A Divalent Recombinant Immunotoxin Formed by a Disulfide Bond between the Extension Peptide Chains

Ji Hae Park; Hye Won Kwon; Hee Kyoung Chung; Ik Hwan Kim; Kwangseog Ahn; Eui Ju Choi; Ira Pastan; Mu Hyeon Choe

A Divalent Recombinant Immunotoxin Formed by a Disulfide Bond between the Extension Peptide Chains

Ji Hae Park, Hye Won Kwon, Hee Kyoung Chung, Ik Hwan Kim, Kwangseog Ahn, Eui Ju Choi, Ira Pastan, Mu Hyeon Choe

Research output: Contribution to journal › Article › peer-review

Abstract

Recombinant immunotoxin for the treatment of cancer was made by connecting toxins to 'carcinoma-specific' antibodies that selectively bind to cancer cells, then kills them without harming the normal cells. The divalent recombinant immunotoxin, [B3(Fab)-ext-PE38]₂, is a derivative of B3(Fab)-PE38. B3(Fab)-PE38 was made by fusing the Fab domain of the monoclonal antibody (MAb) B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE). In this study, B3(Fab)-ext-PE38 was constructed, which has the hinge region of the B3(Fab)-PE38 extended with the peptide extension, G₄C(G₄S)₂, and connected to the C3 connector. The Cys residue of the extension peptide chain makes the disulfide bond between the two Fab domains. The extension sequence (ext) makes the dimerization of B3(Fab)-ext-PE38 easier to form the divalent immunotoxin, because it decreases the steric hindrance between the two PE38s. The constructed genes were expressed in E. coli as inclusion bodies. Polypeptides that were obtained from the inclusion body were refolded, and the active forms were purified. The ID₅₀ values of the divalent molecule, [B3(Fab)-ext-PE38]₂, were about 4 ng/ml on A431 cell lines, about 1 ng/ml on CRL1739 cell lines, and 5 ng/ml on MCF-7 cell lines. The [B3(Fab)-ext-PE38]₂ showed about a 12-fold higher cytotoxicity on CRL1739 cell lines than B3(scFv)-PE40 did.

Original language	English
Pages (from-to)	398-402
Number of pages	5
Journal	Molecules and cells
Volume	12
Issue number	3
Publication status	Published - 2001 Dec 31

Keywords

Antibody Refolding
B3 Antibody
Cytotoxicity
Divalent Immunotoxin
Pseudomonas Exotoxin A
[B3(Fab)-ext-PE38]

ASJC Scopus subject areas

Molecular Biology
Cell Biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Cite this

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title = "A Divalent Recombinant Immunotoxin Formed by a Disulfide Bond between the Extension Peptide Chains",

abstract = "Recombinant immunotoxin for the treatment of cancer was made by connecting toxins to 'carcinoma-specific' antibodies that selectively bind to cancer cells, then kills them without harming the normal cells. The divalent recombinant immunotoxin, [B3(Fab)-ext-PE38]2, is a derivative of B3(Fab)-PE38. B3(Fab)-PE38 was made by fusing the Fab domain of the monoclonal antibody (MAb) B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE). In this study, B3(Fab)-ext-PE38 was constructed, which has the hinge region of the B3(Fab)-PE38 extended with the peptide extension, G4C(G4S)2, and connected to the C3 connector. The Cys residue of the extension peptide chain makes the disulfide bond between the two Fab domains. The extension sequence (ext) makes the dimerization of B3(Fab)-ext-PE38 easier to form the divalent immunotoxin, because it decreases the steric hindrance between the two PE38s. The constructed genes were expressed in E. coli as inclusion bodies. Polypeptides that were obtained from the inclusion body were refolded, and the active forms were purified. The ID50 values of the divalent molecule, [B3(Fab)-ext-PE38]2, were about 4 ng/ml on A431 cell lines, about 1 ng/ml on CRL1739 cell lines, and 5 ng/ml on MCF-7 cell lines. The [B3(Fab)-ext-PE38]2 showed about a 12-fold higher cytotoxicity on CRL1739 cell lines than B3(scFv)-PE40 did.",

keywords = "Antibody Refolding, B3 Antibody, Cytotoxicity, Divalent Immunotoxin, Pseudomonas Exotoxin A, [B3(Fab)-ext-PE38]",

author = "Park, {Ji Hae} and Kwon, {Hye Won} and Chung, {Hee Kyoung} and Kim, {Ik Hwan} and Kwangseog Ahn and Choi, {Eui Ju} and Ira Pastan and Choe, {Mu Hyeon}",

year = "2001",

month = dec,

day = "31",

language = "English",

volume = "12",

pages = "398--402",

journal = "Molecules and cells",

issn = "1016-8478",

publisher = "Elsevier B.V.",

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TY - JOUR

T1 - A Divalent Recombinant Immunotoxin Formed by a Disulfide Bond between the Extension Peptide Chains

