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  • 1. Altai, Mohamed
    et al.
    Honarvar, Hadis
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Protein Technology.
    Strand, Joanna
    Varasteh, Zohreh
    Rosestedt, Maria
    Orlova, Anna
    Dunås, Finn
    Sandström, Mattias
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.2014In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 87, p. 519-28Article in journal (Refereed)
    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 (188)Re-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 (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-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 (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

  • 2. Altai, Mohamed
    et al.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, Hadis
    Strand, Joanna
    Orlova, Anna
    Varasteh, Zohreh
    Sandström, Mattias
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Larsson, Erik
    Strand, Sven-Erik
    Lubberink, Mark
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology. Uppsala University, Sweden.
    Tolmachev, Vladimir
    Re-188-Z(HER2:V2), a Promising Affibody-Based Targeting Agent Against HER2-Expressing Tumors: Preclinical Assessment2014In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 55, no 11, p. 1842-1848Article in journal (Refereed)
    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 Tc-99m-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated Z(HER2:V2)) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate Re-188-Z(HER2:v2) as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors. Methods: Z(HER2:V2) was labeled with Re-188 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 Re-188-Z(HER2: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 Re-188-Z(HER2: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: (188)ReZ(HER2:v2) can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.

  • 3. Altai, Mohamed
    et al.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Rosestedt, Maria
    Hosseinimehr, Seyed Jalal
    Tolmachev, Vladimir
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of Tc-99m-labeled recombinant Affibody molecules2012In: Amino Acids, ISSN 0939-4451, E-ISSN 1438-2199, Vol. 42, no 5, p. 1975-1985Article in journal (Refereed)
    Abstract [en]

    Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide Tc-99m. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of Tc-99m-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied Tc-99m-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of Tc-99m. The biodistribution of all Tc-99m-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of Tc-99m was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

  • 4. Ekerljung, L.
    et al.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Sohrabian, A.
    Andersson, K.
    Friedman, M.
    Frejd, F. Y.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gedda, L.
    Generation and evaluation of bispecific affibody molecules for simultaneous targeting of EGFR and HER22012In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 9, p. 1802-1811Article in journal (Refereed)
    Abstract [en]

    Coexpression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here, we have generated six such bispecific targeting proteins, each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly 4Ser) 3 or (Ser 4Gly) 3, and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays, it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the inherent monomeric affibody molecules was favorable to promote the binding to the respective target. Interestingly, bispecific constructs containing the novel (Ser 4Gly) 3 linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly 4Ser) 3. It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimer formation and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.

  • 5.
    Jonsson, Andreas
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Herne, N.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Frejd, F. Y.
    Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, p. 93-103Article in journal (Refereed)
    Abstract [en]

    Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For Z(TNF alpha:185), subnanomolar affinity (K-D = 0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the Z(TNF-alpha:185) affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dinners with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

  • 6.
    Lindberg, Hanna
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Altai, Mohamed
    Honorvar, Hadis
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Orlova, Anna
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Tolmachev, Vladimir
    Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for Tc-99m-labelling at the C terminus2012In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 33, no 3, p. 641-651Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide Tc-99m. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with Tc-99m. Tc-99m-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, Tc-99m-Z(HER2:2395)-VDC and Tc-99m-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of Tc-99m-Z(HER2:2395)-VDC, but it was substantially higher than uptake of Tc-99m-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of Tc-99m was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of Tc-99m labelling due to a partial loss of site-specificity of nuclide chelation.

  • 7. Orlova, Anna
    et al.
    Wållberg, Helena
    Stone-Elander, Sharon
    Tolmachev, Vladimir
    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 Model2009In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 50, no 3, p. 417-425Article in journal (Refereed)
    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.

