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Preclinical Development of Imaging Agents for HER2 Expression in Prostate Cancer Using Radiolabeled Affibody Molecules
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform. (Orlova)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is focused on pre-clinical evaluation of in vivo detection of HER2-expression in prostate cancer (PCa) patients and on the possibility of using targeted molecular imaging to personalize treatment of disseminated PCa. The work is divided into three distinct parts: (1) the establishment of a preclinical model for further studies, (2) imaging of HER2 in a murine model of PCa and (3) exploration of new treatment regimes against PCa. The characterized cell line panel reflect the heterogeneity of PCa in a way that one cell line never could, and is crucial for a better understanding of different developmental stages during the progression toward androgen independence. The possibility of molecular detection of HER2 in PCa was determined in vitro using 111In-labeled CHX-A’’DTPA-trastuzumab and anti-HER2 ABY-025 affibody molecules. A novel real-time assay for radiolabeled tracer kinetics on living cells was evaluated, in an attempt to bring early developmental work a step closer to the target environment (imaging in living systems). The second part demonstrated the possibility of imaging PCa xenografts, despite the low expression levels, and that  ABY-025 is better adapted for this than the therapeutic anti-HER2 antibody trastuzumab. The study also demonstrated that a residualizing radiometal-label further improves imaging contrast. A comparative study of a HER2-binding affibody molecule N-terminally conjugated to DOTA, NOTA or NODAGA highlighted the influence of the chelator on biodistribution and emphasized the importance of taking into account potential metastatic sited during tracer development. The final study used the previously established in vitro model to explore the hypothesis of using molecular imaging of HER2 to identify PCa patients that may benefit from complemental treatment.

One conclusion from this thesis is that for imaging of PCa, molecular biological context and expression of the molecular target are equally important to consider. Another, that evaluation of response to treatment also need to consider the effect on the overall phenotypic profile, and consequently what this could mean for the efficacy of the treatment. The results of this thesis are in a larger perspective related to how the heterogeneity of tumors may affect the models used for diagnostics and monitoring of cancer in general.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 66 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 179
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science; Biomedical Radiation Science
Identifiers
URN: urn:nbn:se:uu:diva-207256ISBN: 978-91-554-8764-5 (print)OAI: oai:DiVA.org:uu-207256DiVA: diva2:650777
Public defence
2013-11-08, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-10-17 Created: 2013-09-11 Last updated: 2014-01-23
List of papers
1. Imaging agents for in vivo molecular profiling of disseminated prostate cancer: Cellular processing of [In-111]-labeled CHX-A "DTPA-trastuzumab and anti-HER2 ABY-025 Affibody in prostate cancer cell lines
Open this publication in new window or tab >>Imaging agents for in vivo molecular profiling of disseminated prostate cancer: Cellular processing of [In-111]-labeled CHX-A "DTPA-trastuzumab and anti-HER2 ABY-025 Affibody in prostate cancer cell lines
2011 (English)In: Experimental and Therapeutic Medicine, ISSN 1792-0981, Vol. 2, no 3, 523-528 p.Article in journal (Refereed) Published
Abstract [en]

The treatment of disseminated prostate cancer remains a great challenge in current oncology practice. The proliferation of prostate cancer cells is testosterone-driven, but clonal selection during androgen deprivation therapy promotes the development of androgen-independent (hormone-refractory) cells, which become phenotypically dominant. Human epidermal growth factor receptor type 2 (HER2) is capable of activating the androgen receptor pathway, even in the absence of the ligand. The detection of phenotypic changes associated with the development of androgen independence may influence patient management, suggesting the initiation of a second-line therapy. This study aimed to establish the level of HER2 expression in a number of prostate cancer cell lines (LNCaP, PC3 and DU145) in order that they be used as models in further studies, and to evaluate the binding and cellular processing of [In-111]-labeled trastuzumab and the anti-HER2 synthetic Affibody molecule ABY-025 in these cell lines. The expression of HER2 was demonstrated and quantified in all three tested prostate cancer cell-lines. Studies on cellular processing demonstrated that internalization of both conjugates increased continuously during the whole incubation. The internalization rate was approximately equal for both monoclonal antibodies and Affibody molecules. In both cases, internalization was moderately rapid. Such features would definitely favor the use of radiometal labels for trastuzumab and, most likely, for affibody molecules. The level of HER2 expression in these cell lines is sufficient for in vivo molecular imaging.

