International journal of molecular sciences, Apr 11, 2024
Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 k... more Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with 177 Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [ 177 Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.
The epidermal growth factor receptor (EGFR) is often overexpressed during prostate cancer (PCa) p... more The epidermal growth factor receptor (EGFR) is often overexpressed during prostate cancer (PCa) progression towards androgen-independence after hormone therapy, but the overexpression is lower than in other types of cancers. Despite the low expression, EGFR has emerged as a promising therapeutic target for patients with castration-resistant PCa. Non-invasive methods for determination of EGFR expression in PCa can serve for patient stratification and therapy response monitoring. Radionuclide imaging probes based on affibody molecules (7 kDa) provide high contrast imaging of cancer-associated molecular targets. We hypothesized that the anti-EGFR affibody molecule DOTA-Z EGFR:2377 labeled with 55 Co (positron-emitter, T1/2=17.5 h) would enable imaging of EGFR expression in PCa xenografts. The human PCa cell line DU-145 was used for in vitro and in vivo experiments and 57 Co was used as a surrogate for 55 Co in the present study. Binding of 57 Co-DOTA-Z EGFR:2377 to EGFR-expressing xenografts was saturable with anti-EGFR monoclonal antibody cetuximab, which would motivate the use of this tracer for monitoring the receptor occupancy during treatment. A significant dose-dependent difference in radioactivity accumulation in tumors and normal organs was observed when the biodistribution was studied 3 h after the injection of 10 and 35 µg of 57 Co-DOTA-Z EGFR:2377 : At lower doses the tumor uptake was 2-fold higher although tumor-to-organ ratios were not altered. For clinically relevant organs for PCa, tumor-to-organ ratios increased with time, and at 24 h pi were 2.2±0.5 for colon, 7±2 for muscle, and 4.0±0.7 for bones. Small animal SPECT/CT images confirmed the capacity of radiocobalt labeled DOTA-Z EGFR:2377 to visualize EGFR expression in PCa. In conclusion, the present study demonstrated the feasibility of using the radiocobalt labeled anti-EGFR affibody conjugate Z EGFR:2377 as an imaging agent for in vivo visualization of low EGFR-expressing tumors, like PCa, and for monitoring of receptor occupancy during cetuximab therapy as well as the importance of optimal dosing in order to achieve higher sensitivity molecular imaging.
Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) express... more Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [ 99m Tc]Tc-(HE) 3 -G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE) 3 ), provided clear imaging of HER2 expressing breast cancer 2-4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E 3 C), Gly-Gly-Gly-Cys (G 3 C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser) 3 -linker (designated as G3-(G 3 S) 3 C) would further improve the contrast of imaging using 99m Tc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9-5 nM. Biodistribution of [ 99m Tc]Tc-G3-G 3 C, [ 99m Tc]Tc-G3-(G 3 S) 3 C, and [ 99m Tc]Tc-G3-E 3 C in mice was compared with the biodistribution of [ 99m Tc]Tc-(HE) 3 -G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [ 99m Tc]Tc-(HE) 3 -G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.
European Journal of Nuclear Medicine and Molecular Imaging, 2019
N-terminal position of histidine-glutamate-containing tag improves biodistribution of [Tc-99m]Tc-... more N-terminal position of histidine-glutamate-containing tag improves biodistribution of [Tc-99m]Tc-labeled DARPin G3
Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gas... more Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gastroesophageal cancer is used for the stratification of patients for HER2-targeting therapies. The use of radionuclide molecular imaging may facilitate such evaluation in a non-invasive way. Designed ankyrin repeat proteins (DARPins) are engineered scaffold proteins with high potential as probes for radionuclide molecular imaging. DARPin G3 binds with high affinity to HER2 and may be used to visualize this important therapeutic target. Studies on other engineered scaffold proteins have demonstrated that selection of the optimal labeling approach improves the sensitivity and specificity of radionuclide imaging. The present study compared two methods of labeling G3, direct and indirect radioiodination, to select an approach providing the best imaging contrast. G3-H 6 was labeled with iodine-124, iodine-125 and iodine-131 using a direct method. A novel construct bearing a C-terminal cysteine, G3-GGGC, was site-specifically labeled using [ 125 I]I-iodo-[(4-hydroxyphenyl)ethyl]maleimide (HPEM). The two radiolabeled G3 variants preserved binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was demonstrated. Biodistribution comparison of [ 131 I]I-G3-H 6 and [ 125 I]I-HPEM-G3-GGGC in mice, bearing HER2-expressing SKOV3 xenografts, demonstrated an appreciable contribution of hepatobiliary excretion to the clearance of [ 125 I]I-HPEM-G3-GGGC and a decreased tumor uptake compared to [ 131 I]I-G3-H 6 . The direct label provided higher tumor-to-blood and tumor-to-organ ratios compared with the indirect label at 4 h post-injection. The feasibility of high contrast PET/CT imaging of HER2 expression in SKOV3 xenografts in mice using [ 124 I]I-G3-H 6 was demonstrated. In conclusion, direct radioiodination is the preferable approach for labeling DARPin G3 with iodine-123 and iodine-124 for clinical single photon emission computed tomography and positron emission tomography imaging.
