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  • 1. Ahlstedt, J.
    et al.
    Orbom, A.
    Akesson, A.
    Frejd, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Strand, S.
    Tran, T.
    Simultaneous dual-radionuclide SPECT-imaging of HER2 expression using 99mTc-Affibody/111In-trastuzumab2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S274-S274, article id OP522Article in journal (Other academic)
  • 2.
    Alhuseinalkhudhur, Ali
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Lubberink, Mark
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Velikyan, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Frejd, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Feldwisch, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Lindman, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Sörensen, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Physiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Kinetic Analysis of the HER2-binding ABY-025 Affibody Using Dynamic PET in Patients with Metastatic Breast Cancer2018In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, p. S457-S457Article in journal (Other academic)
  • 3.
    Bass, Tarek Z.
    et al.
    KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, SE-10691 Stockholm, Sweden..
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, SE-17163 Solna, Sweden.
    Löfblom, John
    KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, SE-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.
    Ståhl, Stefan
    KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, SE-10691 Stockholm, Sweden..
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    In vivo evaluation of a novel format of a bivalent HER3-targeting and albumin- binding therapeutic affibody construct2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 43118Article in journal (Refereed)
    Abstract [en]

    Overexpression of human epidermal growth factor receptor 3 (HER3) is involved in resistance to several therapies for malignant tumours. Currently, several anti-HER3 monoclonal antibodies are under clinical development. We introduce an alternative approach to HER3-targeted therapy based on engineered scaffold proteins, i.e. affibody molecules. We designed a small construct (22.5 kDa, denoted 3A3), consisting of two high-affinity anti-HER3 affibody molecules flanking an albumin-binding domain ABD, which was introduced for prolonged residence in circulation. In vitro, 3A3 efficiently inhibited growth of HER3-expressing BxPC-3 cells. Biodistribution in mice was measured using 3A3 that was site-specifically labelled with In-111 via a DOTA chelator. The residence time of In-111-DOTA-3A3 in blood was extended when compared with the monomeric affibody molecule. In-111-DOTA-3A3 accumulated specifically in HER3-expressing BxPC-3 xenografts in mice. However, In-111-DOTA-3A3 cleared more rapidly from blood than a size-matched control construct In-111-DOTA-TAT, most likely due to sequestering of 3A3 by mErbB3, the murine counterpart of HER3. Repeated dosing and increase of injected protein dose decreased uptake of In-111-DOTA-3A3 in mErbB3-expressing tissues. Encouragingly, growth of BxPC-3 xenografts in mice was delayed in an experimental (pilot-scale) therapy study using 3A3. We conclude that the 3A3 affibody format seems promising for treatment of HER3-overexpressing tumours.

  • 4.
    Frejd, Fredrik Y.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, Gunnar Asplunds Alle 24, SE-17169 Solna, Sweden..
    Kim, Kyu-Tae
    AbClon Inc, Seoul, South Korea..
    Affibody molecules as engineered protein drugs2017In: Experimental and Molecular Medicine, ISSN 1226-3613, E-ISSN 2092-6413, Vol. 49, article id e306Article, review/survey (Refereed)
    Abstract [en]

    Affibody molecules can be used as tools for molecular recognition in diagnostic and therapeutic applications. There are several preclinical studies reported on diagnostic and therapeutic use of this molecular class of alternative scaffolds, and early clinical evidence is now beginning to accumulate that suggests the Affibody molecules to be efficacious and safe in man. The small size and ease of engineering make Affibody molecules suitable for use in multispecific constructs where AffiMabs is one such that offers the option to potentiate antibodies for use in complex disease.

