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  • 1.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Wållberg, Helena
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Varasteh, Zohreh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Dunås, Finn
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Löfblom, John
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Ståhl, Stefan
    Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with 188-Re2013In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, no Suppl. 2, p. S219-S220Article in journal (Other academic)
    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 188Re-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 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1±0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-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 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

  • 2.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Wållberg, Helena
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Varasteh, Zohreh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Dunås, Finn
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Medical Radiation Sciences.
    Löfblom, John
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Ståhl, Stefan
    Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with 188Re2014In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 87, p. 519-528Article 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 188Re-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 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-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 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

  • 3.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Leitao, Charles Dahlsson
    KTH Royal Inst Technol, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-10691 Stockholm, Sweden.
    Rinne, Sara S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Vorobyeva, Anzhelika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Atterby, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Ståhl, Stefan
    KTH Royal Inst Technol, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-10691 Stockholm, Sweden.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Löfblom, John
    KTH Royal Inst Technol, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-10691 Stockholm, Sweden.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs2018In: CELLS, ISSN 2073-4409, Vol. 7, no 10, article id 164Article in journal (Refereed)
    Abstract [en]

    Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD 035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.

  • 4.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Liu, H.
    KTH, Div Prot Technol, Stockholm, Sweden..
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Gräslund, T.
    KTH, Div Prot Technol, Stockholm, Sweden..
    Improving of molecular design of a novel Affibody-fused HER2-recognising anticancer toxin using radionuclide-based techniques2016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S178-S178Article in journal (Refereed)
  • 5.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Liu, Hao
    KTH Royal Inst Technol, Dept Prot Sci, Roslagstullsbacken 21, S-11417 Stockholm, Sweden.
    Ding, Haozhong
    KTH Royal Inst Technol, Dept Prot Sci, Roslagstullsbacken 21, S-11417 Stockholm, Sweden.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Edqvist, Per-Henrik D
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Gräslund, Torbjorn
    KTH Royal Inst Technol, Dept Prot Sci, Roslagstullsbacken 21, S-11417 Stockholm, Sweden.
    Affibody-derived drug conjugates: Potent cytotoxic molecules for treatment of HER2 over-expressing tumors2018In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 288, p. 84-95Article in journal (Refereed)
    Abstract [en]

    Patients with HER2-positive tumors often suffer resistance to therapy, warranting development of novel treatment modalities. Affibody molecules are small affinity proteins which can be engineered to bind to desired targets. They have in recent years been found to allow precise targeting of cancer specific molecular signatures such as the HER2 receptor. In this study, we have investigated the potential of an affibody molecule targeting HER2, Z(HER2:2891), conjugated with the cytotoxic maytansine derivate MC-DM1, for targeted cancer therapy. Z(HER2:2891) was expressed as a monomer (Z(HER2:2891)), dimer ((Z(HER2:2891)) 2) and dimer with an albumin binding domain (ABD) for half-life extension ((Z(HER2:2891)) 2-ABD). All proteins had a unique C-terminal cysteine that could be used for efficient and site-specific conjugation with MC-DM1. The resulting affibody drug conjugates were potent cytotoxic molecules for human cells over-expressing HER2, with sub-nanomolar IC50-values similar to trastuzumab emtansine, and did not affect cells with low HER2 expression. A biodistribution study of a radiolabeled version of (Z(HER2:2891))(2)-ABD-MC-DM1, showed that it was taken up by the tumor. The major site of off-target uptake was the kidneys and to some extent the liver. (Z(HER2:2891)) 2-ABD-MC-DM1 was found to have a half-life in circulation of 14 h. The compound was tolerated well by mice at 8.5 mg/kg and was shown to extend survival of mice bearing HER2 over-expressing tumors. The findings in this study show that affibody molecules are a promising class of engineered affinity proteins to specifically deliver small molecular drugs to cancer cells and that such conjugates are potential candidates for clinical evaluation on HER2-overexpressing cancers.