Analytical RP-HPLC analysis of purified DOTA-C-(HE)3-ADAPT6 ; Mass spectrum of purified DOTA-C-(H... more Analytical RP-HPLC analysis of purified DOTA-C-(HE)3-ADAPT6 ; Mass spectrum of purified DOTA-C-(HE)3-ADAPT6 ; Overlay of circular dichroism spectra from 250-195 nm for DOTA-C-(HE)3-ADAPT6 before (blue) and after (red) heating from 20-90{degree sign}C ; Normalized relative ellipticity of DOTA-C-(HE)3-ADAPT6 at 221 nm as a function of temperature ; SDS-PAGE analysis of 111In-DOTA-C-(HE)3-ADAPT6 ; Representative binding curve of the LigandTracer measurement of 111In-DOTA-C-(HE)3-ADAPT6 affinity using SKOV-3 cells ; InteractionMap of 111In-DOTA-C-(HE)3-ADAPT6 binding to SKOV-3 cells ; In vivo targeting specificity of 111In-DOTA-C-(HE)3-ADAPT6 (3 µg) in mice bearing SKOV-3 xenografts at 1 h p.i ; Biodistribution of 111In-DOTA-C-(HE)3-ADAPT6 in mice bearing SKOV-3 xenografts .
Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cance... more Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cancer is exploited for targeted therapy using monoclonal antibodies and antibody-drug conjugates. Small engineered scaffold proteins, such as the albumin binding domain (ABD) derived affinity proteins (ADAPTs), are a promising new format of targeting probes for development of drug conjugates with well-defined structure and tunable pharmacokinetics. Radiolabeled ADAPT6 has shown excellent tumor-targeting properties in clinical trials. Recently, we developed a drug conjugate based on the HER2-targeting ADAPT6 fused to an albumin binding domain (ABD) for increased bioavailability and conjugated to DM1 for cytotoxic action, designated as ADAPT6-ABD-mcDM1. In this study, we investigated the therapeutic efficacy of this conjugate in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. A secondary aim was to evaluate several formats of imaging probes for visualization of HER2 expression in...
European Journal of Nuclear Medicine and Molecular Imaging, 2017
Radiocobalt-labeled anti-HER1 affibody molecule DOTA-Z(EGFR:2377) for imaging of low HER1 express... more Radiocobalt-labeled anti-HER1 affibody molecule DOTA-Z(EGFR:2377) for imaging of low HER1 expression in prostate cancer pre-clinical model
Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted ther... more Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted therapy extends survival of patients with disseminated cancers. Radionuclide molecular imaging of EGFR expression would make EGFR-directed treatment more personalized and therefore more efficient. A previous study demonstrated that affibody molecule [68Ga]Ga-DFO-ZEGFR:2377 permits specific positron-emission tomography (PET) imaging of EGFR expression in xenografts at 3 h after injection. We anticipated that imaging at 24 h after injection would provide higher contrast, but this is prevented by the short half-life of 68Ga (67.6 min). Here, we therefore tested the hypothesis that the use of the non-conventional long-lived positron emitter 66Ga (T1/2 = 9.49 h, β+ = 56.5%) would permit imaging with higher contrast. 66Ga was produced by the 66Zn(p,n)66Ga nuclear reaction and DFO-ZEGFR:2377 was efficiently labelled with 66Ga with preserved binding specificity in vitro and in vivo. At 24 h after in...