  • 5.
    Garousi, Javad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Andersson, K.
    KTH Royal Inst Technol, Stockholm, Sweden..
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Orlova, Anna
    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.
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala Univ, Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Evaluation of Affibody Molecules for Radionuclide Imaging of Carbonic Abhydrase IX Expression In Vivo2016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S428-S428Article in journal (Refereed)
  • 6.
    Garousi, Javad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Andersson, Ken G.
    KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, SE-10691 Stockholm, Sweden.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Buijs, Jos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Ridgeview Instruments AB, SE-74020 Vange, Sweden.
    Löfblom, John
    KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, SE-10691 Stockholm, Sweden.
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, SE-17163 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 Affibody molecules for radionuclide imaging of in vivo expression of carbonic anhydrase IX2016In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 13, no 11, p. 3676-3687Article in journal (Refereed)
    Abstract [en]

    Overexpression of the enzyme carbonic anhydrase IX (CAIX) is documented for chronically hypoxic malignant tumors as well as for normoxic renal cell carcinoma. Radionuclide molecular imaging of CAIX would be useful for detection of hypoxic areas in malignant tumors, for patients' stratification of CAIX-targeted therapies and for discrimination of primary malignant and benign renal tumors. Earlier, we have reported feasibility of in vivo radionuclide based imaging of CAIX expressing tumors using Affibody molecules, small affinity proteins based on a non-immunoglobulin scaffold. In this study, we compared imaging properties of several anti-CAIX Affibody molecules having identical scaffold parts and competing for the same epitope on CAIX, but having different binding paratopes. Four variants were labeled using residualizing 99mTc and non-residualizing 125I labels. All radiolabeled variants demonstrated high-affinity detection of CAIX-expressing cell line SK-RC-52 in vitro and specific accumulation in SK-RC-52 xenografts in vivo. 125I-labeled conjugates demonstrated much lower radioactivity uptake in kidneys but higher radioactivity concentration in blood compared with 99mTc-labed counterparts. Although all variants cleared rapidly from blood and non-specific compartments, there was noticeably difference in their biodistribution. The best variant for imaging of expression of CAIX- in disseminated cancer was 99mTc-(HE)3-ZCAIX:2 providing tumor uptake of 16.3±0.9 %ID/g and tumor-to-blood ratio of 44±7 at 4 h after injection. For primary renal cell carcinoma, the most promising imaging candidate was 125I-ZCAIX:4 providing tumor-kidney ratio of 2.1±0.5. In conclusion, several clones of scaffold proteins should be evaluated to select the best variant for development of an imaging probe with optimal sensitivity for the intended application.

  • 7.
    Garousi, Javad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Huizing, Fokko J.
    Radboud Univ Nijmegen, Dept Radiat Oncol, Med Ctr, Nijmegen, Netherlands.
    Vorobyeva, Anzhelika
    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, Theranostics.
    Andersson, Ken G.
    KTH Royal Inst Technol, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, Stockholm, Sweden.
    Leitao, Charles Dahlsson
    KTH Royal Inst Technol, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, Stockholm, Sweden.
    Frejd, Fredrik Y.
    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, Stockholm, Sweden.
    Bussink, Johan
    Radboud Univ Nijmegen, Dept Radiat Oncol, Med Ctr, Nijmegen, Netherlands.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Heskamp, Sandra
    Radboud Univ Nijmegen, Dept Radiol & Nucl Med, Med Ctr, Nijmegen, Netherlands.
    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 affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 14907Article in journal (Refereed)
    Abstract [en]

    Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE3-ZCAIX:2, was labeled with In-111 and characterized in vitro. Tumor-targeting properties of [In-111]In-DOTA-HE3-ZCAIX:2 were compared head-to-head with properties of the parental variant, [(99)mTc]Tc(CO)(3)-HE3-ZCAIX:2, and the most promising antibody fragment-based tracer, [In-111]In-DTPA-G250(Fab')(2), in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the (99)mTc-labeled parental variant, [In-111]In-DOTA-HE3-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [In-111]In-DOTA-HE3-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [In-111]In-G250(Fab']2. In conclusion, [In-111]In-DOTA-HE3-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.