  • 8. Tolmachev, V.
    et al.
    Orlova, A.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Abrahmsén, L.
    Feldwisch, J.
    EGFR targeting with site-specifically In-111-labeled second generation Affibody molecules2010In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 37, no Suppl. 2, p. S264-S264Article in journal (Other academic)
  • 9. Tolmachev, V.
    et al.
    Rosik, D.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Sjöberg, A.
    Sandström, M.
    Hansson, M.
    Wennborg, A.
    Orlova, A.
    Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR In-111-DOTA-Z(EGFR:2377) Affibody molecule: aspect of the amount of injected tracer2009In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 36, p. S251-S252Article in journal (Other academic)
  • 10. Tolmachev, Vladimir
    et al.
    Rosik, Daniel
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Sjoberg, Anna
    Sandstrom, Mattias
    Hansson, Monika
    Wennborg, Anders
    Orlova, Anna
    Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR In-111-DOTA-Z(EGFR:2377) Affibody molecule: aspect of the injected tracer amount2010In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 37, no 3, p. 613-622Article in journal (Refereed)
    Abstract [en]

    Overexpression of epidermal growth factor receptor (EGFR) is a prognostic and predictive biomarker in a number of malignant tumours. Radionuclide molecular imaging of EGFR expression in cancer could influence patient management. However, EGFR expression in normal tissues might complicate in vivo imaging. The aim of this study was to evaluate if optimization of the injected protein dose might improve imaging of EGFR expression in tumours using a novel EGFR-targeting protein, the DOTA-Z(EGFR:2377) Affibody molecule. An anti-EGFR Affibody molecule, Z(EGFR:2377), was labelled with In-111 via the DOTA chelator site-specifically conjugated to a C-terminal cysteine. The affinity of DOTA-Z(EGFR:2377) for murine and human EGFR was measured by surface plasmon resonance. The cellular processing of In-111-DOTA-Z(EGFR:2377) was evaluated in vitro. The biodistribution of radiolabelled Affibody molecules injected in a broad range of injected Affibody protein doses was evaluated in mice bearing EGFR-expressing A431 xenografts. Site-specific coupling of DOTA provided a uniform conjugate possessing equal affinity for human and murine EGFR. The internalization of In-111-DOTA-Z(EGFR:2377) by A431 cells was slow. In vivo, the conjugate accumulated specifically in xenografts and in EGFR-expressing tissues. The curve representing the dependence of tumour uptake on the injected Affibody protein dose was bell-shaped. The highest specific radioactivity (lowest injected protein dose) provided a suboptimal tumour-to-blood ratio. The results of the biodistribution study were confirmed by gamma-camera imaging. The In-111-DOTA-Z(EGFR:2377) Affibody molecule is a promising tracer for radionuclide molecular imaging of EGFR expression in malignant tumours. Careful optimization of protein dose is required for high-contrast imaging of EGFR expression in vivo.

  • 11. Tran, T. A.
    et al.
    Rosik, D.
    Abrahmsen, L.
    Sandström, M.
    Sjöberg, A.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Ahlgren, S.
    Orlova, A.
    Tolmachev, V.
    Molecular imaging of HER2 expression using a multifunctional Affibody molecule2009In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 36, p. S233-S233Article in journal (Other academic)
  • 12. Tran, Thuy A.
    et al.
    Rosik, Daniel
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Abrahmsen, Lars
    Sandstrom, Mattias
    Sjoberg, Anna
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ahlgren, Sara
    Orlova, Anna
    Tolmachev, Vladimir
    Design, synthesis and biological evaluation of a multifunctional HER2-specific Affibody molecule for molecular imaging2009In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 36, no 11, p. 1864-1873Article in journal (Refereed)
    Abstract [en]

    The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling both recombinant and synthetic production and site-specific labelling with Tc-99m or trivalent radiometals. The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow Tc-99m labelling. The cysteine can alternatively serve as a conjugation site of the chelator DOTA for indium labelling. The resulting Tc-99m- and In-111-labelled Affibody molecules were evaluated both in vitro and in vivo. Both conjugates retained their capacity to bind to HER2 receptors in vitro and in vivo. The tumour to blood ratio in LS174T xenografts was 30 at 4 h post-injection for both conjugates. Biodistribution data showed that the Tc-99m-labelled Affibody molecule had a fourfold lower kidney accumulation compared with the In-111-labelled Affibody molecule while the accumulation in other organs was similar. Gamma camera imaging of the conjugates could clearly visualise the tumours 4 h after injection. Incorporation of the C-terminal SECG sequence in Affibody molecules provides a general multifunctional platform for site-specific labelling with different nuclides (technetium, indium, gallium, cobalt or yttrium) and for a flexible production (chemical synthesis or recombinant).