  • 6.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Membreno, Rosemery
    CUNY Hunter Coll, Dept Chem, New York, NY 10021 USA.;CUNY, Grad Ctr, PhD Program Chem, New York, NY USA.;Mem Sloan Kettering Canc Ctr, Dept Radiol, 1275 York Ave, New York, NY 10021 USA..
    Cook, Brendon
    CUNY Hunter Coll, Dept Chem, New York, NY 10021 USA.;CUNY, Grad Ctr, PhD Program Chem, New York, NY USA.;Mem Sloan Kettering Canc Ctr, Dept Radiol, 1275 York Ave, New York, NY 10021 USA..
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Zeglis, Brian M.
    CUNY Hunter Coll, Dept Chem, New York, NY 10021 USA.;CUNY, Grad Ctr, PhD Program Chem, New York, NY USA.;Mem Sloan Kettering Canc Ctr, Dept Radiol, 1275 York Ave, New York, NY 10021 USA..
    Pretargeted Imaging and Therapy2017In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 58, no 10, p. 1553-1559Article in journal (Refereed)
    Abstract [en]

    In vivo pretargeting stands as a promising approach to harnessing the exquisite tumor-targeting properties of antibodies for nuclear imaging and therapy while simultaneously skirting their pharmacokinetic limitations. The core premise of pretargeting lies in administering the targeting vector and radioisotope separately and having the 2 components combine within the body. In this manner, pretargeting strategies decrease the circulation time of the radioactivity, reduce the uptake of the radionuclide in healthy nontarget tissues, and facilitate the use of short-lived radionuclides that would otherwise be incompatible with antibody-based vectors. In this short review, we seek to provide a brief yet informative survey of the 4 preeminent mechanistic approaches to pretargeting, strategies predicated on streptavidin and biotin, bispecific antibodies, complementary oligonucleotides, and bioorthogonal click chemistry.

  • 7.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Radiolabeled Probes Targeting Tyrosine-Kinase Receptors For Personalized Medicine2014In: Current pharmaceutical design, ISSN 1381-6128, E-ISSN 1873-4286, Vol. 20, no 14, p. 2275-2292Article in journal (Refereed)
    Abstract [en]

    Receptor tyrosine kinases (RTK) are transmembrane receptors regulating cellular proliferation, differentiation, apoptosis, motility and recruitment of the vasculature. Aberrant expression and/or function of RTK have been detected in many malignant tumors and are considered to be a part of the transformed phenotype. The action of several classes of anti-cancer drugs is based on specific recognition of RTK. Monoclonal antibodies target extracellular binding domains, while tyrosine kinase inhibitors (TKI) bind to intracellular kinase domains to suppress RTK signaling. The issues regarding the efficient use of RTK targeting are the inter- and intra-patient heterogeneity of RTK expression and the changes of expression levels during the course of disease and in response to therapy. Radionuclide molecular imaging of RTK expression may aid in selecting patients who would benefit from RTK-targeting therapy and in identifying non-responders. Therefore, the therapy would be more personalized. Currently, radiolabeled proteins (monoclonal antibodies and their fragments, natural peptides ligands to RTK and de novo selected affinity proteins) and TKI and their analogues are under development for the visualization of RTK. In this review, we discuss the advantages and disadvantages of these approaches.

  • 8.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Perols, Anna
    Eriksson Karlström, Amelie
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Medical Physics.
    Boschetti, Frederic
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA2012In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 39, no 4, p. 518-529Article in journal (Refereed)
    Abstract [en]

    Introduction

    Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule.

    Methods

    The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to ZHER2:2395 Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 were studied. Biodistribution of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 was compared in mice.

    Results

    The affinity of [MMA-NODAGA-Cys61]-ZHER2:2395 binding to HER2 was 67 pM. The 111In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [111In-MMA-NODAGA-Cys61]-ZHER2:2395 bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [111In-MMA-DOTA-Cys61]-ZHER2:2395 (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [111In-MMA-NODAGA-Cys61]-ZHER2:2395 due to higher clearance rate from normal tissues.

    Conclusions

    MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.

  • 9.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Perols, Anna
    Tsourma, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Robillard, Marc
    Rossin, Raffaella
    Ten Hoeve, Wolter
    Lubberink, Mark
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Eriksson Karlström, Amelie
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Feasibility of affibody-based bioorthogonal chemistry-mediated radionuclide pretargeting2016In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 57, no 3, p. 431-436Article in journal (Refereed)
    Abstract [en]

    Affibody molecules constitute a new class of probes for radionuclide tumor targeting. The small size of affibody molecules is favorable for rapid localization in tumors and clearance from circulation. However, high renal re-absorption of affibody molecules prevents the use of residualizing radiometals, including a number of promising low energy beta- and alpha-emitters, for radionuclide therapy. We tested a hypothesis that affibody-based pretargeting mediated by a bioorthogonal interaction between trans-cyclooctene (TCO) and tetrazine would provide higher accumulation of radiometals in tumor xenografts than in the kidneys.

    Methods:

    TCO was conjugated to the anti-HER2 affibody molecule Z2395. DOTA-tetrazine was labeled with indium-111 and lutetium-177. In vitro pretargeting was studied in HER2-expressing SKOV-3 and BT474 cell lines. In vivo studies were performed on BALB/C nu/nu mice bearing SKOV-3 xenografts.