  • 8.
    Göstring, Lovisa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Malm, Magdalena
    KTH, Skolan för bioteknologi, Avdelningen för molekylär bioteknologi.
    Höidén-Guthenberg, Ingmarie
    Affibody AB.
    Frejd, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi, Avdelningen för molekylär bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi, Avdelningen för molekylär bioteknologi.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Cellular effects of HER3-specific affibody molecules2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 6, p. e40023-Article in journal (Refereed)
    Abstract [en]

    Recent discoveries have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. Although practically devoid of kinase activity, signaling via this receptor is often seen in tumours resistant to EGFR or HER2 therapy. Here, we show that two HER3-specific affibody molecules, Z5416 and Z5417, reduce heregulin-induced cell growth of the breast cancer cells MCF-7 and, to a lesser extent, SKBR‑3 cells. These affibody molecules have earlier been shown to block binding of the natural ligand heregulin (HRG) to HER3, which was confirmed here in cellular studies. Further, both Z5416 and Z5417 blocked HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, but only HER3 phosphorylation in SKBR-3 cells which have constantly active HER2.. These findings demonstrate that Z5416 and Z5417 exert an anti-proliferative effect on two breast cancer cells with either high or low HER2 expression, by inhibiting HRG-induced phosphorylation of HER3. The promising results presented in this study indicate that the HER3-binding affibody molecules may be suitable candidates for future therapy of cancers in which the interaction between HER3 and HRG plays an important role.

  • 9.
    Honarvar, Hadis
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Garousi, Javad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Gunneriusson, E.
    Hoiden-Guthenberg, I.
    Altai, Mohamed
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Widström, Charles
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Medical Physics.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Frejd, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Imaging of CAIX-expressing xenografts in vivo using 99mTc-HEHEHE-Z09781 Affibody molecule2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no S2, p. S176-S176, article id OP071Article in journal (Other academic)
  • 10. Krasniqi, Ahmet
    et al.
    D'Huyvetter, Matthias
    Devoogdt, Nick
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, Solna, Sweden.
    Sörensen, Jens
    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, Theranostics.
    Keyaerts, Marleen
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Same-day imaging using small proteins: Clinical experience and translational prospects in oncology.2018In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 59, no 6, p. 885-891Article in journal (Refereed)
    Abstract [en]

    Imaging of expression of therapeutic targets may enable patients' stratification for targeted treatments. The use of small radiolabeled probes based on the heavy-chain variable region of heavy-chain-only immunoglobulins or non-immunoglobulin scaffolds permits rapid localization of radiotracers in tumors and rapid clearance from normal tissues. This makes high-contrast imaging possible on the day of injection. This mini-review focuses on small proteins for radionuclide-based imaging that would allow same-day imaging, with the emphasis on clinical applications and promising preclinical developments within the field of oncology.

  • 11. Liu, Hao
    et al.
    Seijsing, Johan
    Frejd, Fredrik Y.
    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, Torbjörn
    Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin Z(HER2:2891)-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain2015In: International Journal of Oncology, ISSN 1019-6439, Vol. 47, no 2, p. 601-609Article in journal (Refereed)
    Abstract [en]

    Development of cancer treatment regimens including immunotoxins is partly hampered by their immunogenicity. Recently, deimmunized versions of toxins have been described, potentially being better suited for translation to the clinic. In this study, a recombinant tripartite fusion toxin consisting of a deimmunized version of exotoxin A from Pseudomonas aeruginosa (PE38) genetically fused to an affibody molecule specifically interacting with the human epidermal growth factor receptor 2 (HER2), and also an albumin binding domain (ABD) for half-life extension, has been produced and characterized in terms of functionality of the three moieties. Biosensor based assays showed that the fusion toxin was able to interact with human and mouse serum albumin, but not with bovine serum albumin and that it interacted with HER2 (K-D=5 nM). Interestingly, a complex of the fusion toxin and human serum albumin also interacted with HER2 but with a somewhat weaker affinity (K-D=12 nM). The IC50-values of the fusion toxin ranged from 6 to 300 pM on SKOV-3, SKBR-3 and A549 cells and was lower for cells with higher surface densities of HER2. The fusion toxin was found specific for HER2 as shown by blocking available HER2 receptors with free affibody molecule before subjecting the cells to the toxin. Analysis of contact time showed that 10 min was sufficient to kill 50% of the cells. In conclusion, all three regions of the fusion toxin were found to be functional.

