TY - JOUR
T1 - A Novel High-Throughput Screening Method for a Human Multicentric Osteosarcoma-Specific Antibody and Biomarker Using a Phage Display-Derived Monoclonal Antibody
AU - Hayashi, Takuma
AU - Yamamoto, Naoki
AU - Kurosawa, Gene
AU - Tajima, Kaori
AU - Kondo, Mariko
AU - Hiramatsu, Noriko
AU - Kato, Yu
AU - Tanaka, Miho
AU - Yamaguchi, Hisateru
AU - Kurosawa, Yoshikazu
AU - Yamada, Harumoto
AU - Fujita, Nobuyuki
N1 - Funding Information:
This work was supported by JSPS KAKENHI (grant number JP23592201). This research was supported by JST START University Ecosystem Promotion Type (Supporting Creation of Startup Ecosystem in Startup Cities), Grant Number JPMJST2183, Japan.
Funding Information:
We established two new HMOS cell lines (), one of which was derived from the anterior (i.e., primary) lesion after an incisional biopsy of the left humerus (designated as HMOS-A), and the other was derived from one of the posterior (i.e., secondary) lesions of the sternum (designated as HMOS-P) []. To compare the cell lines, we used the following HOS cell lines: MNNG-HOS (EC87070201; European Collection of Authenticated Cell Cultures: ECACC, Salisbury, UK), MG63 (EC86051601; ECACC), and Saos2 (EC89050205; ECACC). MNNG-HOS, MG63, and Saos2 cells were cultured in accordance with the culture conditions of ECACC. HOS is defined as malignant tumors that form osteoids. Per this definition, NOS-1 (RCB1032) and NOS-2 (RCB1033) HOS cell lines that form osteoids in vitro or in xenograft models were used to validate HMOS-specific proteins. NOS-1 and NOS-2 cells were provided by the Riken BioResource Research Center (RIKEN BRC) through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Mycoplasma contamination in the cell lines was inspected using a mycoplasma detection kit (EZ-PCR Mycoplasma Test Kit, Biological Industries, Cromwell, CT, USA), according to the manufacturer’s instructions. This study was approved by the Ethics Committee of Fujita Health University (approval number: 15-181). TM
Publisher Copyright:
© 2022 by the authors.
PY - 2022/12
Y1 - 2022/12
N2 - Osteosarcoma is a malignant tumor that produces neoplastic bone or osteoid osteoma. In human multicentric osteosarcoma (HMOS), a unique variant of human osteosarcoma (HOS), multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HMOS is associated with an extremely poor prognosis, and effective treatment options are lacking. Using the proteins in our previously generated HMOS cell lines as antigens, we generated antibodies using a human antibody phage library. We obtained antibody clones recognizing 95 independent antigens and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) technique capable of evaluating the reactivity of these antibodies by fluorescence intensity, allowing simple, rapid, and high-throughput selection of antibody clones. These results were highly correlated with those using flow cytometry. Subsequently, the HMOS cell lysate was incubated with the antibody, the antigen–antibody complex was recovered with magnetic beads, and the protein bands from electrophoresis were analyzed using liquid chromatography-mass spectrometry (LC/MS). CAVIN1/polymerase I transcript release factor was specifically detected in the HMOS cells. In conclusion, we found via a novel high-throughput screening method that CAVIN1/PTRF is an HMOS-specific cell membrane biomarker and an antigen capable of producing human antibodies. In the future, antibody–drug conjugate targeting of these specific proteins may be promising for clinical applications.
AB - Osteosarcoma is a malignant tumor that produces neoplastic bone or osteoid osteoma. In human multicentric osteosarcoma (HMOS), a unique variant of human osteosarcoma (HOS), multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HMOS is associated with an extremely poor prognosis, and effective treatment options are lacking. Using the proteins in our previously generated HMOS cell lines as antigens, we generated antibodies using a human antibody phage library. We obtained antibody clones recognizing 95 independent antigens and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) technique capable of evaluating the reactivity of these antibodies by fluorescence intensity, allowing simple, rapid, and high-throughput selection of antibody clones. These results were highly correlated with those using flow cytometry. Subsequently, the HMOS cell lysate was incubated with the antibody, the antigen–antibody complex was recovered with magnetic beads, and the protein bands from electrophoresis were analyzed using liquid chromatography-mass spectrometry (LC/MS). CAVIN1/polymerase I transcript release factor was specifically detected in the HMOS cells. In conclusion, we found via a novel high-throughput screening method that CAVIN1/PTRF is an HMOS-specific cell membrane biomarker and an antigen capable of producing human antibodies. In the future, antibody–drug conjugate targeting of these specific proteins may be promising for clinical applications.
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U2 - 10.3390/cancers14235829
DO - 10.3390/cancers14235829
M3 - Article
AN - SCOPUS:85143597654
SN - 2072-6694
VL - 14
JO - Cancers
JF - Cancers
IS - 23
M1 - 5829
ER -