Development and characterization of an αvβ6-specific diabody and a disulfide-stabilized αvβ6-specific cys-diabody

Jason B. White, David L. Boucher, Kirstin A. Zettlitz, Anna M. Wu, Julie Sutcliffe

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Introduction: This work describes the development and characterization of two antibody fragments that specifically target the αvβ6 integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound αvβ6. Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[18F]fluorobenzoate ([18F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([64Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [18F]-FB-αvβ6 diabody, [18F]-FB-αvβ6 cys-diabody, and the [64Cu]-NOTA-αvβ6 cys-diabody. Methods: Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-αvβ6 intact antibody. The anti-αvβ6 cys-diabody was engineered with C-terminal cysteines to enable covalent dimerization and site-specific modification. Biochemical characterization included SDS-PAGE, Western blot, and electrospray ionization to confirm MW, and flow cytometry and ELISA experiments were used to determine binding affinity and specificity to αvβ6. The diabodies were radiolabeled with [18F]-SFB and in addition, the anti-αvβ6 cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [64Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroβ6vβ6+) and DX3Puro (αvβ6-) cell lines. Results: The diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards αvβ6 was confirmed by ELISA (diabody IC50=0.8nM, cys-diabody IC50=0.6nM) and flow cytometry revealed high specificity only to the DX3Puroβ6 cell line for both diabodies. RCYs were 22.6%±3.6% for the [18F]-FB-αvβ6 diabody, 8.3%±1.7% for the [18F]-FB-αvβ6 cys-diabody and 43.5%±5.5% for the [64Cu]-NOTA-αvβ6 cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([18F]-FB-αvβ6 diabody=58.7%±6.7%, [18F]-FB-αvβ6 cys-diabody=80.4%±4.4%, [64Cu]-NOTA-αvβ6 cys-diabody=59.4%±0.6%) regardless of the radiolabeling method used. Conclusions: Two novel diabodies with excellent binding affinity and specificity for the αvβ6 integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([18F]) and [64Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.

Original languageEnglish (US)
Pages (from-to)945-957
Number of pages13
JournalNuclear Medicine and Biology
Volume42
Issue number12
DOIs
StatePublished - Dec 1 2015

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Disulfides
Integrins
Inhibitory Concentration 50
Flow Cytometry
Enzyme-Linked Immunosorbent Assay
Cell Line
Immobilized Cells
Immunoglobulin Fragments
Acids
Fluorine
Dimerization
Chelating Agents
Cysteine
Copper
Polyacrylamide Gel Electrophoresis
Cell Culture Techniques
Western Blotting
Antibodies
In Vitro Techniques
N-succinimidyl-4-fluorobenzoate

Keywords

  • Copper-64
  • Diabody
  • Fluorine-18
  • Radiolabeling
  • Site-specific conjugation
  • αβ integrin

ASJC Scopus subject areas

  • Cancer Research
  • Molecular Medicine
  • Radiology Nuclear Medicine and imaging

Cite this

Development and characterization of an αvβ6-specific diabody and a disulfide-stabilized αvβ6-specific cys-diabody. / White, Jason B.; Boucher, David L.; Zettlitz, Kirstin A.; Wu, Anna M.; Sutcliffe, Julie.

In: Nuclear Medicine and Biology, Vol. 42, No. 12, 01.12.2015, p. 945-957.

Research output: Contribution to journalArticle

White, Jason B. ; Boucher, David L. ; Zettlitz, Kirstin A. ; Wu, Anna M. ; Sutcliffe, Julie. / Development and characterization of an αvβ6-specific diabody and a disulfide-stabilized αvβ6-specific cys-diabody. In: Nuclear Medicine and Biology. 2015 ; Vol. 42, No. 12. pp. 945-957.
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abstract = "Introduction: This work describes the development and characterization of two antibody fragments that specifically target the αvβ6 integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound αvβ6. Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[18F]fluorobenzoate ([18F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([64Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [18F]-FB-αvβ6 diabody, [18F]-FB-αvβ6 cys-diabody, and the [64Cu]-NOTA-αvβ6 cys-diabody. Methods: Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-αvβ6 intact antibody. The anti-αvβ6 cys-diabody was engineered with C-terminal cysteines to enable covalent dimerization and site-specific modification. Biochemical characterization included SDS-PAGE, Western blot, and electrospray ionization to confirm MW, and flow cytometry and ELISA experiments were used to determine binding affinity and specificity to αvβ6. The diabodies were radiolabeled with [18F]-SFB and in addition, the anti-αvβ6 cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [64Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroβ6 (αvβ6+) and DX3Puro (αvβ6-) cell lines. Results: The diabodies were purified from cell culture supernatants with purities >98{\%}. Subnanomolar binding affinity towards αvβ6 was confirmed by ELISA (diabody IC50=0.8nM, cys-diabody IC50=0.6nM) and flow cytometry revealed high specificity only to the DX3Puroβ6 cell line for both diabodies. RCYs were 22.6{\%}±3.6{\%} for the [18F]-FB-αvβ6 diabody, 8.3{\%}±1.7{\%} for the [18F]-FB-αvβ6 cys-diabody and 43.5{\%}±5.5{\%} for the [64Cu]-NOTA-αvβ6 cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([18F]-FB-αvβ6 diabody=58.7{\%}±6.7{\%}, [18F]-FB-αvβ6 cys-diabody=80.4{\%}±4.4{\%}, [64Cu]-NOTA-αvβ6 cys-diabody=59.4{\%}±0.6{\%}) regardless of the radiolabeling method used. Conclusions: Two novel diabodies with excellent binding affinity and specificity for the αvβ6 integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([18F]) and [64Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.",
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TY - JOUR