  • 13. Wallberg, Helena
    et al.
    Ahlgren, Sara
    Widstrom, Charles
    Orlova, Anna
    Evaluation of the Radiocobalt-Labeled [MMA-DOTA-Cys(61)]-Z(HER2:2395)-Cys Affibody Molecule for Targeting of HER2-Expressing Tumors2010In: Molecular Imaging and Biology, ISSN 1536-1632, E-ISSN 1860-2002, Vol. 12, no 1, p. 54-62Article in journal (Refereed)
    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.

  • 14.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Design and Evaluation of Radiolabeled Affibody Tracers for Imaging of HER2-expressing Tumors2011Doctoral 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.

  • 15.
    Wållberg, Helena
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Grafström, Jonas
    Cheng, Qing
    Lu, Li
    Ahlzén, Hanna-Stina Martinsson
    Samén, Erik
    Thorell, Jan-Olov
    Johansson, Katarina
    Dunås, Finn
    Olofsson, Maria Hägg
    Stone-Elander, Sharon
    Arnér, Elias S. J.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    HER2-Positive Tumors Imaged Within 1 Hour Using a Site-Specifically C-11-Labeled Sel-Tagged Affibody Molecule2012In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 53, no 9, p. 1446-1453Article in journal (Refereed)
    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 individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. Methods: A HER2-binding Affibody molecule, Z(HER2:342), was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either C-11 or Ga-68, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the C-11-labeled Affibody molecule. Results: Both the C-11- and Ga-68-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4-5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (>5-fold) for the C-11-labeled protein, and its overall absorbed dose was considerably lower. C-11-Z(HER2:342) showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P = 0.002), comparable to uptake in tumors with low HER2 expression. Conclusion: To our knowledge, the Sel-tagging technique is the first that enables site-specific C-11-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, C-11-labeled Set-tagged Z(HER2:342) can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.

  • 16.
    Wållberg, Helena
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Grafström, Jonas
    Cheng, Qing
    Lu, Li
    Martinsson Ahlzén, Hanna-Stina
    Samén, Erik
    Thorell, Jan-Olov
    Johansson, Katarina
    Dunås, Finn
    Hägg Olofsson, Maria
    Stone-Elander, Sharon
    Arnér, Elias
    Shåhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Specific in vivo imaging of HER2-positive tumors within one hour using a site-specifically 11C-labeled Sel-tagged Affibody moleculeManuscript (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.

  • 17. Wållberg, Helena
    et al.
    Löfdahl, Per-Åke
    Tschapalda, Kirsten
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Tolmachev, Vladimir
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand2011In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 76, no 1, p. 127-135Article in journal (Refereed)
    Abstract [en]

    Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new beta-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (Tc-99m)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.

  • 18. Wållberg, Helena
    et al.
    Orlova, Anna
    Slow internalization of anti-HER2 synthetic affibody monomer 111In-DOTA-ZHER2: 342-pep22008In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 23, no 4, p. 435-42Article in journal (Refereed)
    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.

  • 19.
    Wållberg, Helena
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Altai, Mohammed
    Hosseinimehr, Seyed Jalal
    Widström, Charles
    Malmberg, Jennie
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Tolmachev, Vladimir
    Molecular Design and Optimization of Tc-99m-Labeled Recombinant Affibody Molecules Improves Their Biodistribution and Imaging Properties2011In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 52, no 3, p. 461-469Article in journal (Refereed)
    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.

  • 20.
    Wållberg, Helena
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Design and evaluation of radiolabeled tracers for tumor imaging2013In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 60, no 4, p. 365-383Article in journal (Refereed)
    Abstract [en]

    The growing understanding of tumor biology and the identification of tumor-specific genetic and molecular alterations, such as the overexpression of membrane receptors and other proteins, allows for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools used to identify patients who are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising noninvasive method to visualize and characterize the expression of such targets. A number of different proteins, from full-length antibodies and their derivatives to small scaffold proteins and peptide receptor-ligands, have been applied to molecular imaging, each demonstrating strengths and weaknesses. Here, we discuss the concept of molecular targeting and, in particular, molecular imaging of cancer-associated targets. Additionally, we describe important biotechnological considerations and desired features when designing and developing tracers for radionuclide molecular imaging.

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