  • 12. Löfblom, John
    et al.
    Feldwisch, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Ståhl, Stefan
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications2010In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 584, no 12, p. 2670-2680Article, review/survey (Refereed)
    Abstract [en]

    Affibody molecules are a class of engineered affinity proteins with proven potential for therapeutic, diagnostic and biotechnological applications. Affibody molecules are small (6.5 kDa) single domain proteins that can be isolated for high affinity and specificity to any given protein target. Fifteen years after its discovery, the Affibody technology is gaining use in many groups as a tool for creating molecular specificity wherever a small, engineering compatible tool is warranted. Here we summarize recent results using this technology, propose an Affibody nomenclature and give an overview of different HER2-specific Affibody molecules. Cumulative evidence suggests that the three helical scaffold domain used as basis for these molecules is highly suited to create a molecular affinity handle for vastly different applications.

  • 13. Malm, Magdalena
    et al.
    Bass, Tarek
    Gudmundsdotter, Lindvi
    Lord, Martin
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Stahl, Stefan
    Lofblom, John
    Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension2014In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 9, p. 1215-1222Article in journal (Refereed)
    Abstract [en]

    Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.

  • 14.
    Malm, Magdalena
    et al.
    KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, Stockholm, Sweden..
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, Stockholm, Sweden..
    Ståhl, Stefan
    KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, Stockholm, Sweden..
    Löfblom, John
    KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, Stockholm, Sweden..
    Targeting HER3 using mono- and bispecific antibodies or alternative scaffolds2016In: mAbs, ISSN 1942-0862, E-ISSN 1942-0870, Vol. 8, no 7, p. 1195-1209Article, review/survey (Refereed)
    Abstract [en]

    The human epidermal growth factor receptor 3 (HER3) has in recent years been recognized as a key node in the complex signaling network of many different cancers. It is implicated in de novo and acquired resistance against therapies targeting other growth factor receptors, e.g., EGFR, HER2, and it is a major activator of the PI3K/Akt signaling pathway. Consequently, HER3 has attracted substantial attention, and is today a key target for drugs in clinical development. Sophisticated protein engineering approaches have enabled the generation of a range of different affinity proteins targeting this receptor, including antibodies and alternative scaffolds that are either mono- or bispecific. Here, we describe HER3 and its role as a key tumor target, and give a comprehensive review of HER3-targeted proteins currently in development, including discussions on the opportunities and challenges of targeting this receptor.

  • 15. Malm, Magdalena
    et al.
    Kronqvist, Nina
    Lindberg, Hanna
    Gudmundsdotter, Lindvi
    Bass, Tarek
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Hoiden-Guthenberg, Ingmarie
    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.
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Stahl, Stefan
    Lofblom, John
    Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, p. e62791-Article in journal (Refereed)
    Abstract [en]

    The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 PM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

  • 16.
    Orlova, Anna
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Bass, Tarek Z.
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, SE-10691 Stockholm, Sweden.
    Rinne, Sara S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Leitao, Charles Dahlsson
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, SE-10691 Stockholm, Sweden.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Atterby, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Gudmundsdotter, Lindvi
    Affibody AB, Solna, Sweden.
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, Solna, Sweden.
    Löfhlom, John
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, SE-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.
    Ståhl, Stefan
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, SE-10691 Stockholm, Sweden.
    Evaluation of the Therapeutic Potential of a HER3-Binding Affibody Construct TAM-HER3 in Comparison with a Monoclonal Antibody, Seribantumab2018In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 8, p. 3394-3403Article in journal (Refereed)
    Abstract [en]

