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A novel PET imaging using ⁶⁴Cu-labeled monoclonal antibody against mesothelin commonly expressed on cancer cells.

Kobayashi K, Sasaki T, Takenaka F, Yakushiji H, Fujii Y, Kishi Y, Kita S, Shen L, Kumon H, Matsuura E - J Immunol Res (2015)

Bottom Line: Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum.Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN.In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan ; Department of Urology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan.

ABSTRACT
Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with (64)Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that (64)Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The (64)Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than (18)F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

No MeSH data available.


Related in: MedlinePlus

In vitro and in vivo stability of 64Cu-DOTA-11-25 mAb and 64DOTA-anti-KLH mAb. (a) In vitro stability of 64Cu-DOTA-11-25 mAb (black bars) and 64DOTA-anti-KLH mAb (white bars) after 24 hours and 48 hours of incubation at 37°C. Radioactivity derived from antibody-bound 64Cu stays at the origin and unbound 64Cu goes to the top of the chromatogram. The percentage of radioactivity that remained in the origin of TLC was shown as the stability. (b) Representative TLC-ARG of plasma and supernatant of homogenate from liver and kidney 24 hours after injection of 64Cu-DOTA-11-25 mAb. Aliquot of blood, liver, and kidney was taken from mice 24 hours after injection and analyzed by TLC-ARG as described in Section 2. (c) In vivo stability of 64Cu-DOTA-11-25 mAb (black bars) and 64Cu-DOTA-anti-KLH mAb (white bars) 24 and 48 hours after injection.
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fig5: In vitro and in vivo stability of 64Cu-DOTA-11-25 mAb and 64DOTA-anti-KLH mAb. (a) In vitro stability of 64Cu-DOTA-11-25 mAb (black bars) and 64DOTA-anti-KLH mAb (white bars) after 24 hours and 48 hours of incubation at 37°C. Radioactivity derived from antibody-bound 64Cu stays at the origin and unbound 64Cu goes to the top of the chromatogram. The percentage of radioactivity that remained in the origin of TLC was shown as the stability. (b) Representative TLC-ARG of plasma and supernatant of homogenate from liver and kidney 24 hours after injection of 64Cu-DOTA-11-25 mAb. Aliquot of blood, liver, and kidney was taken from mice 24 hours after injection and analyzed by TLC-ARG as described in Section 2. (c) In vivo stability of 64Cu-DOTA-11-25 mAb (black bars) and 64Cu-DOTA-anti-KLH mAb (white bars) 24 and 48 hours after injection.

Mentions: DOTA-conjugated 11-25 mAb and anti-KLH mAb were incubated with 64Cu and purified as described in Section 2. The radiochemical purity of resultant 64Cu-DOTA-mAbs determined by HPLC was 97.9% for 11-25 mAb and 100% for anti-KLH mAb. The specific activities of 64Cu-DOTA-11-25 mAb and 64Cu-DOTA-aKLH mAb were 0.74 and 0.82 MBq/μg, respectively. The immunoreactivity retention after radiolabeling of 11-25 mAb against MSLN protein was measured by ELISA (Figure 4). At the concentration of 1000 ng/mL, the OD value of 64Cu-DOTA-11-25 mAb was 66.0% compared to that of native 11-25 mAb. In vitro stability in plasma after 24 and 48 hours of incubation analyzed by HPLC was 78.0% and 76.9% for 64Cu-DOTA-11-25 mAb and 82.8% and 77.9% for 64Cu-DOTA-anti-KLH mAb. The same preparations were also analyzed by TLC ARG (Figure 5(a)) and the results tended to be higher (94.3% and 92.6% for 64Cu-DOTA-11-25 mAb) than those analyzed by HPLC.


A novel PET imaging using ⁶⁴Cu-labeled monoclonal antibody against mesothelin commonly expressed on cancer cells.

Kobayashi K, Sasaki T, Takenaka F, Yakushiji H, Fujii Y, Kishi Y, Kita S, Shen L, Kumon H, Matsuura E - J Immunol Res (2015)

In vitro and in vivo stability of 64Cu-DOTA-11-25 mAb and 64DOTA-anti-KLH mAb. (a) In vitro stability of 64Cu-DOTA-11-25 mAb (black bars) and 64DOTA-anti-KLH mAb (white bars) after 24 hours and 48 hours of incubation at 37°C. Radioactivity derived from antibody-bound 64Cu stays at the origin and unbound 64Cu goes to the top of the chromatogram. The percentage of radioactivity that remained in the origin of TLC was shown as the stability. (b) Representative TLC-ARG of plasma and supernatant of homogenate from liver and kidney 24 hours after injection of 64Cu-DOTA-11-25 mAb. Aliquot of blood, liver, and kidney was taken from mice 24 hours after injection and analyzed by TLC-ARG as described in Section 2. (c) In vivo stability of 64Cu-DOTA-11-25 mAb (black bars) and 64Cu-DOTA-anti-KLH mAb (white bars) 24 and 48 hours after injection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390102&req=5

fig5: In vitro and in vivo stability of 64Cu-DOTA-11-25 mAb and 64DOTA-anti-KLH mAb. (a) In vitro stability of 64Cu-DOTA-11-25 mAb (black bars) and 64DOTA-anti-KLH mAb (white bars) after 24 hours and 48 hours of incubation at 37°C. Radioactivity derived from antibody-bound 64Cu stays at the origin and unbound 64Cu goes to the top of the chromatogram. The percentage of radioactivity that remained in the origin of TLC was shown as the stability. (b) Representative TLC-ARG of plasma and supernatant of homogenate from liver and kidney 24 hours after injection of 64Cu-DOTA-11-25 mAb. Aliquot of blood, liver, and kidney was taken from mice 24 hours after injection and analyzed by TLC-ARG as described in Section 2. (c) In vivo stability of 64Cu-DOTA-11-25 mAb (black bars) and 64Cu-DOTA-anti-KLH mAb (white bars) 24 and 48 hours after injection.
Mentions: DOTA-conjugated 11-25 mAb and anti-KLH mAb were incubated with 64Cu and purified as described in Section 2. The radiochemical purity of resultant 64Cu-DOTA-mAbs determined by HPLC was 97.9% for 11-25 mAb and 100% for anti-KLH mAb. The specific activities of 64Cu-DOTA-11-25 mAb and 64Cu-DOTA-aKLH mAb were 0.74 and 0.82 MBq/μg, respectively. The immunoreactivity retention after radiolabeling of 11-25 mAb against MSLN protein was measured by ELISA (Figure 4). At the concentration of 1000 ng/mL, the OD value of 64Cu-DOTA-11-25 mAb was 66.0% compared to that of native 11-25 mAb. In vitro stability in plasma after 24 and 48 hours of incubation analyzed by HPLC was 78.0% and 76.9% for 64Cu-DOTA-11-25 mAb and 82.8% and 77.9% for 64Cu-DOTA-anti-KLH mAb. The same preparations were also analyzed by TLC ARG (Figure 5(a)) and the results tended to be higher (94.3% and 92.6% for 64Cu-DOTA-11-25 mAb) than those analyzed by HPLC.

Bottom Line: Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum.Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN.In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan ; Department of Urology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan.

ABSTRACT
Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with (64)Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that (64)Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The (64)Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than (18)F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

No MeSH data available.


Related in: MedlinePlus