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Molecular Radionuclide Imaging Using Site-specifically Labelled Recombinant Affibody Molecules: Preparation and Preclinical Evaluation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Radionuclide molecular imaging is an emerging multidisciplinary technique that is used in modern medicine to visualise diseases at cellular and molecular levels. This thesis is based on five papers (I-V) and focuses on the development of site-specific radiolabelled recombinant anti-HER2 Affibody molecules and preclinical evaluations in vitro and in vivo of the labelled conjugates. This work is part of a preclinical development of an Affibody molecule-based tracer for molecular imaging of HER2 expressing tumours.

Papers I and II report the evaluation of the Affibody molecule ZHER2:2395-C, site-specifically labelled with the radiometals 111In (for SPECT) and 57Co (as a surrogate for 55Co, suitable for PET applications) using a thiol reactive DOTA derivative as a chelator. Both conjugates demonstrated very suitable biodistribution properties, enabling high contrast imaging just a few hours after injection.

Papers III and IV report the development and optimization of a technique for site-specific labelling of ZHER2:2395-C with 99mTc using an N3S chelating peptide sequence. 99mTc-ZHER2:2395-C demonstrated high and specific tumour uptake and rapid clearance of non-bound tracer from the blood, resulting in high tumour-to-non-tumour ratios shortly after injection, enabling high contrast imaging. In addition, in the study described in paper IV, freeze-dried kits previously developed for 99mTc-labelling were optimised, resulting in the development of a kit in which all the reagents and protein needed for labelling of ZHER2:2395-C with 99mTc were contained in a single vial.

Paper V reports the evaluation of an anti-HER2 Affibody molecule, ABY-025, with a fundamentally re-engineered scaffold. Despite the profound re-engineering, the biodistribution pattern of 111In-ABY-025 was very similar to that of two variants of the parental molecule.

It seems reasonable to believe that these results will also be applicable to Affibody molecules towards other targets. Hopefully, this work will also be helpful in the development of other small proteinaceous tracers.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 70
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 553
Keywords [en]
molecular radionuclide imaging, Affibody molecules, HER2, cancer detection, radiolabelling, technetium, indium, cobalt, SPECT, PET
National Category
Cancer and Oncology Radiology, Nuclear Medicine and Medical Imaging Radiology, Nuclear Medicine and Medical Imaging
Research subject
Biomedical Radiation Science; Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-122177ISBN: 978-91-554-7787-5 (print)OAI: oai:DiVA.org:uu-122177DiVA, id: diva2:309711
Public defence
2010-05-22, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-04-29 Created: 2010-04-07 Last updated: 2010-04-29Bibliographically approved
List of papers
1. Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules
Open this publication in new window or tab >>Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules
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2008 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, no 1, p. 235-243Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-104530 (URN)10.1021/bc700307y (DOI)000252520300030 ()18163536 (PubMedID)
Available from: 2009-05-29 Created: 2009-05-28 Last updated: 2017-12-13Bibliographically approved
2. Evaluation of the Radiocobalt-Labeled [MMA-DOTA-Cys61]-ZHER2:2395-Cys Affibody Molecule for Targeting of HER2-Expressing Tumors
Open this publication in new window or tab >>Evaluation of the Radiocobalt-Labeled [MMA-DOTA-Cys61]-ZHER2:2395-Cys Affibody Molecule for Targeting of HER2-Expressing Tumors
2010 (English)In: Molecular Imaging and Biology, ISSN 1536-1632, E-ISSN 1860-2002, Vol. 12, no 1, p. 54-62Article in journal (Refereed) Published
Abstract [en]

PURPOSE: Imaging using positron emission tomography (PET) in the field of nuclear medicine is becoming increasingly important. The aim of this study was to develop a method for labeling of affibody molecules with radiocobalt for PET applications. PROCEDURES: The human epidermal growth factor receptors type 2 (HER2) binding affibody molecule DOTA-Z(2395)-C was radiolabeled with (57)Co (used as a surrogate of (55)Co). The binding specificity and cellular processing of the labeled compound was studied in vitro followed by in vivo characterization in normal and tumor-bearing mice. Furthermore, a comparative biodistribution study was performed with a (111)In-labeled counterpart. RESULTS: DOTA-Z(2395)-C was successfully labeled with radiocobalt with nearly quantitative yield. The compound displayed good retention on cells over time and high tumor accumulation of radioactivity in animal studies. Imaging studies showed clear visualization of HER2-positive tumors. Furthermore, the radiocobalt label provided better tumor-to-organ ratios than (111)In. CONCLUSIONS: Radiocobalt is a promising label for affibody molecules for future PET applications.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-122173 (URN)10.1007/s11307-009-0238-8 (DOI)000273479300008 ()19557480 (PubMedID)
Available from: 2010-04-07 Created: 2010-04-07 Last updated: 2017-12-12Bibliographically approved
3. Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine
Open this publication in new window or tab >>Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine
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2009 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 50, no 5, p. 781-789Article in journal (Refereed) Published
Abstract [en]

The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced (99m)Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. METHODS: A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with (99m)Tc. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K(D)], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K(D), 27 pM), were labeled with (99m)Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts. RESULTS: (99m)Tc-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. (99m)Tc-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of (99m)Tc-Z(HER2:2395)-Cys. CONCLUSION: The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.

