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Design and Evaluation of Radiolabeled Affibody Tracers for Imaging of HER2-expressing Tumors
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The growing understanding of tumor biology and the identification of tumor specificgenetic and molecular alterations, such as the overexpression of human epidermal growthfactor receptor 2 (HER2), opens up for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools usedto identify patients that are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising non-invasive method to visualize and characterize the expression of such targets. This thesis, based on five papers, is focused on the development of radiolabeled Affibody molecules for imaging of HER2-expression in malignant tumors. Affibody molecules, which represent a rather novel class of affinity proteins developed by combinatorial protein engineering of the protein A derived Z-domain, display manyfeatures that make them promising tracers for molecular imaging applications. The aim of the work presented here was to further develop the tracer format for improved in vivo properties and flexibility in the choice of radionuclide.

In paper I, the development of an assay that enables quantitative studies of the internalization rate and cellular processing of high affinity Affibody molecules is described. The assay was applied to a HER2-binding Affibody variant that was efficiently retained by HER2-expressing cells, although characterized by a slow internalization rate. This may have implications for the choice of label for Affibody molecules since high affinity to the target may be equally, or more, important for good imaging quality than residualizing properties of the radiolabel. In paper II, a HER2-binding Affibody molecule and the monoclonal antibody trastuzumab were labeled with positron emitting 124I, for a head-to-head in vivocomparison of the two tracer formats. The effects of tracer size and presence of an Fc region on the biodistribution profile were investigated. In paper III, a HER2-binding Affibody molecule was site-specifically labeled with radiocobalt and evaluated in vitro and in vivo.A head-to-head in vivo comparison with the well-studied 111In-labeled counterpart was performed, revealing promising potential for the cobalt-labeled molecule as a PET-tracerfor visualization of HER2. Paper IV describes the in vitro and in vivo evaluation of a panel of Affibody molecules with different C-terminal peptide-based chelators for the coordination of 99mTc. Even small changes in the C-terminal sequence had appreciable impact on the biodistribution of the Affibody molecules and by optimizing the design of the chelator, the kidney uptake of 99mTc could be significantly reduced. Finally, in paper V we describe the development of a HER2-targeting Affibody variant equipped with a Sel-tag for site-specific labeling with the short-lived positron emitter 11C. This novel Affibody tracer could be used to image HER2-expressing tumors in vivo within one hour after injection.

Taken together, Affibody molecules show great promise as targeting tracers for radionuclide molecular imaging of HER2. Careful design and optimization of the tracer protein is important and can be used to improve the biodistribution and targeting properties of Affibody molecules.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology , 2011. , xi, 79 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:21
Keyword [en]
Affibody molecule, radionuclide molecular imaging, HER2, radiotracer, SPECT, PET, biodistribution, protein engineering, radiolabeling
National Category
Medical Biotechnology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-40890ISBN: 978-91-7501-092-2 (print)OAI: oai:DiVA.org:kth-40890DiVA: diva2:442649
Public defence
2011-10-21, FD5, AlbaNova University center, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20110922Available from: 2011-09-22 Created: 2011-09-22 Last updated: 2011-09-22Bibliographically approved
List of papers
1. Slow internalization of anti-HER2 synthetic affibody monomer 111In-DOTA-ZHER2: 342-pep2
Open this publication in new window or tab >>Slow internalization of anti-HER2 synthetic affibody monomer 111In-DOTA-ZHER2: 342-pep2
2008 (English)In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 23, no 4, 435-42 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a novel class of targeting proteins, demonstrating promising results in the molecular imaging of tumor markers. The aim of this study was to investigate the cellular processing of Affibody molecules bound to human epidermal growth-factor-receptor type 2 (HER2). Cellular processing of the synthetic Affibody molecule, DOTA-Z(HER2:342-pep2) (K(D) = 65 (p)M) labeled with indium-111, was studied both during continuous and interrupted incubation with HER2-expressing cell lines (SKOV-3, SKBR-3, and BT474). The internalized and membrane bound fractions of Affibody molecule were discriminated by treatment with 4 M of urea solution in 0.2 M of glycine buffer (pH 2.0). Incubation media collected after an interrupted incubation was analyzed for the presence of radiocatabolites. Continuous incubation of tumor cells with (111)In-DOTA-Z(HER2:342-pep2) led to the saturation of HER2 and slow internalization. Sixty (60)- to 80% of the radioactivity remained cell associated 24 hours after interrupted incubation. The rate of Affibody molecule internalization was the same after interrupted incubation, as in the continuous incubation experiments. Internalization of (111) In-DOTA-Z(HER2:342-pep2) was relatively slow. A high level of cellular retention of the tracer was provided by strong binding to cell-surface receptors. These data suggest that good tumor targeting with anti-HER Affibody molecules may be obtained by using short-lived, nonresidualizing labels.

