Applying Phage Antibodies to Proteomics: Selecting Single Chain Fv Antibodies to Antigens Blotted on Nitrocellulose

Liu, Bin; Marks, James D.

doi:10.1006/abio.2000.4788

Cited by 48 publications

(42 citation statements)

References 34 publications

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“…The library was created by subcloning human scFv gene repertoires from a naive phagemid (26) into a phage vector for multivalent display (25,27). The library was preincubated with control cells (BPH-1 and LP9/hTERT) at 4jC for 4 h to reduce binders to common cell surface antigens as described (17).…”

Section: Methodsmentioning

confidence: 99%

Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies

An¹,

Drummond²,

Wilson³

et al. 2008

Molecular Cancer Therapeutics

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Mesothelioma is a malignancy of the mesothelium and current treatments are generally ineffective. One promising area of anticancer drug development is to explore tumor susceptibility to targeted therapy. To achieve efficient, targeted intracellular delivery of therapeutic agents to mesothelioma cells, we selected a naive human singlechain (scFv) phage antibody display library directly on the surface of live mesothelioma cells to identify internalizing antibodies that target mesothelioma-associated cell surface antigens. We have identified a panel of internalizing scFvs that bind to mesothelioma cell lines derived from both epithelioid (M28) and sarcomatous (VAMT-1) types of this disease. Most importantly, these antibodies stain mesothelioma cells in situ and therefore define a panel of clinically represented tumor antigens. We have further exploited the internalizing function of these scFvs to achieve targeted intracellular drug delivery to mesothelioma cells. We showed that scFv-targeted immunoliposomes were efficiently and specifically taken up by both epithelioid and sarcomatous mesothelioma cells, but not control cells, and immunoliposomes encapsulating the small-molecule drug topotecan caused targeted killing of both types of mesothelioma cells in vitro. [Mol Cancer Ther 2008;7(3):569 -78]

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Section: Methodsmentioning

confidence: 99%

Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies

An¹,

Drummond²,

Wilson³

et al. 2008

Molecular Cancer Therapeutics

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“…recAbs are usually selected by utilizing phage display technologies (21), or other selection principles, such as ribosomal display (20). Much of the in vitro selection technology has been based on recAb fragments, especially scFvs and Fabs (24), but alternative protein scaffolds have also been used (25). Nonproteinaceous binding ligands, such as the in vitro selected aptamers (26), have also been successfully utilized for specific recognition of target molecules.…”

Section: Generation Of Antibodiesmentioning

confidence: 99%

Antibody-based Proteomics for Human Tissue Profiling

Uhlén

Pontén

2005

Molecular & Cellular Proteomics

263

161

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Here, we describe the use of antibody-based proteomics involving the generation of protein-specific antibodies to functionally explore the human proteome. The antibodies can be used for analysis of corresponding proteins in a wide range of assay platforms, including i) immunohistochemistry for detailed tissue profiling, ii) specific affinity reagents for various functional protein assays, and iii) capture ("pull-down") reagents for purification of specific proteins and their associated complexes for structural and biochemical analyses. In this review, the use of antibodies for such analysis will be discussed with focus on the possibility to create a descriptive and comprehensive protein atlas for tissue distribution and subcellular localization of human proteins in both normal and disease tissues.

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“…A multivalent fd phage display library consisting of 5 ϫ 10 8 members was derived from a 7 ϫ 10 9 -member phagemid library (16) by subcloning the SfiI/NotI scFv insert from pHEN1 into bacterial vector fd-SfiI/NotI (25)(26)(27). Phage were produced via growth in culture of Escherichia coli TG1, concentrated by precipitation with polyethylene glycol 8000, and purified by CsCl gradient centrifugation, as reported previously (25)(26)(27).…”

Section: Methodsmentioning

confidence: 99%

“…We applied this approach to breast tumor cells and generated mAbs to a number of known internalizing receptors, including epidermal growth factor receptor and ErbB2 (23,24). To broaden applicability, libraries of phage displaying multiple copies of scFv were engineered (25)(26)(27), which unlike existing phage libraries can cross-link receptors, allowing more efficient phage endocytosis (21).…”

Section: Introductionmentioning

confidence: 99%

Mapping Tumor Epitope Space by Direct Selection of Single-Chain Fv Antibody Libraries on Prostate Cancer Cells

Liu¹,

Conrad²,

Cooperberg

et al. 2004

Cancer Research

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111

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The identification of tumor-specific cell surface antigens is a critical step toward the development of targeted therapeutics for cancer. The epitope space at the tumor cell surface is highly complex, composed of proteins, carbohydrates, and other membrane-associated determinants including post-translational modification products, which are difficult to probe by approaches based on gene expression. This epitope space can be efficiently mapped by complementary monoclonal antibodies. By selecting human antibody gene diversity libraries directly on the surface of prostate cancer cells, we have taken a functional approach to identifying fully human, tumor-specific monoclonal antibodies without prior knowledge of their target antigens. Selection conditions have been optimized to favor tumor-specific antibody binding and internalization. To date, we have discovered >90 monoclonal antibodies that specifically bind and enter prostate cancer cells, with little or no binding to control cells. These antibodies are able to efficiently deliver intracellular payloads when attached to nanoparticles such as liposomes. In addition, a subset of the antibodies displayed intrinsic antiproliferative activity. These tumorspecific internalizing antibodies are likely to be useful for targeted therapeutics either alone or in combination with effector molecules. The antigens they bind constitute a tumor-specific internalizing epitope space that is likely to play a significant role in cancer cell homeostasis. Targeting components of this epitope space may facilitate development of immunotherapeutic and small molecule-based strategies as well as the use of other therapeutic agents that rely upon delivery to the interior of the tumor cell.

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Applying Phage Antibodies to Proteomics: Selecting Single Chain Fv Antibodies to Antigens Blotted on Nitrocellulose

Cited by 48 publications

References 34 publications

Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies

Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies

Antibody-based Proteomics for Human Tissue Profiling

Mapping Tumor Epitope Space by Direct Selection of Single-Chain Fv Antibody Libraries on Prostate Cancer Cells

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