    Results:

    125I-Z2395-TCO bound specifically to HER2-expressing cells in vitro with an affinity of 45±16 pM. 111In-tetrazine bound specifically and selectively to Z2395-TCO pre-treated cells. In vivo studies demonstrated HER2-specific 125I-Z2395-TCO accumulation in xenografts. TCO-mediated 111In-tetrazine localization was shown in tumors, when the radiolabeled tracer was injected 4 h after an injection of Z2395-TCO. At 1 h post injection, the tumor uptake of 111In-tetrazine and 177Lu-tetrazine was ca. 2-fold higher than the renal uptake. Pretargeting provided more than a 56-fold reduction of renal uptake of 111In in comparison with direct targeting.

    Conclusion:

    The feasibility of affibody-based bioorthogonal chemistry-mediated pretargeting was demonstrated. The use of pretargeting provides a substantial reduction of radiometal accumulation in kidneys, creating preconditions for palliative radionuclide therapy.

  • 10.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Rosik, D.
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Karlstrom, A. Eriksson
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Comparative evaluation of anti-HER2 affibody molecules labeled with 68Ga and 111In using maleimido derivatives of DOTA and NODAGA2012In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, no S2, p. S299-S299Article in journal (Other academic)
  • 11.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Rosik, Daniel
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Eriksson Karlström, Amelie
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Influence of Nuclides and Chelators on Imaging Using Affibody Molecules: Comparative Evaluation of Recombinant Affibody Molecules Site-Specifically Labeled with 68Ga and 111In via Maleimido Derivatives of DOTA and NODAGA2013In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 6, p. 1102-1109Article in journal (Refereed)
    Abstract [en]

    Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use of the generator-produced positron-emitting radionuclide 68Ga should increase sensitivity of HER2 imaging. The chemical nature of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant ZHER2:2395 HER2-binding Affibody molecule with 68Ga. DOTA and NODAGA were site-specifically conjugated to the ZHER2:2395 Affibody molecule having a C-terminal cysteine and labeled with 68Ga and 111In. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were clearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for 68Ga than for 111In. The tumor uptake of 68Ga-NODAGA-ZHER2:2395 and 68Ga-DOTA-ZHER2:2395 and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for 68Ga-NODAGA- ZHER2:2395 (8 ± 2 vs 5.0 ± 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.

  • 12.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Tsourma, M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform. Preclin PET Platform, Uppsala, Sweden..
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Perols, A.
    KTH, Div Prot Technol, Stockholm, Sweden..
    Robillard, M.
    Tagworks Pharmaceut, Eindhoven, Netherlands..
    Rossin, R.
    Tagworks Pharmaceut, Eindhoven, Netherlands..
    ten Hoeve, W.
    Syncom BV, Groningen, Netherlands..
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Karlstrom, A. Eriksson
    KTH, Div Prot Technol, Stockholm, Sweden..
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Affibody-based bioorthogonal chemistry-mediated radionuclide pretargeting: proof-of-principle2015In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, no S1, p. S246-S246Article in journal (Other academic)
  • 13.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Varasteh, Zohreh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Eek, Annemarie
    Boerman, Otto
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    In Vivo and In Vitro Studies on Renal Uptake of Radiolabeled Affibody Molecules for Imaging of HER2 Expression in Tumors2013In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 28, no 3, p. 187-195Article in journal (Refereed)
    Abstract [en]

    Affibody molecules (6-7 kDa) are a new class of small robust three-helical scaffold proteins. Radiolabeled subnanomolar anti-HER2 affibody Z(HER2:342) was developed for imaging of HER2 expression in tumors, and a clinical study has demonstrated that the In-111- and Ga-68-labeled affibody molecules can efficiently detect HER2 expressing metastases in breast cancer patients. However, a significant renal accumulation of radioactivity after systemic injection of a radiolabeled anti-HER2 affibody conjugate is observed. The aim of this study was to investigate the mechanism of renal reabsorption of anti-HER2 affibody at the molecular level. Renal accumulation of radiolabeled anti-HER2 affibody molecules was studied in a murine model and in vitro using opossum-derived proximal tubule (OK) cells. It was found that kidney reabsorption of affibody molecule was not driven by megalin/cubilin. Amino acids in the target-binding side of affibody molecule were involved in binding to OK cells. On OK cells, two types of receptors for anti-HER2 affibody molecule were found: K-D1 = 0.8 nM, B-max1 = 71,500 and K-D2 = 9.2 nM, B-max2 = 367,000. The results of the present study indicate that affibody molecule and other scaffold-based targeting proteins with a relatively low kidney uptake can be selected using in vitro studies with tubular kidney cells.