International journal of molecular sciences, Apr 11, 2024
Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 k... more Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with 177 Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [ 177 Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.
The epidermal growth factor receptor (EGFR) is often overexpressed during prostate cancer (PCa) p... more The epidermal growth factor receptor (EGFR) is often overexpressed during prostate cancer (PCa) progression towards androgen-independence after hormone therapy, but the overexpression is lower than in other types of cancers. Despite the low expression, EGFR has emerged as a promising therapeutic target for patients with castration-resistant PCa. Non-invasive methods for determination of EGFR expression in PCa can serve for patient stratification and therapy response monitoring. Radionuclide imaging probes based on affibody molecules (7 kDa) provide high contrast imaging of cancer-associated molecular targets. We hypothesized that the anti-EGFR affibody molecule DOTA-Z EGFR:2377 labeled with 55 Co (positron-emitter, T1/2=17.5 h) would enable imaging of EGFR expression in PCa xenografts. The human PCa cell line DU-145 was used for in vitro and in vivo experiments and 57 Co was used as a surrogate for 55 Co in the present study. Binding of 57 Co-DOTA-Z EGFR:2377 to EGFR-expressing xenografts was saturable with anti-EGFR monoclonal antibody cetuximab, which would motivate the use of this tracer for monitoring the receptor occupancy during treatment. A significant dose-dependent difference in radioactivity accumulation in tumors and normal organs was observed when the biodistribution was studied 3 h after the injection of 10 and 35 µg of 57 Co-DOTA-Z EGFR:2377 : At lower doses the tumor uptake was 2-fold higher although tumor-to-organ ratios were not altered. For clinically relevant organs for PCa, tumor-to-organ ratios increased with time, and at 24 h pi were 2.2±0.5 for colon, 7±2 for muscle, and 4.0±0.7 for bones. Small animal SPECT/CT images confirmed the capacity of radiocobalt labeled DOTA-Z EGFR:2377 to visualize EGFR expression in PCa. In conclusion, the present study demonstrated the feasibility of using the radiocobalt labeled anti-EGFR affibody conjugate Z EGFR:2377 as an imaging agent for in vivo visualization of low EGFR-expressing tumors, like PCa, and for monitoring of receptor occupancy during cetuximab therapy as well as the importance of optimal dosing in order to achieve higher sensitivity molecular imaging.
Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) express... more Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [ 99m Tc]Tc-(HE) 3 -G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE) 3 ), provided clear imaging of HER2 expressing breast cancer 2-4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E 3 C), Gly-Gly-Gly-Cys (G 3 C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser) 3 -linker (designated as G3-(G 3 S) 3 C) would further improve the contrast of imaging using 99m Tc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9-5 nM. Biodistribution of [ 99m Tc]Tc-G3-G 3 C, [ 99m Tc]Tc-G3-(G 3 S) 3 C, and [ 99m Tc]Tc-G3-E 3 C in mice was compared with the biodistribution of [ 99m Tc]Tc-(HE) 3 -G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [ 99m Tc]Tc-(HE) 3 -G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.
European Journal of Nuclear Medicine and Molecular Imaging, 2019
N-terminal position of histidine-glutamate-containing tag improves biodistribution of [Tc-99m]Tc-... more N-terminal position of histidine-glutamate-containing tag improves biodistribution of [Tc-99m]Tc-labeled DARPin G3
Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gas... more Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gastroesophageal cancer is used for the stratification of patients for HER2-targeting therapies. The use of radionuclide molecular imaging may facilitate such evaluation in a non-invasive way. Designed ankyrin repeat proteins (DARPins) are engineered scaffold proteins with high potential as probes for radionuclide molecular imaging. DARPin G3 binds with high affinity to HER2 and may be used to visualize this important therapeutic target. Studies on other engineered scaffold proteins have demonstrated that selection of the optimal labeling approach improves the sensitivity and specificity of radionuclide imaging. The present study compared two methods of labeling G3, direct and indirect radioiodination, to select an approach providing the best imaging contrast. G3-H 6 was labeled with iodine-124, iodine-125 and iodine-131 using a direct method. A novel construct bearing a C-terminal cysteine, G3-GGGC, was site-specifically labeled using [ 125 I]I-iodo-[(4-hydroxyphenyl)ethyl]maleimide (HPEM). The two radiolabeled G3 variants preserved binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was demonstrated. Biodistribution comparison of [ 131 I]I-G3-H 6 and [ 125 I]I-HPEM-G3-GGGC in mice, bearing HER2-expressing SKOV3 xenografts, demonstrated an appreciable contribution of hepatobiliary excretion to the clearance of [ 125 I]I-HPEM-G3-GGGC and a decreased tumor uptake compared to [ 131 I]I-G3-H 6 . The direct label provided higher tumor-to-blood and tumor-to-organ ratios compared with the indirect label at 4 h post-injection. The feasibility of high contrast PET/CT imaging of HER2 expression in SKOV3 xenografts in mice using [ 124 I]I-G3-H 6 was demonstrated. In conclusion, direct radioiodination is the preferable approach for labeling DARPin G3 with iodine-123 and iodine-124 for clinical single photon emission computed tomography and positron emission tomography imaging.