T1 - Development and characterization of an αvβ6-specific diabody and a disulfide-stabilized αvβ6-specific cys-diabody

AU - White, Jason B.

AU - Boucher, David L.

AU - Zettlitz, Kirstin A.

AU - Wu, Anna M.

AU - Sutcliffe, Julie

PY - 2015/12/1

Y1 - 2015/12/1

N2 - Introduction: This work describes the development and characterization of two antibody fragments that specifically target the αvβ6 integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound αvβ6. Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[18F]fluorobenzoate ([18F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([64Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [18F]-FB-αvβ6 diabody, [18F]-FB-αvβ6 cys-diabody, and the [64Cu]-NOTA-αvβ6 cys-diabody. Methods: Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-αvβ6 intact antibody. The anti-αvβ6 cys-diabody was engineered with C-terminal cysteines to enable covalent dimerization and site-specific modification. Biochemical characterization included SDS-PAGE, Western blot, and electrospray ionization to confirm MW, and flow cytometry and ELISA experiments were used to determine binding affinity and specificity to αvβ6. The diabodies were radiolabeled with [18F]-SFB and in addition, the anti-αvβ6 cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [64Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroβ6 (αvβ6+) and DX3Puro (αvβ6-) cell lines. Results: The diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards αvβ6 was confirmed by ELISA (diabody IC50=0.8nM, cys-diabody IC50=0.6nM) and flow cytometry revealed high specificity only to the DX3Puroβ6 cell line for both diabodies. RCYs were 22.6%±3.6% for the [18F]-FB-αvβ6 diabody, 8.3%±1.7% for the [18F]-FB-αvβ6 cys-diabody and 43.5%±5.5% for the [64Cu]-NOTA-αvβ6 cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([18F]-FB-αvβ6 diabody=58.7%±6.7%, [18F]-FB-αvβ6 cys-diabody=80.4%±4.4%, [64Cu]-NOTA-αvβ6 cys-diabody=59.4%±0.6%) regardless of the radiolabeling method used. Conclusions: Two novel diabodies with excellent binding affinity and specificity for the αvβ6 integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([18F]) and [64Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.

AB - Introduction: This work describes the development and characterization of two antibody fragments that specifically target the αvβ6 integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound αvβ6. Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[18F]fluorobenzoate ([18F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([64Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [18F]-FB-αvβ6 diabody, [18F]-FB-αvβ6 cys-diabody, and the [64Cu]-NOTA-αvβ6 cys-diabody. Methods: Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-αvβ6 intact antibody. The anti-αvβ6 cys-diabody was engineered with C-terminal cysteines to enable covalent dimerization and site-specific modification. Biochemical characterization included SDS-PAGE, Western blot, and electrospray ionization to confirm MW, and flow cytometry and ELISA experiments were used to determine binding affinity and specificity to αvβ6. The diabodies were radiolabeled with [18F]-SFB and in addition, the anti-αvβ6 cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [64Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroβ6 (αvβ6+) and DX3Puro (αvβ6-) cell lines. Results: The diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards αvβ6 was confirmed by ELISA (diabody IC50=0.8nM, cys-diabody IC50=0.6nM) and flow cytometry revealed high specificity only to the DX3Puroβ6 cell line for both diabodies. RCYs were 22.6%±3.6% for the [18F]-FB-αvβ6 diabody, 8.3%±1.7% for the [18F]-FB-αvβ6 cys-diabody and 43.5%±5.5% for the [64Cu]-NOTA-αvβ6 cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([18F]-FB-αvβ6 diabody=58.7%±6.7%, [18F]-FB-αvβ6 cys-diabody=80.4%±4.4%, [64Cu]-NOTA-αvβ6 cys-diabody=59.4%±0.6%) regardless of the radiolabeling method used. Conclusions: Two novel diabodies with excellent binding affinity and specificity for the αvβ6 integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([18F]) and [64Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.

KW - Copper-64

KW - Diabody

KW - Fluorine-18

KW - Radiolabeling

KW - Site-specific conjugation

KW - αβ integrin

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