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Tumour Targeting using Radiolabelled Affibody Molecules: Influence of Labelling Chemistry
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affibody molecules are promising candidates for targeted radionuclide-based imaging and therapy applications. Optimisation of targeting properties would permit the in vivo visualization of cancer-specific surface receptors with high contrast. In therapy, this may increase the ratio of radioactivity uptake between tumour and normal tissues.  This thesis work is based on 5 original research articles (papers I-V) and focuses on optimisation of targeting properties of anti-HER2 affibody molecules by optimising the labelling chemistry.

Paper I and II report the comparative evaluation of the anti-HER2 ZHER2:2395 affibody molecule site specifically labelled with 111In (suitable for SPECT imaging) and 68Ga (suitable for PET imaging) using the thiol reactive derivatives of DOTA and NODAGA as chelators. The incorporation of different macrocyclic chelators and labelling with different radionuclides modified the biodistribution properties of affibody molecules. This indicates that the labelling strategy may have a profound effect on the targeting properties of radiotracers and must be carefully optimized.

Paper III reports the study of the mechanism of renal reabsorption of anti-HER2 ZHER2:2395 affibody molecule. An unknown receptor (not HER2) is suspected to be responsible for the high reabsorption of ZHER2:2395 molecules in the kidneys.

Paper IV reports the optimization and development of in vivo targeting properties of 188Re-labelled anti-HER2 affibody molecules. By using an array of peptide based chelators, it was found that substitution of one amino acid by another or changing its position can have a dramatic effect on the biodistribution properties of 188Re-labelled affibody molecules. This permitted the selection of –GGGC chelator whichdemonstrated the lowest retention of radioactivity in kidneys compared to other variants and showed excellent tumour targeting properties.

Paper V reports the preclinical evaluation of 188Re-ZHER2:V2 as a potential candidate for targeted radionuclide therapy of HER2-expressing tumours. In vivo experiments in mice along with dosimetry assessment in both murine and human models revealed that future human radiotherapy studies using 188Re-ZHER2:V2 may be feasible.

It would be reasonable to believe that the results of optimisation of anti-HER2 affibody molecules summarized in this thesis can be of importance for the development of other scaffold protein-based targeting agents.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 77 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1013
Keyword [en]
HER2, Affibody molecule, Radionuclide molecular maging, Targeted radionuclide therapy, Labeling chemistry
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-229090ISBN: 978-91-554-8983-0 (print)OAI: oai:DiVA.org:uu-229090DiVA: diva2:735588
Public defence
2014-09-20, Rudbecksalen, DagHammarskjölds väg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2014-08-28 Created: 2014-07-29 Last updated: 2014-09-08
List of papers
1. Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA
Open this publication in new window or tab >>Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA
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2012 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 39, no 4, 518-529 p.Article in journal (Refereed) Published
Abstract [en]

Introduction

Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule.

Methods

The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to ZHER2:2395 Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 were studied. Biodistribution of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 was compared in mice.

Results

The affinity of [MMA-NODAGA-Cys61]-ZHER2:2395 binding to HER2 was 67 pM. The 111In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [111In-MMA-NODAGA-Cys61]-ZHER2:2395 bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [111In-MMA-DOTA-Cys61]-ZHER2:2395 (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [111In-MMA-NODAGA-Cys61]-ZHER2:2395 due to higher clearance rate from normal tissues.

Conclusions

MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-164424 (URN)10.1016/j.nucmedbio.2011.10.013 (DOI)000303790600008 ()22172396 (PubMedID)
Available from: 2012-05-04 Created: 2011-12-20 Last updated: 2017-12-07Bibliographically approved
2. Influence of Nuclides and Chelators on Imaging Using Affibody Molecules: Comparative Evaluation of Recombinant Affibody Molecules Site-Specifically Labeled with 68Ga and 111In via Maleimido Derivatives of DOTA and NODAGA
Open this publication in new window or tab >>Influence of Nuclides and Chelators on Imaging Using Affibody Molecules: Comparative Evaluation of Recombinant Affibody Molecules Site-Specifically Labeled with 68Ga and 111In via Maleimido Derivatives of DOTA and NODAGA
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2013 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 6, 1102-1109 p.Article in journal (Refereed) Published
Abstract [en]

Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use of the generator-produced positron-emitting radionuclide 68Ga should increase sensitivity of HER2 imaging. The chemical nature of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant ZHER2:2395 HER2-binding Affibody molecule with 68Ga. DOTA and NODAGA were site-specifically conjugated to the ZHER2:2395 Affibody molecule having a C-terminal cysteine and labeled with 68Ga and 111In. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were clearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for 68Ga than for 111In. The tumor uptake of 68Ga-NODAGA-ZHER2:2395 and 68Ga-DOTA-ZHER2:2395 and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for 68Ga-NODAGA- ZHER2:2395 (8 ± 2 vs 5.0 ± 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.

National Category
Basic Medicine
Identifiers
urn:nbn:se:uu:diva-203054 (URN)10.1021/bc300678y (DOI)000320898900030 ()23705574 (PubMedID)
Note

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2013-07-02 Created: 2013-07-02 Last updated: 2017-12-06Bibliographically approved
3. In Vivo and In Vitro Studies on Renal Uptake of Radiolabeled Affibody Molecules for Imaging of HER2 Expression in Tumors
Open this publication in new window or tab >>In Vivo and In Vitro Studies on Renal Uptake of Radiolabeled Affibody Molecules for Imaging of HER2 Expression in Tumors
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2013 (English)In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 28, no 3, 187-195 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules (6-7 kDa) are a new class of small robust three-helical scaffold proteins. Radiolabeled subnanomolar anti-HER2 affibody Z(HER2:342) was developed for imaging of HER2 expression in tumors, and a clinical study has demonstrated that the In-111- and Ga-68-labeled affibody molecules can efficiently detect HER2 expressing metastases in breast cancer patients. However, a significant renal accumulation of radioactivity after systemic injection of a radiolabeled anti-HER2 affibody conjugate is observed. The aim of this study was to investigate the mechanism of renal reabsorption of anti-HER2 affibody at the molecular level. Renal accumulation of radiolabeled anti-HER2 affibody molecules was studied in a murine model and in vitro using opossum-derived proximal tubule (OK) cells. It was found that kidney reabsorption of affibody molecule was not driven by megalin/cubilin. Amino acids in the target-binding side of affibody molecule were involved in binding to OK cells. On OK cells, two types of receptors for anti-HER2 affibody molecule were found: K-D1 = 0.8 nM, B-max1 = 71,500 and K-D2 = 9.2 nM, B-max2 = 367,000. The results of the present study indicate that affibody molecule and other scaffold-based targeting proteins with a relatively low kidney uptake can be selected using in vitro studies with tubular kidney cells.

Keyword
affibody molecules, HER2, OK cells, megalin, renal reabsorption
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-200076 (URN)10.1089/cbr.2012.1304 (DOI)000317478200002 ()
Note

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2013-05-23 Created: 2013-05-20 Last updated: 2017-12-06Bibliographically approved
4. Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with 188-Re
Open this publication in new window or tab >>Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with 188-Re
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2013 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, no Suppl. 2, S219-S220 p.Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1±0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172±32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-211592 (URN)10.1007/s00259-013-2535-3 (DOI)000325853400417 ()
Conference
Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), Lyon 19-23 oktober 2013.
Available from: 2013-12-03 Created: 2013-11-27 Last updated: 2017-12-06Bibliographically approved
5. 188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors: preclinical assessment
Open this publication in new window or tab >>188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors: preclinical assessment
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2014 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 55, no 11, 1842-1848 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of 99mTc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide–based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate 188Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)–expressing tumors.

Methods:

ZHER2:V2 was labeled with 188Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment.

Results:

Binding of 188Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that 188Re-ZHER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible.

Conclusion:

188Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.

National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-211593 (URN)10.2967/jnumed.114.140194 (DOI)000344209200015 ()25278516 (PubMedID)
Available from: 2013-12-03 Created: 2013-11-27 Last updated: 2017-12-06Bibliographically approved

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