    Human epidermal growth factor receptor type 3 (HER3) is recognized to be involved in resistance to HER targeting therapies. A number of HER3-targeting monoclonal antibodies are under clinical investigation as potential cancer therapeutics. Smaller high-affinity scaffold proteins are attractive non-Fc containing alternatives to antibodies. A previous study indicated that anti-HER3 affibody molecules could delay the growth of xenografted HER3-positive tumors. Here, we designed a second-generation HER3-targeting construct (TAM-HER3), containing two HER3-specific affibody molecules bridged by an albumin-binding domain (ABD) for extension of blood circulation. Receptor blocking activity was demonstrated in vitro. In mice bearing BxPC-3 xenografts, the therapeutic efficacy of TAM-HER3 was compared to the HER3-specific monoclonal antibody seribantumab (MM-121). TAM-HER3 inhibited heregulin-induced phosphorylation in a panel of HER3-expressing cancer cells and was found to be equally as potent as seribantumab in terms of therapeutic efficacy in vivo and with a similar safety profile. Median survival times were 60 days for TAM-HER3, 54 days for seribantumab, and 41 days for the control group. No pathological changes were observed in cytopathological examination. The multimeric HER3-binding affibody molecule in fusion to ABD seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.

  • 17.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Jonsson, Andreas
    Rosik, Daniel
    Lundqvist, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Lindborg, Malin
    Abrahmsen, Lars
    Ekblad, Caroline
    Frejd, Fredrik Y
    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.
    Site-Specific Radiometal Labeling and Improved Biodistribution Using ABY-027, A Novel HER2-Targeting Affibody Molecule-Albumin-Binding Domain Fusion Protein2013In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 54, no 6, p. 961-968Article in journal (Refereed)
    Abstract [en]

    Because of their better penetration, smaller targeting proteins may be superior to antibodies for radioimmunotherapy of solid tumors. Therefore, Affibody molecules (6.5 kDa) have a potential for being suitable as targeted moiety for radiolabeled therapeutic proteins. Previous studies have demonstrated that a fusion of an Affibody molecule with an albumin-binding domain (ABD) provides a strong noncovalent binding to albumin in vivo. This strong noncovalent binding can be used for reduction of the renal uptake of the Affibody molecule while maintaining a size smaller than that of an antibody, which is important when using residualizing radionuclide labels conjugated to Affibody molecules. The goal of this study was to design and evaluate a new targeting Affibody–ABD fusion protein with improved biodistribution properties for radionuclide therapy.

    Methods:

    A novel Affibody-based construct, ZHER2:2891-ABD035-DOTA (ABY-027), was created by fusion of the reengineered HER2-binding Affibody molecule ZHER2:2891 to the N terminus of the high-affinity ABD035, and a maleimido-derivative of DOTA was conjugated at the C terminus of the construct. Binding and processing of 177Lu-ABY-027 by HER2-expressing cells were evaluated in vitro. Targeting of HER2-expressing SKOV-3 xenografts was evaluated in BALB/C nu/nu mice and compared with targeting of previously reported ABD-(ZHER2:342)2.

    Results:

    The binding affinity (dissociation constant) of ABY-027 to HER2 (74 pM) was the same as for the parental ZHER2:2891 (76 pM). ABY-027 was stably labeled with 177Lu and 111In with preserved specific binding to HER2-expressing cells in vitro. In vivo receptor saturation experiments demonstrated that targeting of SKOV-3 xenografts in BALB/C nu/nu mice was HER2-specific. 177Lu-ABY-027 demonstrated substantially (2- to 3-fold) lower renal and hepatic uptake than previously assessed HER2-specific Affibody-based albumin-binding agents. Tumor uptake of radiolabeled ABY-027 at 48 h after injection was 2-fold higher than that for previously reported ABD-(ZHER2:342)2.

    Conclusion:

    An optimized molecular design of an ABD fusion protein resulted in an Affibody molecule construct with better properties for therapy. Fully preserved in vivo targeting of the fusion protein was shown in xenografted mice. Site-specific coupling of DOTA provides a uniform conjugate and creates the potential for labeling with a broad range of therapeutic radionuclides. The biodistribution of 177Lu-ABY-027 in a murine model suggests it is more suitable for therapy than alternative approaches.

