| 1. |
- Barta, Pavel, et al.
(författare)
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Protein interactions with HER-family receptors can have different characteristics depending on the hosting cell line
- 2012
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 40:5, s. 1677-1682
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Hofström, Camilla, et al.
(författare)
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HAHAHA, HEHEHE, HIHIHI, or HKHKHK : Influence of Position and Composition of Histidine Containing Tags on Biodistribution of [Tc-99m(CO)(3)](+)-Labeled Affibody Molecules
- 2013
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 56:12, s. 4966-4974
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Tidskriftsartikel (refereegranskat)abstract
- Engineered affibody molecules can be used for high contrast in vivo molecular imaging. Extending a recombinantly produced HER2 binding affibody molecule with a hexa-histidine tag allows for convenient purification by immobilized metal-ion affinity chromatography and labeling with [Tc-99m(CO)(3)](+) but increases radioactivity uptake in the liver. To investigate the impact of charge, lipophilicity, and position on biodistribution, 10 variants of a histidine-based tag was attached to a HER2 binding affibody molecule. The biochemical properties and the HER2 binding affinity appeared to be similar for all variants. In vivo, positive charge promoted liver uptake. For N-terminally placed tags, promoted liver uptake and decreased kidney uptake. Kidney uptake was higher for C-terminally placed tags compared to their N-terminal counterparts. The variant with the amino acid composition HEHEHE placed in the N-terminus gave the lowest nonspecific uptake.
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| 3. |
- Honarvar, Hadis, et al.
(författare)
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Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging
- 2013
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Ingår i: Nuclear Medicine and Biology. - : Elsevier BV. - 0969-8051 .- 1872-9614. ; 40:3, s. 378-386
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Affibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL). Methods: The HER2-targeting NCL-cyclized Affibody molecule Z(HER2:342min) has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-Z(HER2:342min) was labeled with In-111 and (68) Ga. The binding affinity of DOTA-Z(HER2:342min) was evaluated in vitro. The targeting properties of In-111- and (68) Ga-DOTA-Z(HER2:342min) were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of In-111- and (68) Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge. Results: The dissociation constant (K-D) for DOTA-Z(HER2:342min) binding to HER2 was 18 nM according to SPR measurements. DOTA-Z(HER2:342min) was labeled with In-111 and (68) Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with K-D1 in low nanbmolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 +/- 1.3 for In-111-DOTA-Z(HER2:342min) and 4.6 +/- 0.7 for (68) Ga-DOTA-Z(HER2:342min). However, the uptake of DOTA-Z(HER2:342min) in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts. Conclusions: Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (Z(HER2:342min)) with low nanomolar target affinity and specific tumor uptake.
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| 4. |
- Malmberg, Jennie, et al.
(författare)
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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
- 2011
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Ingår i: Nuclear Medicine and Biology. - : Elsevier BV. - 0969-8051 .- 1872-9614. ; 38:8, s. 1093-1102
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Malmberg, Jennie, et al.
(författare)
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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.
- 2012
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 39:3, s. 481-492
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Malmberg, Jennie, et al.
(författare)
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Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with In-111 using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts
- 2012
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 39:3, s. 481-492
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Tidskriftsartikel (refereegranskat)abstract
- 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 In-111, 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 In-111-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. In-111-NODAGA-Z(HER2:S1) had the most rapid clearance from blood and healthy tissues. In-111-NOTA-Z(HER2:S1) showed high hepatic uptake and was excluded from further evaluation. In-111-DOTA-Z(HER2:S1) and In-111-NODAGAZHER2: S1 demonstrated specific uptake in DU-145 prostate cancer xenografts in nude mice. The tumour uptake of In-111-NODAGA-Z(HER2:S1), 5.6 +/- 0.4% ID/g, was significantly lower than the uptake of In-111-DOTA-Z(HER2:S1), 7.4 +/- 0.5% ID/g, presumably because of lower bioavailability due to more rapid clearance. In-111-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 In-111-DOTA-Z(HER2:S1) a preferable agent for imaging of HER2 expression in disseminated prostate cancer.
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| 7. |
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| 8. |
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| 9. |
- Malmberg, Jennie, et al.
(författare)
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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
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Ingår i: Experimental and Therapeutic Medicine. - : Spandidos Publications. - 1792-0981 .- 1792-1015. ; 2:3, s. 523-528
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Malmberg, Jennie, et al.
(författare)
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Imaging agents for in vivo molecular profiling of disseminated prostate cancer - targeting EGFR receptors in prostate cancer : Comparison of cellular processing of [In-111]-labeled affibody molecule Z(EGFR:2377) and cetuximab
- 2011
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 38:4, s. 1137-1143
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Tidskriftsartikel (refereegranskat)abstract
- Expression of receptor tyrosine-kinase (RTK) EGFR is low in normal prostate, but increases in prostate cancer. This receptor is significantly up-regulated as tumors progress into higher grade, androgen-insensitive and metastatic lesions. The up-regulated receptors could serve as targets for novel selective anti-cancer drugs, e.g. antibodies and tyrosine kinase inhibitors. Radionuclide imaging of RTK can facilitate patient stratification and monitoring of anti-RTK therapy of prostate cancer. The goal of the study was to evaluate binding and cellar processing of radiolabeled EGFR-targeting conjugates by prostate cancer cell lines. Receptor expression of EGFR was studied in three prostate cancer cell lines: DU145 (brain metastasis of PC, hormone insensitive), PC3 (bone metastasis of PC) and LNCaP (lymph node metastasis of PC, androgen and estrogen receptor positive). Uptake and internalization of anti-EGFR mAbs (cetuximab) and affibody molecule (Z2377) labeled with indium-111 was investigated. EGFR expression on prostate cancer cell lines was clearly demonstrated. Both labelled conjugates 111In-Z2377 and 111In-cetuximab bound to prostate cancer cells in the receptor mediated model. Expression levels were modest but correlate with degree of hormone independence. Internalization of Affibody molecules was relatively slow in all cell lines. Internalization of mAbs was more rapid. The level of EGFR expression in these cell lines is sufficient for in vivo molecular imaging. Slow internalization indicates possibility of the use of non-residualizing labels for affibody molecules.
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