  • 14.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Westerlund, K.
    KTH, Div Prot Technol, Stockholm, Sweden..
    Velletta, J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Eriksson-Karlström, A.
    KTH, Div Prot Technol, Stockholm, Sweden..
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Comparative evaluation of Lu-177-HP2 and In-111-HP2, secondary agents for affibody-based PNA-mediated radionuclide pretargeting2016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S237-S237Article in journal (Refereed)
  • 15.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Westerlund, Kristina
    KTH Royal Inst Technol, Div Prot Technol, Sch Biotechnol, Stockholm, Sweden..
    Velletta, Justin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Eriksson Karlström, Amelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Evaluation of affibody molecule-based PNA-mediated radionuclide pretargeting: Development of an optimized conjugation protocol and 177Lu labeling2017In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 54, p. 1-9Article in journal (Refereed)
    Abstract [en]

    Introduction: We have previously developed a pretargeting approach for affibody-mediated cancer therapy based on PNA-PNA hybridization. In this article we have further developed this approach by optimizing the production of the primary agent, Z(HER2.342)-SR-HP1, and labeling the secondary agent, HP2, with the therapeutic radionuclide Lu-177. We also studied the biodistribution profile of Lu-177-HP2 in mice, and evaluated pretargeting with Lu-177-HP2 in vitro and in vivo.

    Methods: The biodistribution profile of Lu-177-HP2 was evaluated in NMRI mice and compared to the previously studied In-111-HP2. Pretargeting using Lu-177-HP2 was studied in vitro using the HER2-expressing cell lines BT-474 and SKOV-3, and in vivo in mice bearing SKOV-3 xenografts.

    Results and conclusion: Using an optimized production protocol for Z(HER2:342)-SR-HP1 the ligation time was reduced from 15 h to 30 min, and the yield increased from 45% to 70%. Lu-177-labeled HP2 binds specifically in vitro to BT474 and SKOV-3 cells pre-treated with Z(HER2:342)-SR-HP1.Lu-177-HP2 was shown to have a more rapid blood clearance compared to In-111-HP2 in NMRI mice, and the measured radioactivity in blood was 0.22 +/- 0.1 and 0.68 +/- 0.07%ID/g for Lu-177- and In-111-HP2, respectively, at 1 h p.i. In contrast, no significant difference in kidney uptake was observed (4.47 +/- 1.17 and 3.94 +/- 0.58%ID/g for Lu-177- and In-111-HP2, respectively, at I h p.i.). Co-injection with either Gelofusine or lysine significantly reduced the kidney uptake for Lu-177-HP2 (1.0 +/- 0.1 and 1.6 +/- 0.2, respectively, vs. 2.97 +/- 0.87%ID/g in controls at 4 h p.i.). Lu-177-HP2 accumulated in SKOV-3 xenografts in BALB/C nu/nu mice when administered after injection of Z(HER2:342)-SR-HP1. Without pre-injection of Z(HER2:342)-SR-HP1, the uptake of Lu-177-HP2 was about 90-fold lower in tumor (0.23 +/- 0.08 vs. 20.7 +/- 3.5%ID/g). The tumor-to-kidney radioactivity accumulation ratio was almost 5-fold higher in the group of mice pre-injected with Z(HER2:342)-SR-HP1. In conclusion, (177)LuHP2 was shown to be a promising secondary agent for affibody-mediated tumor pretargeting in vivo.

  • 16.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Wållberg, Helena
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Strand, Joanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Varasteh, Zohreh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Sandström, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Medical Radiation Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Löfblom, John
    Larsson, Erik
    Strand, Sven-Erik
    Lubberink, Mark
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Medical Physics.
    Ståhl, Stefan
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    188Re-ZHER2: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 99mTc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide–based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate 188Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)–expressing tumors.

    Methods:

    ZHER2:V2 was labeled with 188Re 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 188Re-ZHER2: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 188Re-ZHER2: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:

    188Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.

  • 17.
    Altai, Mohamed
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Wållberg, Helena
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Hosseinimehr, Seyed Jalal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Ståhl, Stefan
    Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of (99m)Tc-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 (99m)Tc. 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 (99m)Tc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied (99m)Tc-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 (99m)Tc. The biodistribution of all (99m)Tc-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 (99m)Tc 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.