  • 18.
    Orlova, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Rosestedt, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Mitran, Bogdan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging.
    Bass, T.
    Royal Inst Technol, Stockholm, Sweden..
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
    Löfblom, J.
    Royal Inst 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.
    Ståhl, S.
    Royal Inst Technol, Stockholm, Sweden..
    In vivo evaluation of pharmacokinetics, tumors targeting and therapeutic efficacy of a novel format of HER3-targeting affibody molecule with prolonged blood circulation2016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S237-S237Article in journal (Refereed)
  • 19. Seijsing, Johan
    et al.
    Lindborg, Malin
    Hoiden-Guthenberg, Ingmarie
    Bonisch, Heiko
    Guneriusson, Elin
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Abrahmsen, Lars
    Ekblad, Caroline
    Lofblom, John
    Uhlen, Mathias
    Graslund, Torbjron
    An engineered affibody molecule with pH-dependent binding to FcRn mediates extended circulatory half-life of a fusion protein2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 48, p. 17110-17115Article in journal (Refereed)
    Abstract [en]

    Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased halflife, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.

  • 20.
    Ståhl, Stefan
    et al.
    KTH Royal Inst Technol, Div Prot Technol, SE-10691 Stockholm, Sweden..
    Gräslund, Torbjörn
    KTH Royal Inst Technol, Div Prot Technol, SE-10691 Stockholm, Sweden..
    Karlström, Amelie Eriksson
    KTH Royal Inst Technol, Div Prot Technol, SE-10691 Stockholm, Sweden..
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, Gunnar Asplunds Alle 24, SE-17169 Solna, Sweden..
    Nygren, Per-Åke
    KTH Royal Inst Technol, Div Prot Technol, SE-10691 Stockholm, Sweden..
    Löfblom, John
    KTH Royal Inst Technol, Div Prot Technol, SE-10691 Stockholm, Sweden..
    Affibody Molecules in Biotechnological and Medical Applications2017In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 35, no 8, p. 691-712Article, review/survey (Refereed)
    Abstract [en]

    Affibody molecules are small (6.5-kDa) affinity proteins based on a three-helix bundle domain framework. Since their introduction 20 years ago as an alternative to antibodies for biotechnological applications, the first therapeutic affibody molecules have now entered clinical development and more than 400 studies have been published in which affibody molecules have been developed and used in a variety of contexts. In this review, we focus primarily on efforts over the past 5 years to explore the potential of affibody molecules for medical applications in oncology, neurodegenerative, and inflammation disorders, including molecular imaging, receptor signal blocking, and delivery of toxic payloads. In addition, we describe recent examples of biotechnological applications, in which affibody molecules have been exploited as modular affinity fusion partners.

  • 21.
    Tolmachev, Vladimir
    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.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Eriksson, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Jonasson, Per
    Frejd, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Abrahmsen, Lars
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    In vivo imaging of PDGFR beta expression using In-111-DOTA-Z09591 Affibody molecule2013In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 56, no S1, p. S387-S387Article in journal (Other academic)
  • 22.
    Tolmachev, Vladimir
    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.
    Honarvar, Hadis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Hosseinimehr, S. Jalal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Eriksson, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Jonasson, P.
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Abrahmsen, L.
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Imaging of PDGFRbeta expression in vivo using Affibody molecule In-111-DOTA-Z095912013In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, no Suppl. 2, p. S220-S220Article in journal (Other academic)
  • 23.
    Tolmachev, Vladimir
    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.
    Honarvar, Hadis
    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.
    Eriksson, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Jonasson, Per
    Frejd, Fredrik Y
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Abrahmsen, Lars
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Imaging of platelet-derived growth factor receptor β expression in glioblastoma xenografts using affibody molecule 111In-DOTA-Z095912014In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 55, no 2, p. 294-300Article in journal (Refereed)
    Abstract [en]

    The overexpression and excessive signaling of platelet-derived growth factor receptor β (PDGFRβ) has been detected in cancers, atherosclerosis, and a variety of fibrotic diseases. Radionuclide in vivo visualization of PDGFRβ expression might help to select PDGFRβ targeting treatment for these diseases. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of PDGFRβ expression using an Affibody molecule, a small nonimmunoglobulin affinity protein.