Keyword
cellular processing, HER2, affibody molecule, indium-111, internalization
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-40854 (URN)10.1089/cbr.2008.0464 (DOI)000259167300005 ()18771347 (PubMedID)
Note
QC 20110921Available from: 2011-09-21 Created: 2011-09-21 Last updated: 2017-12-08Bibliographically approved
2. On the Selection of a Tracer for PET Imaging of HER2-Expressing Tumors: Direct Comparison of a (124)I-Labeled Affibody Molecule and Trastuzumab in a Murine Xenograft Model
Open this publication in new window or tab >>On the Selection of a Tracer for PET Imaging of HER2-Expressing Tumors: Direct Comparison of a (124)I-Labeled Affibody Molecule and Trastuzumab in a Murine Xenograft Model
2009 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 50, no 3, 417-425 p.Article in journal (Refereed) Published
Abstract [en]

Human epidermal growth factor receptor type 2 (HER2) is a tyrosine kinase, which is often overexpressed in many carcinomas. Imaging HER2 expression in malignant tumors can provide important prognostic and predictive diagnostic information. The use of anti-HER2 tracers labeled with positron-emitting radionuclides may increase the sensitivity of HER2 imaging. The goal of this study was to compare directly 2 approaches for developing anti-HER2 PET tracers: a (124)I-labeled monoclonal antibody and a small (7-kDa) scaffold protein, the Affibody molecule, Methods: The anti-HER2 Affibody Z(HER2:342) and humanized monoclonal antibody trastuzumab were labeled with (124/125)I using p-iodobenzoate (PIB) as a linker. Cellular processing of both tracers by HER2-expressing cells was investigated. The biodistributions of (124)I-PIB-Z(HER2:342) and (125)I-PIB-trastuzumab were compared in BALB/C nu/nu mice bearing HER2-expressing NCI-N87 xenografts using paired labels. Small-animal PET of (124)I-PIB-Z(HER2:342) and (124)I-PIB-trastuzumab in tumor-bearing mice was performed at 6, 24, and 72 h after injection. Results: Both radioiodinated Z(HER2:342) and trastuzumab bound specifically to HER2-expressing cells in vitro and specifically targeted HER2-expressing xenografts in vivo. Radioiodinated trastuzumab was more rapidly internalized and degraded, which resulted in better retention of radioactivity delivered by Z(HER2:342). Total uptake of trastuzumab in tumors was higher than that of (124)I-PIB-Z(HER2:342). However, tumor-to-organ ratios were appreciably higher for (124)I-PIB-Z(HER2:342) due to the more rapid clearance of radioactivity from blood and normal organs. The ex vivo results were confirmed by small-animal PET. Conclusion: The use of the small scaffold targeting Affibody provides better contrast in HER2 imaging than does the monoclonal antibody.

Keyword
Affibody molecules, imaging, targeting, xenografts, HER2
National Category
Medical and Health Sciences Natural Sciences
Identifiers
urn:nbn:se:kth:diva-40873 (URN)10.2967/jnumed.108.057919 (DOI)000264084500021 ()19223403 (PubMedID)
Note
QC 20110922Available from: 2011-09-22 Created: 2011-09-22 Last updated: 2017-12-08Bibliographically approved
3. Evaluation of the Radiocobalt-Labeled [MMA-DOTA-Cys(61)]-Z(HER2:2395)-Cys Affibody Molecule for Targeting of HER2-Expressing Tumors
Open this publication in new window or tab >>Evaluation of the Radiocobalt-Labeled [MMA-DOTA-Cys(61)]-Z(HER2:2395)-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, 54-62 p.Article in journal (Refereed) Published
Abstract [en]

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. 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. 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. Radiocobalt is a promising label for affibody molecules for future PET applications.