Keyword
human epidermal growth factor receptor type 2, prostate cancer, trastuzumab, Affibody molecule, DU145, PC3, LNCaP, indium-111, in vitro, cellular processing
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-153296 (URN)10.3892/etm.2011.217 (DOI)000289818200023 ()
Available from: 2011-05-10 Created: 2011-05-10 Last updated: 2015-03-24Bibliographically approved
2. Protein interactions with HER-family receptors can have different characteristics depending on the hosting cell line
Open this publication in new window or tab >>Protein interactions with HER-family receptors can have different characteristics depending on the hosting cell line
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2012 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 40, no 5, 1677-1682 p.Article in journal (Refereed) Published
Abstract [en]

Cell lines are common model systems in the development of therapeutic proteins and in the research on cellular functions and dysfunctions. In this field, the protein interaction assay is a frequently used tool for assessing the adequacy of a protein for diagnostic and therapeutic purposes. In this study, we investigated the extent to which the interaction characteristics depend on the choice of cell line for HER-family receptors. The interaction characteristics of two therapeutic antibodies (trastuzumab and cetuximab) and one Affibody molecule (ZHER2:342), interacting with the intended receptor were characterized with high precision using an automated real-time interaction method, in different cell lines (HaCaT, A431, HEP-G2, SKOV3, PC3, DU-145). Clear differences in binding affinity and kinetics, up to one order of magnitude, were found for the interaction of the same protein binding to the same receptor on different cells for all three proteins. For HER-family receptors, it is therefore important to refer to the measured affinity for a protein-receptor interaction together with the hosting cell line. The ability to accurately measure affinity and kinetics of a protein-receptor interaction on cell lines of different origins may increase the understanding of underlying receptor biology, and impact the selection of candidates in the development of therapeutic or diagnostic agents.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-164998 (URN)10.3892/ijo.2011.1307 (DOI)000302273400041 ()22200885 (PubMedID)
Available from: 2012-01-01 Created: 2012-01-01 Last updated: 2017-12-08Bibliographically approved
3. Comparative biodistribution of imaging agents for in vivo molecular profiling of disseminated prostate cancer in mice bearing prostate cancer xenografts: focus on (111)In- and (125)I-labeled anti-HER2 humanized monoclonal trastuzumab and ABY-025 Affibody
Open this publication in new window or tab >>Comparative biodistribution of imaging agents for in vivo molecular profiling of disseminated prostate cancer in mice bearing prostate cancer xenografts: focus on (111)In- and (125)I-labeled anti-HER2 humanized monoclonal trastuzumab and ABY-025 Affibody
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2011 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 38, no 8, 1093-1102 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: Human epidermal growth factor receptor type 2 (HER2) overexpression supports proliferation of androgen-independent prostate cancer (PC). Radionuclide molecular imaging of HER2 expression in disseminated PC would aid in the selection of patients who are likely responders to HER2 targeting therapy. In this study, we evaluated whether ABY-025 Affibody molecule, a small (similar to 7-kDa) HER2-binding scaffold protein, produces superior tumor-to-nontumor ratios compared with those obtained through the use of radiolabeled humanized anti-HER2 antibody, trastuzumab. The influence of (111)In vs. (125)I radiolabel was evaluated for both tracers.

Methods: ABY-025 was labeled with (111)In using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid chelator, site-specifically coupled to the C-terminus via the maleimido derivative. Trastuzumab was labeled with (111)In using a CHX-A" diethylene triamine pentaacetic acid (DTPA) chelator. An indirect radioiodination with [(125)I]-N-succinimidyl-para-iodobenzoate was used for both targeting proteins. Biodistribution of all labeled targeting proteins was evaluated in mice bearing DU-145 PC xenografts.

Results: The use of residualizing (111)In-label facilitated better tumor uptake and better tumor-to-nontumor ratios for both targeting agents. [(111)In]-ABY-025 provided tumor uptake of 7.1 +/- 0.8% injected dose per gram of tissue (% ID/g) and tumor-to-blood ratio of 47 +/- 13 already at 6 h postinjection. The maximum tumor-to-nontumor ratios with [(111)In]-CHX-DTPA-trastuzumab were achieved at 72 h postinjection, whereas tumor uptake was 11 +/- 4% ID/g and tumor-to-blood ratio was 18 +/- 7. The biodistribution data were confirmed with gamma-camera imaging.

Conclusions: Radiolabeled ABY-025 Affibody molecule provides higher contrast in imaging of HER2-expressing PC xenografts than radiolabeled trastuzumab. Residualizing radiometal label for ABY-025 provides better contrast in imaging of HER2-expressing PC xenografts than nonresidualizing radiohalogen.

Keyword
HER2, Prostate cancer, Trastuzumab, Affibody molecule, Biodistribution, Radiolabeling
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-166866 (URN)10.1016/j.nucmedbio.2011.04.005 (DOI)000298070400003 ()22137850 (PubMedID)
Available from: 2012-01-16 Created: 2012-01-16 Last updated: 2017-12-08Bibliographically approved
4. Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with 111In using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts.
Open this publication in new window or tab >>Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with 111In using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts.
Show others...
2012 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, no 3, 481-492 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: In disseminated prostate cancer, expression of human epidermal growth factor receptor type 2 (HER2) is one of the pathways to androgen independence. Radionuclide molecular imaging of HER2 expression in disseminated prostate cancer might identify patients for HER2-targeted therapy. Affibody molecules are small (7 kDa) targeting proteins with high potential as tracers for radionuclide imaging. The goal of this study was to develop an optimal Affibody-based tracer for visualization of HER2 expression in prostate cancer.