    Methods

    The PDGFRβ-binding Z09591 Affibody molecule was site-specifically conjugated with a maleimido derivative of DOTA and labeled with 111In. Targeting of the PDGFRβ-expressing U-87 MG glioblastoma cell line using 111In-DOTA-Z09591 was evaluated in vitro and in vivo.

    Results

    DOTA-Z09591 was stably labeled with 111In with preserved specific binding to PDGFRβ-expressing cells in vitro. The dissociation constant for 111In-DOTA-Z09591 binding to U-87 MG cells was determined to be 92 ± 10 pM. In mice bearing U-87 MG xenografts, the tumor uptake of 111In-DOTA-Z09591 was 7.2 ± 2.4 percentage injected dose per gram and the tumor-to-blood ratio was 28 ± 14 at 2 h after injection. In vivo receptor saturation experiments demonstrated that targeting of U-87 MG xenografts in mice was PDGFRβ-specific. U-87 MG xenografts were clearly visualized using small-animal SPECT/CT at 3 h after injection.

    Conclusion

    This study demonstrates the feasibility of in vivo visualization of PDGFRβ-expressing xenografts using an Affibody molecule. Further development of radiolabeled Affibody molecules might provide a useful clinical imaging tool for PDGFRβ expression during various pathologic conditions.

  • 24.
    Trotter, Dinko E. Gonzalez
    et al.
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    Meng, Xiangjun
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    McQuade, Paul
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    Rubins, Daniel
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    Klimas, Michael
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    Zeng, Zhizhen
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    Connolly, Brett M.
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    Miller, Patricia J.
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    O'Malley, Stacey S.
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    Lin, Shu-An
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    Getty, Krista L.
    Merck & Co Inc, Screening & Prot Sci Dept, West Point, PA 19486 USA..
    Fayadat-Dilman, Laurence
    Merck & Co Inc, Biol Discovery, Palo Alto, CA USA..
    Liang, Linda
    Merck & Co Inc, Biol Discovery, Palo Alto, CA USA..
    Wahlberg, Elisabet
    Affibody AB, Solna, Sweden..
    Widmark, Olof
    Affibody AB, Solna, Sweden..
    Ekblad, Caroline
    Affibody AB, Solna, Sweden..
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, Solna, Sweden.
    Hostetler, Eric D.
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    Evelhoch, Jeffrey L.
    Merck & Co Inc, Translat Biomarkers Dept, West Point, PA 19486 USA..
    In Vivo Imaging of the Programmed Death Ligand 1 by F-18 PET2017In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 58, no 11, p. 1852-1857Article in journal (Refereed)
    Abstract [en]