AU - Park, Ji Hae

AU - Kwon, Hye Won

AU - Chung, Hee Kyoung

AU - Kim, Ik Hwan

AU - Ahn, Kwangseog

AU - Choi, Eui Ju

AU - Pastan, Ira

AU - Choe, Mu Hyeon

PY - 2001/12/31

Y1 - 2001/12/31

N2 - Recombinant immunotoxin for the treatment of cancer was made by connecting toxins to 'carcinoma-specific' antibodies that selectively bind to cancer cells, then kills them without harming the normal cells. The divalent recombinant immunotoxin, [B3(Fab)-ext-PE38]2, is a derivative of B3(Fab)-PE38. B3(Fab)-PE38 was made by fusing the Fab domain of the monoclonal antibody (MAb) B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE). In this study, B3(Fab)-ext-PE38 was constructed, which has the hinge region of the B3(Fab)-PE38 extended with the peptide extension, G4C(G4S)2, and connected to the C3 connector. The Cys residue of the extension peptide chain makes the disulfide bond between the two Fab domains. The extension sequence (ext) makes the dimerization of B3(Fab)-ext-PE38 easier to form the divalent immunotoxin, because it decreases the steric hindrance between the two PE38s. The constructed genes were expressed in E. coli as inclusion bodies. Polypeptides that were obtained from the inclusion body were refolded, and the active forms were purified. The ID50 values of the divalent molecule, [B3(Fab)-ext-PE38]2, were about 4 ng/ml on A431 cell lines, about 1 ng/ml on CRL1739 cell lines, and 5 ng/ml on MCF-7 cell lines. The [B3(Fab)-ext-PE38]2 showed about a 12-fold higher cytotoxicity on CRL1739 cell lines than B3(scFv)-PE40 did.

AB - Recombinant immunotoxin for the treatment of cancer was made by connecting toxins to 'carcinoma-specific' antibodies that selectively bind to cancer cells, then kills them without harming the normal cells. The divalent recombinant immunotoxin, [B3(Fab)-ext-PE38]2, is a derivative of B3(Fab)-PE38. B3(Fab)-PE38 was made by fusing the Fab domain of the monoclonal antibody (MAb) B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE). In this study, B3(Fab)-ext-PE38 was constructed, which has the hinge region of the B3(Fab)-PE38 extended with the peptide extension, G4C(G4S)2, and connected to the C3 connector. The Cys residue of the extension peptide chain makes the disulfide bond between the two Fab domains. The extension sequence (ext) makes the dimerization of B3(Fab)-ext-PE38 easier to form the divalent immunotoxin, because it decreases the steric hindrance between the two PE38s. The constructed genes were expressed in E. coli as inclusion bodies. Polypeptides that were obtained from the inclusion body were refolded, and the active forms were purified. The ID50 values of the divalent molecule, [B3(Fab)-ext-PE38]2, were about 4 ng/ml on A431 cell lines, about 1 ng/ml on CRL1739 cell lines, and 5 ng/ml on MCF-7 cell lines. The [B3(Fab)-ext-PE38]2 showed about a 12-fold higher cytotoxicity on CRL1739 cell lines than B3(scFv)-PE40 did.

KW - Antibody Refolding

KW - B3 Antibody

KW - Cytotoxicity

KW - Divalent Immunotoxin

KW - Pseudomonas Exotoxin A

KW - [B3(Fab)-ext-PE38]

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M3 - Article

C2 - 11804341

AN - SCOPUS:0035981227

SN - 1016-8478

VL - 12

SP - 398

EP - 402

JO - Molecules and cells

JF - Molecules and cells

IS - 3

ER -

A Divalent Recombinant Immunotoxin Formed by a Disulfide Bond between the Extension Peptide Chains

Abstract

Keywords

ASJC Scopus subject areas

UN SDGs

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