Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display

Ahn, Hyun Joo; Kim, Yong-Sam; Lee, Chul‐Ho; Cho, Eun‐Wie; Yoo, Hyang–Sook; Kim, Seung‐Ho; Ko, Jeong–Heon; Kim, Sang Jick

doi:10.1016/j.jbiotec.2010.12.004

Cited by 7 publications

(16 citation statements)

References 28 publications

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“…The pIII-display phagemid pDR-D1 vector, a gift from Dr. Sang-Jick Kim (KRIBB, Korea), has the carboxy-terminal domain (residues 230–406) of pIII in downstream of the PelB secretion leader sequence with a myc tag sequence (EQKLISEEDL) between the insert and pIII sequences and a 6×His tag in the C-terminus of pIII (PelB- Sfi I-insert- Sfi I-myc- Not I-pIII- Not I-6×his) under the control of lac promoter [29] (Figure S1A). The open reading frame of 3D8 VL transbody [13], [14], TAT peptide (residues 48–60, GRKKRRQRRRPPQ) [23], and anti-death receptor 4 (DR4, TRAIL receptor 1) hAY4 scFv [30] was subcloned with Sfi I into the N-terminus of pIII, resulting in fusion phagemids encoding 3D8 VL-pIII, TAT-pIII, and hAY4 scFv-pIII fusion proteins, respectively (Figure S1A).…”

Section: Methodsmentioning

confidence: 99%

“…VCSM13 helper phage (Stratagene, La Jolla, CA) was prepared as previously described [29]. The recombinant phagemid vectors were electroporated into E. coli ER2738 cells.…”

Section: Methodsmentioning

confidence: 99%

“…The recombinant phagemid vectors were electroporated into E. coli ER2738 cells. Recombinant phages displaying the insert-pIII were recovered by superinfection of phagemid-bearing bacteria with the VCSM13 helper phage at a multiplicity of infection (MOI) of 20∶1 (phage-to-cell ratio) [29], [31]. Briefly, ER2738 cells carrying fusion phagemids were grown at 37°C to mid-log phase (OD 600 of 0.6–0.8) in 2×YT medium containing 100 µg/ml ampicillin and 2% glucose (2×YT/AG) medium.…”

Section: Methodsmentioning

confidence: 99%

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Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

Kim

Shin

et al. 2012

PLoS ONE

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Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

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Section: Methodsmentioning

confidence: 99%

“…VCSM13 helper phage (Stratagene, La Jolla, CA) was prepared as previously described [29]. The recombinant phagemid vectors were electroporated into E. coli ER2738 cells.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

Kim

Shin

et al. 2012

PLoS ONE

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“…The low sensitivity observed especially when monitoring the glycans of sparse candidate biomarkers is attributable to the low affinity of lectins. These obstacles may be overcome by attempts to develop glycan-specific antibodies with sufficient specificity and affinity (51). Finally, the glycan library that has been found to exist in nature is not expansive enough to personally classify every patient by using the glycan alone.…”

Section: Promises and Limitations In The Use Of Aberrant Glycosylatiomentioning

confidence: 99%

Pros and cons of using aberrant glycosylation as companion biomarkers for therapeutics in cancer

Kang¹,

Ko²,

Kim³

2011

BMB Reports

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Cancer treatment has been stratified by companion biomarker tests that serve to provide information on the genetic status of cancer patients and to identify patients who can be expected to respond to a given treatment. This stratification guarantees better efficiency and safety during treatment. Cancer patients, however, marginally benefit from the current companion biomarker-aided treatment regimens, presumably because companion biomarker tests are dependent solely on the mutation status of several genes status quo. In the true sense of the term, "personalized medicine", cancer patients are deemed to be identified individually by their molecular signatures, which are not necessarily confined to genetic mutations. Glycosylation is tremendously dynamic and shows alterations in cancer. Evidence is accumulating that aberrant glycosylation contributes to the development and progression of cancer, holding the promise for use of glycosylation status as a companion biomarker in cancer treatment. There are, however, several challenges derived from the lack of a reliable detection system for aberrant glycosylation, and a limited library of aberrant glycosylation. The challenges should be addressed if glycosylation status is to be used as a companion biomarker in cancer treatment and contribute to the fulfillment of personalized medicine. [BMB reports 2011; 44(12): 765-771]

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“…The aberrant glycans on TIMP-1, induced by GlcNAcT-V, may affect the properties of binding with gelatinases, presumably by conferring a steric hindrance arising from the massiveness of glycosylation and an electrostatic repulsion arising from the attachment of acidic residues to the binding to gelatinases[94,95]. Because of the functional role of aberrant glycosylation of TIMP-1, Ahn et al [96] generated antibodies recognizing an aberrant glycoform of TIMP-1. Such efforts will be very helpful in the development of immune-based assays to evaluate the clinical performance and utilities of aberrant glycoforms of TIMP-1 in a personalized approach to cancer diagnosis and treatment.…”

Section: Introductionmentioning

confidence: 99%

Aberrant glycosylation associated with enzymes as cancer biomarkers

2011

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BackgroundOne of the new roles for enzymes in personalized medicine builds on a rational approach to cancer biomarker discovery using enzyme-associated aberrant glycosylation. A hallmark of cancer, aberrant glycosylation is associated with differential expressions of enzymes such as glycosyltransferase and glycosidases. The aberrant expressions of the enzymes in turn cause cancer cells to produce glycoproteins with specific cancer-associated aberrations in glycan structures.ContentIn this review we provide examples of cancer biomarker discovery using aberrant glycosylation in three areas. First, changes in glycosylation machinery such as glycosyltransferases/glycosidases could be used as cancer biomarkers. Second, most of the clinically useful cancer biomarkers are glycoproteins. Discovery of specific cancer-associated aberrations in glycan structures of these existing biomarkers could improve their cancer specificity, such as the discovery of AFP-L3, fucosylated glycoforms of AFP. Third, cancer-associated aberrations in glycan structures provide a compelling rationale for discovering new biomarkers using glycomic and glycoproteomic technologies.SummaryAs a hallmark of cancer, aberrant glycosylation allows for the rational design of biomarker discovery efforts. But more important, we need to translate these biomarkers from discovery to clinical diagnostics using good strategies, such as the lessons learned from translating the biomarkers discovered using proteomic technologies to OVA 1, the first FDA-cleared In Vitro Diagnostic Multivariate Index Assay (IVDMIA). These lessons, providing important guidance in current efforts in biomarker discovery and translation, are applicable to the discovery of aberrant glycosylation associated with enzymes as cancer biomarkers as well.

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Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display

Cited by 7 publications

References 28 publications

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

Pros and cons of using aberrant glycosylation as companion biomarkers for therapeutics in cancer

Aberrant glycosylation associated with enzymes as cancer biomarkers

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