Ying Cong, Rianne Biemans, Natasja G. Lieuwes, Dennis Suijlen, Philippe Lambin, Ingrid Dijkgraaf, Matthias Bauwens, Ala Yaromina, Ludwig J. Dubois
{"title":"一种新型抗ceacam5 VHH用于SPECT成像和潜在的癌症治疗应用","authors":"Ying Cong, Rianne Biemans, Natasja G. Lieuwes, Dennis Suijlen, Philippe Lambin, Ingrid Dijkgraaf, Matthias Bauwens, Ala Yaromina, Ludwig J. Dubois","doi":"10.1007/s00259-025-07321-z","DOIUrl":null,"url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Purpose</h3><p>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.</p><h3 data-test=\"abstract-sub-heading\">Methods</h3><p>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 <sup>99m</sup>Tc-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.</p><h3 data-test=\"abstract-sub-heading\">Result</h3><p>6B11 demonstrated high binding affinity (EC<sub>50</sub> 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 (<i>p</i> < 0.05) in A549-CEA5-OV tumors, resulting in increased tumor-to-blood ratios especially at 4 (2.0016 ± 1.1893, <i>p</i> = 0.035) and 24 (2.9371 ± 2.0683, <i>p</i> = 0.003) hpi compared to A549-CEA5-KO tumors at 4 (0.5640 ± 0.3576) and 24 (0.8051 ± 0.4351) hpi. <sup>99m</sup>Tc-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.</p><h3 data-test=\"abstract-sub-heading\">Conclusion</h3><p>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.</p><h3 data-test=\"abstract-sub-heading\">Graphical Abstract</h3>","PeriodicalId":11909,"journal":{"name":"European Journal of Nuclear Medicine and Molecular Imaging","volume":"123 1","pages":""},"PeriodicalIF":8.6000,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Development of a novel anti-CEACAM5 VHH for SPECT imaging and potential cancer therapy applications\",\"authors\":\"Ying Cong, Rianne Biemans, Natasja G. 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Biodistribution and binding specificity of <sup>99m</sup>Tc-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.</p><h3 data-test=\\\"abstract-sub-heading\\\">Result</h3><p>6B11 demonstrated high binding affinity (EC<sub>50</sub> 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 (<i>p</i> < 0.05) in A549-CEA5-OV tumors, resulting in increased tumor-to-blood ratios especially at 4 (2.0016 ± 1.1893, <i>p</i> = 0.035) and 24 (2.9371 ± 2.0683, <i>p</i> = 0.003) hpi compared to A549-CEA5-KO tumors at 4 (0.5640 ± 0.3576) and 24 (0.8051 ± 0.4351) hpi. <sup>99m</sup>Tc-6B11 was predominantly renally cleared. Autoradiography and immunohistochemistry confirmed these uptake patterns. 6B11 nor 6F8 did exhibit significant anti-cancer therapeutic efficacy in vitro. 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Development of a novel anti-CEACAM5 VHH for SPECT imaging and potential cancer therapy applications
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.
期刊介绍：
The European Journal of Nuclear Medicine and Molecular Imaging serves as a platform for the exchange of clinical and scientific information within nuclear medicine and related professions. It welcomes international submissions from professionals involved in the functional, metabolic, and molecular investigation of diseases. The journal's coverage spans physics, dosimetry, radiation biology, radiochemistry, and pharmacy, providing high-quality peer review by experts in the field. Known for highly cited and downloaded articles, it ensures global visibility for research work and is part of the EJNMMI journal family.
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