Combining phenotypic and proteomic approaches to identify membrane targets in a 'triple negative' breast cancer cell type.
Combining phenotypic and proteomic approaches to identify membrane targets in a 'triple negative' breast cancer cell type.
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DOI：
10.1186/1476-4598-12-11
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发表时间：
2013-02-13
期刊：
影响因子：
37.3
通讯作者：
Minter R
中科院分区：
文献类型：
--
作者：
Rust S;Guillard S;Sachsenmeier K;Hay C;Davidson M;Karlsson A;Karlsson R;Brand E;Lowne D;Elvin J;Flynn M;Kurosawa G;Hollingsworth R;Jermutus L;Minter R
继续发现治疗性抗体，以解决未满足的医疗需求，需要继续发现易于处理的抗体靶点。多种蛋白水平的靶标发现方法是可用的，这些方法可以结合起来广泛地调查相关的细胞膜。在这项研究中，选择MDA-MB-231细胞系进行膜组调查，因为它是一种“三阴性”乳腺癌细胞系，它代表了一种侵袭性强且治疗选择很少的癌症亚型。以MDA-MB-231乳腺癌细胞系为研究对象，探索三种膜体靶点发现方法，并平行交叉验证鉴定抗原的意义。一种蛋白质组学方法，使用膜蛋白富集，然后通过质谱鉴定蛋白质，与两种表型抗体筛选方法一起使用。第一种表型筛选方法是基于杂交瘤技术，第二种是基于噬菌体展示技术。通过表型方法分离的抗体进行了细胞特异性和内化测试，鉴定的靶标相互比较，以及通过蛋白质组学方法鉴定的靶标。从基于噬菌体展示的表型方法中获得的抗cd73抗体被测试与其他“三阴性”乳腺癌细胞系的结合，并在MDA-MB-231异种移植模型中测试了肿瘤生长抑制活性。所有这些方法都鉴定出多种细胞表面标记物，包括整合素、CD44、EGFR、CD71、半乳糖凝集素-3、CD73和BCAM，其中一些先前已被证实可用于抗体治疗。共鉴定出40个细胞表面标记物供进一步研究。除了细胞表面标记物鉴定外，表型抗体筛选方法为靶标验证研究提供了试剂抗体。这是用抗cd73抗体证明的，该抗体结合其他“三阴性”乳腺癌细胞系，并在MDA-MB-231异种移植模型中产生显著的肿瘤生长抑制活性。这项研究表明，需要多种方法来成功分析所需细胞类型的膜体。它还成功地证明了表型抗体筛选提供了一种快速发现和评估抗体可处理靶点的机制，这可以显着加快治疗发现过程。
The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated using the anti-CD73 antibody, which bound other ‘triple negative’ breast cancer cell lines and produced significant tumour growth inhibitory activity in a MDA-MB-231 xenograft model. This study has demonstrated that multiple methods are required to successfully analyse the membranome of a desired cell type. It has also successfully demonstrated that phenotypic antibody screening provides a mechanism for rapidly discovering and evaluating antibody tractable targets, which can significantly accelerate the therapeutic discovery process.
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DOI：
10.1093/jnci/53.3.661
发表时间：
1974-01-01
期刊：
JOURNAL OF THE NATIONAL CANCER INSTITUTE
影响因子：
--
作者：
CAILLEAU, R;YOUNG, R;REEVES, WJ
通讯作者：
REEVES, WJ
DOI：
10.1073/pnas.0712202105
发表时间：
2008-05-20
影响因子：
11.1
作者：
Kurosawa, Gene;Akahori, Yasushi;Kurosawa, Yoshikazu
通讯作者：
Kurosawa, Yoshikazu
影响因子：
120.1
作者：
Swinney, David C.;Anthony, Jason
通讯作者：
Anthony, Jason
影响因子：
3.8
作者：
Pal, Sumanta Kumar;Childs, Barrett H.;Pegram, Mark
通讯作者：
Pegram, Mark
影响因子：
3.1
作者：
Padliya, Neerav D.;Bhatia, Mohit B.;Hariri, Robert J.
通讯作者：
Hariri, Robert J.