Expression and characterization of a chimeric bispecific antibody against fibrin and against urokinase-type plasminogen activator

Hiroko Tada, Tomofumi Kurokawa, Takeshi Seita, Takeshi Watanabe, Susumu Iwasa

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

We have produced a chimeric bispecific antibody that has dual specificity of human fibrin and urokinase-type plasminogen activator (u-PA). Complementary DNAs for variable regions of both anti-fibrin and anti-u-PA antibodies were cloned from two murine hybridomas secreting respective antibodies using polymerase chain reaction (PCR) techniques, and joined to cDNAs for human constant regions to form chimeric antibody genes. Both of two expression vectors for chimeric anti-fibrin and chimeric anti-u-PA antibodies were sequentially introduced into Chinese hamster ovary cells, and stable transfectants secreting the chimeric bispecific antibody were obtained. The highest producer transfectant (SULF/C2-30) secreted high level (about 40 μg ml-1) of total chimeric IgG and about 2% of the IgG had the bispecific activity of binding with both antigens. The chimeric bispecific antibody was purified by a combination of affinity chromatographies employing antigen-coupled columns and hydroxyapatite high-performance liquid chromatography. The purified chimeric bispecific antibody significantly enhanced the thrombolytic potency of single chain u-PA in an in vitro clot lysis assay as well as the original murine bispecific antibody.

Original languageEnglish
Pages (from-to)157-174
Number of pages18
JournalJournal of Biotechnology
Volume33
Issue number2
DOIs
Publication statusPublished - Mar 31 1994
Externally publishedYes

Fingerprint

Bispecific Antibodies
Urokinase-Type Plasminogen Activator
Fibrin
Antibodies
Complementary DNA
Immunoglobulin G
Affinity chromatography
Antigens
Polymerase chain reaction
Hybridomas
High performance liquid chromatography
Durapatite
Cricetulus
Affinity Chromatography
Ovary
Assays
Genes
High Pressure Liquid Chromatography
Cells
Polymerase Chain Reaction

Keywords

  • Chimeric bispecific antibody
  • Clot lysis
  • Fibrin specificity
  • Polymerase chain reaction
  • Single chain urokinase-type plasminogen activator

ASJC Scopus subject areas

  • Biotechnology

Cite this

Expression and characterization of a chimeric bispecific antibody against fibrin and against urokinase-type plasminogen activator. / Tada, Hiroko; Kurokawa, Tomofumi; Seita, Takeshi; Watanabe, Takeshi; Iwasa, Susumu.

In: Journal of Biotechnology, Vol. 33, No. 2, 31.03.1994, p. 157-174.

Research output: Contribution to journalArticle

Tada, Hiroko ; Kurokawa, Tomofumi ; Seita, Takeshi ; Watanabe, Takeshi ; Iwasa, Susumu. / Expression and characterization of a chimeric bispecific antibody against fibrin and against urokinase-type plasminogen activator. In: Journal of Biotechnology. 1994 ; Vol. 33, No. 2. pp. 157-174.
@article{0ac834889ee6440fbe9161fd6882ca98,
title = "Expression and characterization of a chimeric bispecific antibody against fibrin and against urokinase-type plasminogen activator",
abstract = "We have produced a chimeric bispecific antibody that has dual specificity of human fibrin and urokinase-type plasminogen activator (u-PA). Complementary DNAs for variable regions of both anti-fibrin and anti-u-PA antibodies were cloned from two murine hybridomas secreting respective antibodies using polymerase chain reaction (PCR) techniques, and joined to cDNAs for human constant regions to form chimeric antibody genes. Both of two expression vectors for chimeric anti-fibrin and chimeric anti-u-PA antibodies were sequentially introduced into Chinese hamster ovary cells, and stable transfectants secreting the chimeric bispecific antibody were obtained. The highest producer transfectant (SULF/C2-30) secreted high level (about 40 μg ml-1) of total chimeric IgG and about 2{\%} of the IgG had the bispecific activity of binding with both antigens. The chimeric bispecific antibody was purified by a combination of affinity chromatographies employing antigen-coupled columns and hydroxyapatite high-performance liquid chromatography. The purified chimeric bispecific antibody significantly enhanced the thrombolytic potency of single chain u-PA in an in vitro clot lysis assay as well as the original murine bispecific antibody.",
keywords = "Chimeric bispecific antibody, Clot lysis, Fibrin specificity, Polymerase chain reaction, Single chain urokinase-type plasminogen activator",
author = "Hiroko Tada and Tomofumi Kurokawa and Takeshi Seita and Takeshi Watanabe and Susumu Iwasa",
year = "1994",
month = "3",
day = "31",
doi = "10.1016/0168-1656(94)90108-2",
language = "English",
volume = "33",
pages = "157--174",
journal = "Journal of Biotechnology",
issn = "0168-1656",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Expression and characterization of a chimeric bispecific antibody against fibrin and against urokinase-type plasminogen activator