Analytical RP-HPLC analysis of purified DOTA-C-(HE)3-ADAPT6 ; Mass spectrum of purified DOTA-C-(H... more Analytical RP-HPLC analysis of purified DOTA-C-(HE)3-ADAPT6 ; Mass spectrum of purified DOTA-C-(HE)3-ADAPT6 ; Overlay of circular dichroism spectra from 250-195 nm for DOTA-C-(HE)3-ADAPT6 before (blue) and after (red) heating from 20-90{degree sign}C ; Normalized relative ellipticity of DOTA-C-(HE)3-ADAPT6 at 221 nm as a function of temperature ; SDS-PAGE analysis of 111In-DOTA-C-(HE)3-ADAPT6 ; Representative binding curve of the LigandTracer measurement of 111In-DOTA-C-(HE)3-ADAPT6 affinity using SKOV-3 cells ; InteractionMap of 111In-DOTA-C-(HE)3-ADAPT6 binding to SKOV-3 cells ; In vivo targeting specificity of 111In-DOTA-C-(HE)3-ADAPT6 (3 µg) in mice bearing SKOV-3 xenografts at 1 h p.i ; Biodistribution of 111In-DOTA-C-(HE)3-ADAPT6 in mice bearing SKOV-3 xenografts .
Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cance... more Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cancer is exploited for targeted therapy using monoclonal antibodies and antibody-drug conjugates. Small engineered scaffold proteins, such as the albumin binding domain (ABD) derived affinity proteins (ADAPTs), are a promising new format of targeting probes for development of drug conjugates with well-defined structure and tunable pharmacokinetics. Radiolabeled ADAPT6 has shown excellent tumor-targeting properties in clinical trials. Recently, we developed a drug conjugate based on the HER2-targeting ADAPT6 fused to an albumin binding domain (ABD) for increased bioavailability and conjugated to DM1 for cytotoxic action, designated as ADAPT6-ABD-mcDM1. In this study, we investigated the therapeutic efficacy of this conjugate in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. A secondary aim was to evaluate several formats of imaging probes for visualization of HER2 expression in...
European Journal of Nuclear Medicine and Molecular Imaging, 2017
Radiocobalt-labeled anti-HER1 affibody molecule DOTA-Z(EGFR:2377) for imaging of low HER1 express... more Radiocobalt-labeled anti-HER1 affibody molecule DOTA-Z(EGFR:2377) for imaging of low HER1 expression in prostate cancer pre-clinical model
Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted ther... more Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted therapy extends survival of patients with disseminated cancers. Radionuclide molecular imaging of EGFR expression would make EGFR-directed treatment more personalized and therefore more efficient. A previous study demonstrated that affibody molecule [68Ga]Ga-DFO-ZEGFR:2377 permits specific positron-emission tomography (PET) imaging of EGFR expression in xenografts at 3 h after injection. We anticipated that imaging at 24 h after injection would provide higher contrast, but this is prevented by the short half-life of 68Ga (67.6 min). Here, we therefore tested the hypothesis that the use of the non-conventional long-lived positron emitter 66Ga (T1/2 = 9.49 h, β+ = 56.5%) would permit imaging with higher contrast. 66Ga was produced by the 66Zn(p,n)66Ga nuclear reaction and DFO-ZEGFR:2377 was efficiently labelled with 66Ga with preserved binding specificity in vitro and in vivo. At 24 h after in...
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Papers by Javad Garousi