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Title:
Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth | Cancer Immunology, Immunotherapy
Description:
Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (αIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 αIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92–98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the αIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble αIGF-IR scFvs, a prototype αIGF-IR scFv and its alternative type αIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. αIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A–agarose chromatography. Levels of αIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that αIGF-IR scFv-Fc is a dimeric antibody. αIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 108 M−1, which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of αIGF-IR scFv-Fc (500 μg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the αIGF-IR scFv-Fc is a first-generation recombinant αIGF-IR for the potential development of future αIGF-IR therapeutics.
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Keywords {🔍}
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Topics {✒️}
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headline:Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth
description: Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (αIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 αIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92–98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the αIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble αIGF-IR scFvs, a prototype αIGF-IR scFv and its alternative type αIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. αIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A–agarose chromatography. Levels of αIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that αIGF-IR scFv-Fc is a dimeric antibody. αIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 108 M−1, which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of αIGF-IR scFv-Fc (500 μg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the αIGF-IR scFv-Fc is a first-generation recombinant αIGF-IR for the potential development of future αIGF-IR therapeutics.
datePublished:
dateModified:
pageStart:243
pageEnd:252
sameAs:https://doi.org/10.1007/s002620000115
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Key words IGF
Recombinant antibody
Single-chain antibody
Tumor growth inhibition
Oncology
Immunology
Cancer Research
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headline:Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth
description: Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (αIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 αIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92–98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the αIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble αIGF-IR scFvs, a prototype αIGF-IR scFv and its alternative type αIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. αIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A–agarose chromatography. Levels of αIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that αIGF-IR scFv-Fc is a dimeric antibody. αIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 108 M−1, which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of αIGF-IR scFv-Fc (500 μg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the αIGF-IR scFv-Fc is a first-generation recombinant αIGF-IR for the potential development of future αIGF-IR therapeutics.
datePublished:
dateModified:
pageStart:243
pageEnd:252
sameAs:https://doi.org/10.1007/s002620000115
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Key words IGF
Recombinant antibody
Single-chain antibody
Tumor growth inhibition
Oncology
Immunology
Cancer Research
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1432-0851
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