METHODS: A synthetic variant of the anti-HER2 Z(HER2:342) Affibody molecule, Z(HER2:S1), was N-terminally conjugated with the chelators DOTA, NOTA and NODAGA. The conjugated proteins were biophysically characterized by electrospray ionization mass spectroscopy (ESI-MS), circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR)-based biosensor analysis. After labelling with (111)In, the biodistribution was assessed in normal mice and the two most promising conjugates were further evaluated for tumour targeting in mice bearing DU-145 prostate cancer xenografts.

RESULTS: The HER2-binding equilibrium dissociation constants were 130, 140 and 90 pM for DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1), respectively. A comparative study of (111)In-labelled DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1) in normal mice demonstrated a substantial influence of the chelators on the biodistribution properties of the conjugates. (111)In-NODAGA-Z(HER2:S1) had the most rapid clearance from blood and healthy tissues. (111)In-NOTA-Z(HER2:S1) showed high hepatic uptake and was excluded from further evaluation. (111)In-DOTA-Z(HER2:S1) and (111)In-NODAGA-Z(HER2:S1) demonstrated specific uptake in DU-145 prostate cancer xenografts in nude mice. The tumour uptake of (111)In-NODAGA-Z(HER2:S1), 5.6 ± 0.4%ID/g, was significantly lower than the uptake of (111)In-DOTA-Z(HER2:S1), 7.4 ± 0.5%ID/g, presumably because of lower bioavailability due to more rapid clearance. (111)In-NODAGA-Z(HER2:S1) provided higher tumour-to-blood ratio, but somewhat lower tumour-to-liver, tumour-to-spleen and tumour-to-bone ratios.

CONCLUSION: Since distant prostate cancer metastases are situated in bone or bone marrow, the higher tumour-to-bone ratio is the most important. This renders (111)In-DOTA-Z(HER2:S1) a preferable agent for imaging of HER2 expression in disseminated prostate cancer.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-173741 (URN)10.1007/s00259-011-1992-9 (DOI)000302287200015 ()22322933 (PubMedID)
Available from: 2012-05-04 Created: 2012-05-04 Last updated: 2017-12-07Bibliographically approved
5. In Vitro Therapy Modeling of HER2 Targeting Therapy in Disseminated Prostate Cancer
Open this publication in new window or tab >>In Vitro Therapy Modeling of HER2 Targeting Therapy in Disseminated Prostate Cancer
Show others...
2014 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 45, no 5, 2153-2158 p.Article in journal (Refereed) Published
Abstract [en]

Prostate cancer (PCa) is the most common cancer type among men. Treatments against advanced PCa are limited and in many cases only palliative. In a later, androgent independent, stage of PCa androgen receptors can be activated without interaction with ligand, i.e., by receptors of tyrosine kinase (RTK) family in the outlaw pathway. Human epidermal growth factor receptors HER2 and EGFR belong to RTK-family. HER2 is one of the main actors in the outlaw pathway with EGFR as the preferable heterodimerizing partner. We hypothesized that information on HER2 expression in advanced PCa could be useful for selection of patients for anti-RTK therapy and monitoring of therapy response. A panel of PCa cell lines (LNCap, PC3, DU-145) was subjected to a 8-week treatment using drugs influencing the RTK: trastuzumab (anti‑HER2), 17-DMAG (Hsp90 inhibitor), alone or in combination, and their HER2 and EGFR expressions were compared with non-treated cells. Treatment with trastuzumab decreased proliferation of LNCap and DU-145 cell lines, while 17-DMAG and trastuzumab/17‑DMAG combination affected all three cell lines. HER2 expression was significantly increased in PC3 cells, the most resistant cell line. On the contrary, in responding cells (LNCap and DU-145) HER2 expression decreased, accompanied by increased EGFR expression. However, additional treatment of cells with cetuximab (anti‑EGFR) did not give any additive effect to trastuzumab. In this study the response to anti-RTK therapy proved to vary between different PCa cell lines. We have demonstrated that RTK targeting treatments may affect the phenotypic profile of PCa tumor cells that correlates with therapy outcome. Observation of such changes during treatment could be used for monitoring and an improved therapy outcome.

Keyword
prostate cancer, HER2, targeted therapy, molecular imaging, in vitro modeling
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-207258 (URN)10.3892/ijo.2014.2628 (DOI)000342713900042 ()25176024 (Scopus ID)
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2013-09-23 Created: 2013-09-11 Last updated: 2017-12-06Bibliographically approved

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