 99mTc-6B11 was predominantly renally cleared. Autoradiography and immunohistochemistry confirmed these uptake patterns. 6B11 nor 6F8 did exhibit significant anti-cancer therapeutic efficacy in vitro. OG488-6B11 was effectively internalized and accumulated in cells in a time-dependent manner, to end up in the lysosomes.
Conclusion:
The anti-CEACAM5 VHH 6B11 is a good candidate for SPECT-based cancer diagnosis and can be potentially used as targeting moiety in the development of VHH-based drug conjugates for cancer treatments.
Insights
A novel anti-CEACAM5 VHH (6B11) shows promise for SPECT-based cancer diagnosis due to its high affinity and tumor uptake. While not therapeutically effective alone, it demonstrates potential as a targeting moiety for VHH-drug conjugates in cancer therapy.
Area of Science:
- Biotechnology
- Oncology
- Molecular Imaging
Background:
- Carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5) is a promising biomarker for various cancers.
- Developing targeted diagnostic and therapeutic agents for CEACAM5-expressing tumors remains a critical challenge.
Purpose of the Study:
- To evaluate a novel anti-CEACAM5 VHH (6B11) for cancer diagnosis using SPECT imaging.
- To assess the potential of 6B11 as a targeting moiety for VHH-drug conjugates in cancer therapy.
Main Methods:
- VHH affinity and cellular binding confirmed via ELISA, FACS, and immunofluorescence.
- Tumor spheroid penetration and VHH internalization assays performed using fluorescently labeled VHH.
- Biodistribution and SPECT/CT imaging of 99mTc-labeled 6B11 in tumor-bearing mice with varying CEACAM5 expression.
- In vitro therapeutic efficacy assessed through viability, wound healing, and adhesion assays.
Main Results:
- 6B11 exhibited high binding affinity (EC50 0.5nM) and effective tumor spheroid penetration.
- SPECT imaging revealed significantly higher uptake of 99mTc-6B11 in CEACAM5-overexpressing tumors compared to knockout tumors.
- While 6B11 showed no significant in vitro therapeutic efficacy, it was effectively internalized by cells, accumulating in lysosomes.
Conclusions:
- The anti-CEACAM5 VHH 6B11 is a suitable candidate for SPECT-based cancer diagnosis.
- 6B11 holds potential as a targeting moiety for developing VHH-drug conjugates for cancer treatment.
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