Keyword
PET, Cobalt, Affibody molecule, HER2, Imaging
National Category
Medical and Health Sciences Natural Sciences
Identifiers
urn:nbn:se:kth:diva-40872 (URN)10.1007/s11307-009-0238-8 (DOI)000273479300008 ()19557480 (PubMedID)
Note
QC 20110922Available from: 2011-09-22 Created: 2011-09-22 Last updated: 2017-12-08Bibliographically approved
4. Molecular Design and Optimization of Tc-99m-Labeled Recombinant Affibody Molecules Improves Their Biodistribution and Imaging Properties
Open this publication in new window or tab >>Molecular Design and Optimization of Tc-99m-Labeled Recombinant Affibody Molecules Improves Their Biodistribution and Imaging Properties
Show others...
2011 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 52, no 3, 461-469 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a recently developed class of targeting proteins based on a nonimmunoglobulin scaffold. The small size (7 kDa) and subnanomolar affinity of Affibody molecules enables high-contrast imaging of tumor-associated molecular targets, particularly human epidermal growth factor receptor type 2 (HER2). Tc-99m as a label offers advantages in clinical practice, and earlier studies demonstrated that Tc-99m-labeled recombinant Affibody molecules with a C-terminal cysteine could be used for HER2 imaging. However, the renal retention of radioactivity exceeded tumor uptake, which might complicate imaging of metastases in the lumbar region. The aim of this study was to develop an agent with low renal uptake and preserved tumor targeting. Methods: A series of recombinant derivatives of the HER2-binding Z(HER2:342) Affibody molecule with a C-terminal chelating sequence, -GXXC (X denoting glycine, serine, lysine, or glutamate), was designed. The constructs were labeled with Tc-99m and evaluated in vitro and in vivo. Results: All variants were stably labeled with Tc-99m, with preserved capacity to bind specifically to HER2-expressing cells in vitro and in vivo. The composition of the chelating sequence had a clear influence on the cellular processing and biodistribution properties of the Affibody molecules. The best variant, Tc-99m-Z(HER2:V2), with the C-terminal chelating sequence -GGGC, provided the lowest radioactivity retention in all normal organs and tissues including the kidneys. Tc-99m-Z(HER2:V2) displayed high uptake of radioactivity in HER2-expressing xenografts, 22.6 +/- 4.0 and 7.7 +/- 1.5 percentage injected activity per gram of tissue at 4 h after injection in SKOV-3 (high HER2 expression) and DU-145 (low HER2 expression) tumors, respectively. In both models, the tumor uptake exceeded the renal uptake. Conclusion: These results demonstrate that the biodistribution properties of recombinant Tc-99m-labeled Affibody molecules can be optimized by modification of the C-terminal cysteine-containing chelating sequence. Tc-99m-Z(HER2:V2) is a promising candidate for further development as a diagnostic radiopharmaceutical for imaging of HER2-expressing tumors. These results may be useful for the development of imaging agents based on other Affibody molecules and, hopefully, other scaffolds.

Keyword
Affibody molecule, Tc-99m, molecular imaging, HER2, C-terminal cysteine
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-31327 (URN)10.2967/jnumed.110.083592 (DOI)000287578200029 ()2-s2.0-79952798198 (Scopus ID)
Note
QC 20110318Available from: 2011-03-18 Created: 2011-03-14 Last updated: 2017-12-11Bibliographically approved
5. Specific in vivo imaging of HER2-positive tumors within one hour using a site-specifically 11C-labeled Sel-tagged Affibody molecule
Open this publication in new window or tab >>Specific in vivo imaging of HER2-positive tumors within one hour using a site-specifically 11C-labeled Sel-tagged Affibody molecule
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for improvement of cancer care. In v ivo imaging methods are available, but are not yet in clinical practice; new methodologies improving speed, sensitivity and specificity are required. Here we describe promising results with a HER2‐binding Affibody molecule, ZHER2:342, recombinantly fused with a C‐terminal selenocysteine‐containing tetrapeptide Sel‐tag and site‐specifically labeled with either 11C or 68Ga for molecular imaging applications with positron emissiontomography (PET).

In mice, both the 11C‐ and 68Ga‐labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4‐5 %ID/g at 1 h. Final uptake in kidneys was much lower (> 5‐fold) for the 11C‐labeled protein, leading to markedly reduced background radioactivity in the abdomen. Furthermore, 11C‐labeled Sel‐tagged ZHER2:342 showed excellent tumor targeting capability, with almost 10 %ID/g in HER2 expressing tumors within the first hour. High specificity was demonstrated by preblocking the binding sites with excess ligand, which yielded low radiotracer uptakes, comparable to those in tumors with low endogenous HER2 expression.

To our knowledge the Sel‐tagging technique is the first that enables site‐specific 11C radiolabelingof proteins. Here we present that, in a favorable combination between radionuclide half‐life and in vivo pharmacokinetics of the Affibody molecules, 11C‐labeled Sel taggedZHER2:342 can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.

National Category
Medical and Health Sciences Natural Sciences
Identifiers
urn:nbn:se:kth:diva-40859 (URN)
Note
QC 20110922Available from: 2011-09-21 Created: 2011-09-21 Last updated: 2012-03-19Bibliographically approved

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