AU - Tada, Hiroko

AU - Kurokawa, Tomofumi

AU - Seita, Takeshi

AU - Watanabe, Takeshi

AU - Iwasa, Susumu

PY - 1994/3/31

Y1 - 1994/3/31

N2 - We have produced a chimeric bispecific antibody that has dual specificity of human fibrin and urokinase-type plasminogen activator (u-PA). Complementary DNAs for variable regions of both anti-fibrin and anti-u-PA antibodies were cloned from two murine hybridomas secreting respective antibodies using polymerase chain reaction (PCR) techniques, and joined to cDNAs for human constant regions to form chimeric antibody genes. Both of two expression vectors for chimeric anti-fibrin and chimeric anti-u-PA antibodies were sequentially introduced into Chinese hamster ovary cells, and stable transfectants secreting the chimeric bispecific antibody were obtained. The highest producer transfectant (SULF/C2-30) secreted high level (about 40 μg ml-1) of total chimeric IgG and about 2% of the IgG had the bispecific activity of binding with both antigens. The chimeric bispecific antibody was purified by a combination of affinity chromatographies employing antigen-coupled columns and hydroxyapatite high-performance liquid chromatography. The purified chimeric bispecific antibody significantly enhanced the thrombolytic potency of single chain u-PA in an in vitro clot lysis assay as well as the original murine bispecific antibody.

AB - We have produced a chimeric bispecific antibody that has dual specificity of human fibrin and urokinase-type plasminogen activator (u-PA). Complementary DNAs for variable regions of both anti-fibrin and anti-u-PA antibodies were cloned from two murine hybridomas secreting respective antibodies using polymerase chain reaction (PCR) techniques, and joined to cDNAs for human constant regions to form chimeric antibody genes. Both of two expression vectors for chimeric anti-fibrin and chimeric anti-u-PA antibodies were sequentially introduced into Chinese hamster ovary cells, and stable transfectants secreting the chimeric bispecific antibody were obtained. The highest producer transfectant (SULF/C2-30) secreted high level (about 40 μg ml-1) of total chimeric IgG and about 2% of the IgG had the bispecific activity of binding with both antigens. The chimeric bispecific antibody was purified by a combination of affinity chromatographies employing antigen-coupled columns and hydroxyapatite high-performance liquid chromatography. The purified chimeric bispecific antibody significantly enhanced the thrombolytic potency of single chain u-PA in an in vitro clot lysis assay as well as the original murine bispecific antibody.

KW - Chimeric bispecific antibody

KW - Clot lysis

KW - Fibrin specificity

KW - Polymerase chain reaction

KW - Single chain urokinase-type plasminogen activator

UR - http://www.scopus.com/inward/record.url?scp=0028210277&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028210277&partnerID=8YFLogxK

U2 - 10.1016/0168-1656(94)90108-2

DO - 10.1016/0168-1656(94)90108-2

M3 - Article

C2 - 7764730

AN - SCOPUS:0028210277

VL - 33

SP - 157

EP - 174

JO - Journal of Biotechnology

JF - Journal of Biotechnology

SN - 0168-1656

IS - 2

ER -