    Programmed death ligand 1 (PD-L1) is an immune regulatory ligand that binds to the T-cell immune check point programmed death 1. Tumor expression of PD-L1 is correlated with immune suppression and poor prognosis. It is also correlated with therapeutic efficacy of programmed death 1 and PD-L1 inhibitors. In vivo imaging may enable real-time follow-up of changing PD-L1 expression and heterogeneity evaluation of PD-L1 expression across tumors in the same subject. We have radiolabeled the PD-L1-binding Affibody molecule NOTA-Z(PD-L1_1) with F-18 and evaluated its in vitro and in vivo binding affinity, targeting, and specificity. Methods: The affinity of the PD-L1-binding Affibody ligand Z(PD-L1_1) was evaluated by surface plasmon resonance. Labeling was accomplished by maleimide coupling of NOTA to a unique cysteine residue and chelation of F-18-AlF. In vivo studies were performed in PD-L1-positive, PD-L1-negative, and mixed tumor-bearing severe combined immunodeficiency mice. Tracer was injected via the tail vein, and dynamic PET scans were acquired for 90 min, followed by gamma-counting biodistribution. Immunohistochemical staining with an antibody specific for anti-PD-L1 (22C3) was used to evaluate the tumor distribution of PD-L1. Immunohistochemistry results were then compared with ex vivo autoradiographic images obtained from adjacent tissue sections. Results: NOTA-Z(PD-L1_1) was labeled, with a radiochemical yield of 15.1% +/- 5.6%, radiochemical purity of 96.7% +/- 2.0%, and specific activity of 14.6 +/- 6.5 GBq/mu mol. Surface plasmon resonance showed a NOTA-conjugated ligand binding affinity of 1 nM. PET imaging demonstrated rapid uptake of tracer in the PD-L1-positive tumor, whereas the PD-L1-negative control tumor showed little tracer retention. Tracer clearance from most organs and blood was quick, with biodistribution showing prominent kidney retention, low liver uptake, and a significant difference between PD-L1-positive (percentage injected dose per gram [%ID/g] = 2.56 +/- 0.33) and -negative (% ID/g = 0.32 +/- 0.05) tumors (P = 0.0006). Ex vivo autoradiography showed excellent spatial correlation with immunohistochemistry in mixed tumors. Conclusion: Our results show that Affibody ligands can be effective at targeting tumor PD-L1 in vivo, with good specificity and rapid clearance. Future studies will explore methods to reduce kidney activity retention and further increase tumor uptake.

  • 25.
    Velikyan, Irina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Schweighoefer, Philip
    Eckert & Ziegler Eurotope GmbH, Berlin, Germany.
    Feldwisch, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, Solna, Sweden.
    Seemann, Johanna
    Eckert & Ziegler Eurotope GmbH, Berlin, Germany.
    Frejd, Fredrik Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Affibody AB, Solna, Sweden.
    Lindman, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Sörensen, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Diagnostic HER2-binding radiopharmaceutical, [Ga-68]Ga-ABY-025, for routine clinical use in breast cancer patients2019In: American Journal of Nuclear Medicine and Molecular Imaging, ISSN 2160-8407, Vol. 9, no 1, p. 12-23Article in journal (Refereed)
    Abstract [en]

    [Ga-68]Ga-ABY-025/PET-CT targeting human epidermal growth factor receptor type 2 (HER2) has demonstrated its potential clinical value for the detection and quantification of HER2 in a phase I clinical study with breast cancer patients. Previously, the radiopharmaceutical was prepared manually, however larger scale of multicenter clinical trials and routine healthcare requires automation of the production process to limit the operator radiation dose, improve tracer manufacturing robustness, and provide on-line documentation for good manufacturing practice (GMP) compliance. The production of [Ga-68]Ga-ABY-025 was implemented on the Modular-Lab PharmTrace synthesis platform (Eckert & Ziegler) and disposable cassettes were developed. Pharmaceutical grade Ge-68/Ga-68 generator (GalliaPharm (R)) was used in the study. The active pharmaceutical ingredient starting material ABY-025 (GMP grade) was provided by Affibody AB. The patient examinations were conducted using a Discovery MI PET/CT scanner (20 cm FOV, GE Healthcare). Reproducible and GMP compliant fully automated production of [Ga-68]Ga-ABY-025 was developed. The radiochemical purity of the product was 98.7 +/- 0.6% with total peptide content of 315 +/- 15 mu g (n = 3). Radionuclidic purity, sterility, endotoxin content, residual solvent content, and sterile filter integrity were controlled and met acceptance criteria. The product was stable at ambient temperature for at least 2 h. The primary tumor and metastasis were detected with SUVmax values of 8.3 and 16.0, respectively. Automated production of [Ga-68]Ga-ABY-025 was established and the process was validated enabling standardized multicenter phase II and III clinical trials and routine clinical use. Patient examinations conformed to the radiopharmaceutical biodistribution observed in the previous phase I study.

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