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Highly sensitive detection of invasive lung cancer cells by novel antibody against amino-terminal domain of laminin ?2 chain.

ABSTRACT: The laminin ?2 chain, a subunit of laminin-332 (?3?3?2), is a molecular marker for invasive cancer cells, but its pathological roles in tumor progression remain to be clarified. It was recently found that the most N-terminal, domain V (dV) of ?2 chain has activities to bind CD44 and stimulate tumor cell migration and vascular permeability. In the present study, we prepared a mAb recognizing ?2 dV. Immunoblotting with this antibody, for the first time, showed that proteolytic fragments containing dV in a range of 15-80 kDa were highly produced in various human cancer cell lines and lung cancer tissues. In immunohistochemistry of adenocarcinomas and squamous cell carcinomas of the lung, this antibody immunostained the cytoplasm of invasive tumor cells and adjacent stroma much more strongly than a widely used antibody recognizing the C-terminal core part of the processed ?2 chain. This suggests that the dV fragments are highly accumulated in tumor cells and stroma compared to the processed ?2 protein. The strong tumor cell staining with the dV antibody correlated with the tumor malignancy grade. We also found that the laminin ?3 and ?3 chains were frequently overexpressed in tumor cells and tumor stroma, respectively. The cytoplasmic dV detection was especially prominent in tumor cells infiltrating stroma, but low in the cells surrounded by basement membranes, suggesting that the active tumor-stroma interaction is critical for the aberrant ?2 expression. The present study suggests important roles of laminin ?2 N-terminal fragments in tumor progression.

SUBMITTER: Miyazaki K

PROVIDER: S-EPMC5198959 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Highly sensitive detection of invasive lung cancer cells by novel antibody against amino-terminal domain of laminin γ2 chain.

Miyazaki Kaoru K Oyanagi Jun J Sugino Atsuko A Sato Hiroki H Yokose Tomoyuki T Nakayama Haruhiko H Miyagi Yohei Y

Cancer science 20161212 12

The laminin γ2 chain, a subunit of laminin-332 (α3β3γ2), is a molecular marker for invasive cancer cells, but its pathological roles in tumor progression remain to be clarified. It was recently found that the most N-terminal, domain V (dV) of γ2 chain has activities to bind CD44 and stimulate tumor cell migration and vascular permeability. In the present study, we prepared a mAb recognizing γ2 dV. Immunoblotting with this antibody, for the first time, showed that proteolytic fragments containing ...[more]

PMID: 27685891

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Project description:The rapidly evolving nature of antibody drug development has resulted in technologies that generate vast numbers (hundreds to thousands) of lead antibody candidates during early discovery. These candidates must be rapidly pared down to identify the most drug-like candidates for in-depth analysis of their safety and efficacy, which can only be performed on a limited number of antibodies due to time and resource requirements. One key biophysical property of successful antibody therapeutics is high specificity, defined as low levels of nonspecific binding or polyspecificity. Although there has been some progress in developing assays for detecting antibody polyspecificity, most of these assays are limited by poor sensitivity or assay formats that require proprietary antibody surface display methods, and some of these assays use complex and poorly defined polyspecificity reagents. Here we report the PolySpecificity Particle (PSP) assay, a sensitive flow cytometry assay for evaluating antibody nonspecific interactions that overcomes previous limitations and can be used for evaluating diverse types of IgGs, multispecific antibodies and Fc-fusion proteins. Our approach uses micron-sized magnetic beads coated with Protein A to capture antibodies at extremely dilute concentrations (<0.02 mg/mL). Flow cytometry analysis of polyspecificity reagent binding to these conjugates results in sensitive detection of differences in nonspecific interactions for clinical-stage antibodies. Our PSP assay strongly discriminates between antibodies with different levels of polyspecificity using previously reported polyspecificity reagents that are either well-defined proteins or highly complex protein mixtures. Moreover, we also find that a unique reagent, namely ovalbumin, results in the best assay sensitivity and specificity. Importantly, our assay is much more sensitive than standard assays such as ELISAs. We expect that our simple, sensitive, and high-throughput PSP assay will accelerate the development of safe and effective antibody therapeutics.

Dataset Information

Highly sensitive detection of invasive lung cancer cells by novel antibody against amino-terminal domain of laminin ?2 chain.

Publications

Highly sensitive detection of invasive lung cancer cells by novel antibody against amino-terminal domain of laminin γ2 chain.

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