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Abstract
Surgery is one of the key therapies in colorectal cancer (CRC) treatment. However, intra-operative decision making remains challenging. Fluorescence-guided surgery (FGS) can aid in the visualization of tumor tissue. Nanobodies (Nbs) show promise as tracers for FGS due to their rapid pharmacokinetics and high specificity. Previously, the anti-CEA Nb-IRDye800CW has been validated in vivo to visualize CRC. However, high background signals were observed due to binding of IRDye800CW to serum proteins. To overcome this problem, this study evaluated the anti-CEA Nb-s775z both in vitro and in vivo.
First, the anti-CEA Nb was randomly conjugated to the fluorescent dye s775z via NHS-chemistry. Subsequently, the affinity of the compound was determined by surface plasmon resonance (SPR) and its binding to human colorectal cancer cells was assessed by cell binding assays. Finally, the in vivo biodistribution profile of the anti-CEA Nb-s775z 1h post-injection was evaluated in a subcutaneous human CRC mouse model and compared to an irrelevant control Nb-s775z.
Random labeling of the anti-CEA Nb to the fluorescent dye s775z led to an average degree of labeling of 1,3. Furthermore, a KD of 4,3 ± 0,2 nM was obtained compared to a KD of 0,3 ± 0,0 nM for the unlabeled anti-CEA Nb. The slight decrease in affinity of anti-CEA Nb-s775z did not affect binding to human CRC HT-29 cells as the compound was able to specifically bind to these cells in vitro.
In vivo, high signal in the tumor was observed with the anti-CEA Nb-s775z (7.863 ± 1.935 a.u.) compared to a lower signal using the control Nb-s775z (4.489 ± 198 a.u.). Furthermore, low to no signal was detected in healthy organs except for a slightly increased signal in the liver (5.549 ± 1.887),stomach (3.855 ± 1.299) and kidneys (616.238 ± 116.218) (Fig. 1). The latter is attributed to the renal clearance of Nbs. Finally, an ex vivo TBR of 6,2 ± 2,3 was achieved for the anti-CEA Nb-s775z.
The anti-CEA Nb-s775z was able to specifically highlight subcutaneous colorectal tumors with no background signal except in the liver, stomach and kidneys. To further validate this compound, it should be tested in an orthotopic colorectal tumor model. In addition, the anti-CEA Nb can also be conjugated with radioisotopes such as 18F or 68Ga for PET-imaging which could aid in the follow-up of patients.
First, the anti-CEA Nb was randomly conjugated to the fluorescent dye s775z via NHS-chemistry. Subsequently, the affinity of the compound was determined by surface plasmon resonance (SPR) and its binding to human colorectal cancer cells was assessed by cell binding assays. Finally, the in vivo biodistribution profile of the anti-CEA Nb-s775z 1h post-injection was evaluated in a subcutaneous human CRC mouse model and compared to an irrelevant control Nb-s775z.
Random labeling of the anti-CEA Nb to the fluorescent dye s775z led to an average degree of labeling of 1,3. Furthermore, a KD of 4,3 ± 0,2 nM was obtained compared to a KD of 0,3 ± 0,0 nM for the unlabeled anti-CEA Nb. The slight decrease in affinity of anti-CEA Nb-s775z did not affect binding to human CRC HT-29 cells as the compound was able to specifically bind to these cells in vitro.
In vivo, high signal in the tumor was observed with the anti-CEA Nb-s775z (7.863 ± 1.935 a.u.) compared to a lower signal using the control Nb-s775z (4.489 ± 198 a.u.). Furthermore, low to no signal was detected in healthy organs except for a slightly increased signal in the liver (5.549 ± 1.887),stomach (3.855 ± 1.299) and kidneys (616.238 ± 116.218) (Fig. 1). The latter is attributed to the renal clearance of Nbs. Finally, an ex vivo TBR of 6,2 ± 2,3 was achieved for the anti-CEA Nb-s775z.
The anti-CEA Nb-s775z was able to specifically highlight subcutaneous colorectal tumors with no background signal except in the liver, stomach and kidneys. To further validate this compound, it should be tested in an orthotopic colorectal tumor model. In addition, the anti-CEA Nb can also be conjugated with radioisotopes such as 18F or 68Ga for PET-imaging which could aid in the follow-up of patients.
Original language | English |
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Publication status | Published - 13 Mar 2024 |
Event | EMIM 2024 - Porto, Portugal Duration: 12 Mar 2024 → 15 Mar 2024 |
Conference
Conference | EMIM 2024 |
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Country/Territory | Portugal |
City | Porto |
Period | 12/03/24 → 15/03/24 |
Keywords
- Nanobodies
- CEA
- Fluorescence-guided surgery
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Dive into the research topics of 'In vitro and in vivo validation of a fluorescently-labeled anti-CEA Nanobody for fluorescence guided surgery of colorectal cancer.'. Together they form a unique fingerprint.Activities
- 1 Talk or presentation at a conference
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In vitro and in vivo validation of a fluorescently-labeled anti-CEA Nanobody for fluorescence guided surgery of colorectal cancer.
Sam Floru (Speaker), Lukasz Mateusiak (Contributor), Jelena Saliën (Contributor), Marcus Stroet (Contributor) & Sophie Hernot (Contributor)
13 Mar 2024Activity: Talk or presentation › Talk or presentation at a conference