Keywords
Affibody molecule, technetium, imaging, HER2, C-terminal cysteine
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-122172 (URN)10.2967/jnumed.108.056929 (DOI)000272487900017 ()19372467 (PubMedID)
Available from: 2010-04-07 Created: 2010-04-07 Last updated: 2017-12-12Bibliographically approved
4. Kit formulation for 99mTc-labeling of recombinant anti-HER2 Affibody molecules with a C-terminally engineered cysteine
Open this publication in new window or tab >>Kit formulation for 99mTc-labeling of recombinant anti-HER2 Affibody molecules with a C-terminally engineered cysteine
2010 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 37, no 5, p. 539-546Article in journal (Refereed) Published
Abstract [en]

Introduction: Molecular imaging of HER2-expression in malignant tumors provides potentially important information for patient management. Affibody molecules have shown to be suitable tracers for imaging applications using SPECT or PET. Results from an earlier evaluation of the application of site specific 99mTc-labeling on the Affibody molecule, ZHER2:2395-C, were favorable.

Methods: As a preparation for clinical application of this tracer we have developed and evaluated a robust single-vial freeze-dried kit, allowing labeling of the Affibody molecule, ZHER2:2395-C, with 99mTc.

Results: The composition of the kit (containing glucoheptonate, EDTA and tin(II)-chloride), as well as the protein amount and the pertechnetate volume were optimized for a high labeling yield (> 90 %) and minimal presence of reduced hydrolyzed technetium colloids (< 1 %). The specificity to HER2 receptors, the binding competence and the stability in PBS and murine serum were verified in vitro. The shelf-life was also evaluated in vitro, showing no reduction in labeling yield or binding capacity to HER2-expressing cells after over 400 days of storage of the single-vial freeze-dried kit.

Conclusions: ZHER2:2395-C labeled with 99mTc using the lyophilized kit was stable and resulted in a favorable biodistribution in an in vivo evaluation in normal NMRI mice.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-122175 (URN)10.1016/j.nucmedbio.2010.02.009 (DOI)000279412400002 ()20610158 (PubMedID)
Available from: 2010-04-07 Created: 2010-04-07 Last updated: 2017-12-12Bibliographically approved
5. Targeting of HER2-Expressing Tumors Using 111In-ABY-025, a Second-Generation Affibody Molecule with a Fundamentally Reengineered Scaffold
Open this publication in new window or tab >>Targeting of HER2-Expressing Tumors Using 111In-ABY-025, a Second-Generation Affibody Molecule with a Fundamentally Reengineered Scaffold
Show others...
2010 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 51, no 7, p. 1131-1138Article in journal (Refereed) Published
Abstract [en]

Overexpression of HER2 in breast carcinomas predicts response to trastuzumab therapy. Affibody molecules based on a non-immunoglobulin scaffold have demon-strated high potential for in vivo molecular imaging of HER2-expressing tumors. Re-engineering of the molecular scaffold has led to a second generation of optimized Affibody molecules, having a surface distinctly different from the parental protein domain from staphylococcal protein A. The new tracer showed further increased melting point, stability and overall hydrophilicity compared to the parental molecule, and was shown to be more amenable for chemical peptide synthesis. The goal of this study was to assess potential effects of this extensive re-engineering on HER2 targeting, using ABY-025, a DOTA conjugated variant of the novel tracer.

Methods: 111In-ABY-025 was compared with previously evaluated parent HER2-binding Affibody tracers in vitro and in vivo. The in vivo behavior was further evaluated in mice bearing SKOV-3 xenografts, in rats and in cynomolgus macaques.

Results: 111In-ABY-025 bound specifically to HER2 in vitro and in vivo. Direct comparison with the previous generation of HER2-binding tracers showed that ABY-025 retained excellent targeting properties. Rapid blood clearance was shown in mice, rats and macaques. A highly specific tumor uptake of 16.7 ± 2.5 %IA/g was seen at 4 h after injection. The tumor-to-blood ratio was 6.3 at 0.5 h, 88 at 4 h, and increased up to 3 days after injection. Gamma camera imaging of tumors was already possible 0.5 h after injection. Furthermore, repeated i.v. administration of ABY-025 did not induce antibody formation in rats.

Conclusions: The biodistribution of 111In-ABY-025 was in remarkably good agreement with the parent tracers, despite profound re-engineering of the non-binding surface. The molecule displayed rapid blood clearance in all species investigated and excellent targeting capacity in tumor bearing mice, leading to high tumor-to-organ-ratios and high contrast imaging shortly after injection.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-122176 (URN)10.2967/jnumed.109.073346 (DOI)000279430900021 ()20554729 (PubMedID)
Available from: 2010-04-07 Created: 2010-04-07 Last updated: 2017-12-12Bibliographically approved

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