In vitro cytotoxicity of Auger electron-emitting [67Ga]Ga-trastuzumab

Muhamad Faiz Bin Othman; Elise Verger; Ines Costa; Meena Tanapirakgul; Margaret S. Cooper; Cinzia Imberti; Valerie J. Lewington; Philip J. Blower; Samantha Y. A. Terry

doi:10.1016/j.nucmedbio.2019.12.004

In vitro cytotoxicity of Auger electron-emitting [⁶⁷Ga]Ga-trastuzumab

Muhamad Faiz Bin Othman, Elise Verger, Ines Costa, Meena Tanapirakgul, Margaret S. Cooper, Cinzia Imberti, Valerie J. Lewington, Philip J. Blower, Samantha Y. A. Terry

King's College London (KCL)

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

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Abstract

Introduction: Molecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of ⁶⁷Ga as a therapeutic radionuclide. Methods: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters ⁶⁷Ga and ¹¹¹In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2–negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography. Results: Labelling efficiencies for [ ⁶⁷Ga]Ga-THP-trastuzumab and [ ¹¹¹In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/μg (78–92 GBq/μmol). At 4 nM total antibody concentration and 200 × 10 ³ cells/mL, [ ⁶⁷Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [ ¹¹¹In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [ ⁶⁷Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [ ¹¹¹In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that ⁶⁷Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population. Conclusions: [ ⁶⁷Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting ⁶⁷Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.

Original language	English
Journal	Nuclear Medicine and Biology
Early online date	13 Dec 2019
DOIs	https://doi.org/10.1016/j.nucmedbio.2019.12.004
Publication status	E-pub ahead of print - 13 Dec 2019

Keywords

Gallium-67
molecular radiotherapy
Auger electrons
Tris(hydroxypyridinone)
radiobiology
trastuzumab
Molecular radiotherapy
Radiobiology
Trastuzumab

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In vitro cytotoxicity of_OTHMAN_Accepted12December2019Publishedonline13December2019_GOLD CP (CC BY)Final published version, 971 KBLicence: CC BY

Cite this

@article{616b7f48c11a41a69194856ba5f20653,

title = "In vitro cytotoxicity of Auger electron-emitting [67Ga]Ga-trastuzumab",

abstract = "Introduction: Molecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of 67Ga as a therapeutic radionuclide. Methods: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2–negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography. Results: Labelling efficiencies for [ 67Ga]Ga-THP-trastuzumab and [ 111In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/μg (78–92 GBq/μmol). At 4 nM total antibody concentration and 200 × 10 3 cells/mL, [ 67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [ 111In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [ 67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [ 111In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that 67Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population. Conclusions: [ 67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored. ",

keywords = "Gallium-67, molecular radiotherapy, Auger electrons, Tris(hydroxypyridinone), radiobiology, trastuzumab, Molecular radiotherapy, Radiobiology, Trastuzumab",

author = "{Bin Othman}, {Muhamad Faiz} and Elise Verger and Ines Costa and Meena Tanapirakgul and Cooper, {Margaret S.} and Cinzia Imberti and Lewington, {Valerie J.} and Blower, {Philip J.} and Terry, {Samantha Y. A.}",

year = "2019",

month = dec,

day = "13",

doi = "10.1016/j.nucmedbio.2019.12.004",

language = "English",

journal = "Nuclear Medicine and Biology",

issn = "0969-8051",

publisher = "Elsevier Inc.",

}

TY - JOUR

T1 - In vitro cytotoxicity of Auger electron-emitting [67Ga]Ga-trastuzumab

AU - Bin Othman, Muhamad Faiz

AU - Verger, Elise

AU - Costa, Ines

AU - Tanapirakgul, Meena

AU - Cooper, Margaret S.

AU - Imberti, Cinzia

AU - Lewington, Valerie J.

AU - Blower, Philip J.

AU - Terry, Samantha Y. A.

PY - 2019/12/13

Y1 - 2019/12/13

N2 - Introduction: Molecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of 67Ga as a therapeutic radionuclide. Methods: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2–negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography. Results: Labelling efficiencies for [ 67Ga]Ga-THP-trastuzumab and [ 111In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/μg (78–92 GBq/μmol). At 4 nM total antibody concentration and 200 × 10 3 cells/mL, [ 67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [ 111In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [ 67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [ 111In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that 67Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population. Conclusions: [ 67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.

AB - Introduction: Molecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of 67Ga as a therapeutic radionuclide. Methods: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2–negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography. Results: Labelling efficiencies for [ 67Ga]Ga-THP-trastuzumab and [ 111In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/μg (78–92 GBq/μmol). At 4 nM total antibody concentration and 200 × 10 3 cells/mL, [ 67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [ 111In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [ 67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [ 111In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that 67Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population. Conclusions: [ 67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.

KW - Gallium-67

KW - molecular radiotherapy

KW - Auger electrons

KW - Tris(hydroxypyridinone)

KW - radiobiology

KW - trastuzumab

KW - Molecular radiotherapy

KW - Radiobiology

KW - Trastuzumab

UR - http://www.scopus.com/inward/record.url?scp=85076961002&partnerID=8YFLogxK

U2 - 10.1016/j.nucmedbio.2019.12.004

DO - 10.1016/j.nucmedbio.2019.12.004

M3 - Article

SN - 0969-8051

JO - Nuclear Medicine and Biology

JF - Nuclear Medicine and Biology

ER -