  • 18.
    Deyev, Sergey
    et al.
    Russian Acad Sci, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Mol Immunol Lab, Moscow, Russia;Natl Res Tomsk Polytech Univ, Tomsk, Russia;Natl Res Nucl Univ MEPhI, Inst Engn Phys Biomed PhysBio, Bionanophoton Lab, Moscow, Russia.
    Vorobyeva, Anzhelika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Schulga, Alexey
    Russian Acad Sci, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Mol Immunol Lab, Moscow, Russia.
    Proshkina, Galina
    Russian Acad Sci, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Mol Immunol Lab, Moscow, Russia.
    Guler, Rezan
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, Stockholm, Sweden.
    Lofblom, John
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, Stockholm, Sweden.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Garousi, Javad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Altai, Mohamed
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Buijs, Jos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Chernov, Vladimir
    Russian Acad Sci, Canc Res Inst, Nucl Med Dept, Tomsk Natl Res Med Ctr, Tomsk, Russia.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Comparative Evaluation of Two DARPin Variants: Effect of Affinity, Size, and Label on Tumor Targeting Properties2019In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 16, no 3, p. 995-1008Article in journal (Refereed)
    Abstract [en]

    Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9_29-H-6 and G3-H-6 were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H-6 was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H-6 in comparison to 9_29-H6. Technetium-99m labeled G3-H-6 demonstrated a better biodistribution profile than 9_29-H-6, with several-fold lower uptake in liver. Radioiodinated G3-H-6 showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H-6 with high clinical potential for imaging of HER2.

  • 19.
    Ding, Haozhong
    et al.
    KTH Royal Inst Technol, Dept Prot Sci, Roslagstullsbacken 21, S-11417 Stockholm, Sweden.
    Altai, Mohamed
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Rinne, Sara S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Vorobyeva, Anzhelika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Gräslund, Torbjorn
    KTH Royal Inst Technol, Dept Prot Sci, Roslagstullsbacken 21, S-11417 Stockholm, Sweden.
    Orlova, Anna
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody-DM1 Drug Conjugates2019In: Cancers, ISSN 2072-6694, Vol. 11, no 8, article id 1168Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderateto high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (Z(HER2:2891))(2) -ABD-MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-3-MC-DM1, or a hexaglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-6-MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (Z(HER2:2891))(2)-ABD-MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

  • 20.
    Eriksson, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Selvaraju, Ram Kumar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Mollaret, Marjorie
    Mellitech SAS, Grenoble, France.
    de Boysson, Yann
    Mellitech SAS, Grenoble, France.
    Chimienti, Fabrice
    Mellitech SAS, Grenoble, France;Innovative Medicines and Early Development Biotech Unit (IMED Biotech), AstraZeneca, ABMölndal, Sweden.
    Altai, Mohamed
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Pancreatic imaging using an antibody fragment targeting the zinc transporter type 8: a direct comparison with radio-iodinated Exendin-42018In: Acta Diabetologica, ISSN 0940-5429, E-ISSN 1432-5233, Vol. 55, no 1, p. 49-57Article in journal (Refereed)
    Abstract [en]

    AIM: The zinc transporter 8 (ZnT8) has been suggested as a suitable target for non-invasive visualization of the functional pancreatic beta cell mass, due to both its pancreatic beta cell restricted expression and tight involvement in insulin secretion.

    METHODS: In order to examine the potential of ZnT8 as a surrogate target for beta cell mass, we performed mRNA transcription analysis in pancreatic compartments. A novel ZnT8 targeting antibody fragment Ab31 was radiolabeled with iodine-125, and evaluated by in vitro autoradiography in insulinoma and pancreas as well as by in vivo biodistribution. The evaluation was performed in a direct comparison with radio-iodinated Exendin-4.

    RESULTS: Transcription of the ZnT8 mRNA was higher in islets of Langerhans compared to exocrine tissue. Ab31 targeted ZnT8 in the cytosol and on the plasma membrane with 108 nM affinity. Ab31 was successfully radiolabeled with iodine-125 with high yield and > 95% purity. [(125)I]Ab31 binding to insulinoma and pancreas was higher than for [(125)I]Exendin-4, but could only by partially competed away by 200 nM Ab31 in excess. The in vivo uptake of [(125)I]Ab31 was higher than [(125)I]Exendin-4 in most tissues, mainly due to slower clearance from blood.

    CONCLUSIONS: We report a first-in-class ZnT8 imaging ligand for pancreatic imaging. Development with respect to ligand miniaturization and radionuclide selection is required for further progress. Transcription analysis indicates ZnT8 as a suitable target for visualization of the human endocrine pancreas.

  • 21.
    Garousi, Javad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.