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  • 1.
    Afrasiabi, Roodabeh
    et al.
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Jokilaakso, Nima
    KTH, School of Biotechnology (BIO), Protein Technology.
    Schmidt, Torsten
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Björk, P.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Linnros, Jan
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Effect of microwave-assisted silanization on sensing properties of silicon nanoribbon FETs2015In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 209, p. 586-595Article in journal (Refereed)
    Abstract [en]

    An important concern with using silicon nanoribbon field-effect transistors (SiNR FET) for ion-sensing is the pH-response of the gate oxide surface. Depending on the application of the FET sensor, this response has to be chemically manipulated. Thus in silicon oxide-gated pH-sensors with integrated sensor and reference FETS, a surface with high pH-sensitivity, compared to the bare gate oxide, is required in the sensor FETs (SEFET), whereas in the reference FETs (REFET) the surface has to be relatively pH-insensitive. In order to control the sensitivity and chemistry of the oxide surface of the nanoribbons, a silanization reagent with a functional group is often self-assembled on the SiNR surface. Choice of a silanization reaction that results in a self-assembled layer on a silicon oxide surface has been studied extensively over the past decades. However, the effect of various self-assembled layers such as monolayers or mixed layers on the electrical response of SiNR FETs in aqueous solution needs to be exploited further, especially for future integrated SEFET/REFET systems. In this work, we have performed a comprehensive study on 3-aminopropyltriethoxysilane (APTES) silanization of silicon oxide surfaces using microwave (MW) heating as a new biocompatible route to conventional methods. A set of complementary surface characterization techniques (ellipsometry, AFM and ATR-FTIR) was used to analyze the properties of the APTES layer deposited on the silicon surface. We have found that a uniform monolayer can be achieved within 10 min by heating the silanization solution to 75 degrees C using MW heating. Furthermore, electrical measurements suggest that little change in device performance is observed after exposure to MW irradiation. Real-time pH measurements indicate that a uniform APTES monolayer not only reduces the pH sensitivity of SiNR FET by passivating the surface silanol groups, but also makes the device less sensitive to cation concentration in the background electrolyte. Our silanization route proves promising for future chemical surface modification of on-chip REFETs.

  • 2.
    Afrasiabi, Roodabeh
    et al.
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Jokilaakso, Nima
    KTH, School of Biotechnology (BIO), Protein Technology.
    Schmidt, Torsten
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Linnros, Jan
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Microwave-assisted silanization of SiNW-FET: characterization and effect on sensing propertiesManuscript (preprint) (Other academic)
  • 3.
    Akpe, Victor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Protein Technology.
    Madu, Christian
    Obirai, Joseph C.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Understanding the Photochemical Pathway of In Vitro Target Delivery of Aluminium Phthalocyanine: A Mechanistic Approach Using Radical Reaction Chemistry2014In: ChemPlusChem, ISSN 2192-6506, Vol. 79, no 5, p. 671-679Article in journal (Refereed)
    Abstract [en]

    A classical dye, aluminium phthalocyanine (AlPc), is used to study the photochemical processes involved in the chromophore-assisted laser inactivation technique. Both cell-free and cell-based systems are investigated by novel methods and radical reaction chemistry. Findings on the photochemical pathways in two models representing cell-free and a cell-based systems are reported. In the cell-free system, the unsubstituted, free, fluorescence-active photosensitiser AlPc recovers its fluorescence signal by means of phosphorescence through a reversible photobleaching process. In the cell-based system, photoactivation of substituted AlPc conjugated to an antibody results in the loss of fluorescence signal at the area examined. Reinjection of the AlPc-conjugated antibodies restores the fluorescence signal.

  • 4. Alkharusi, Amira
    et al.
    Yu, Shengze
    KTH, School of Biotechnology (BIO).
    Landazuri, Natalia
    Zadjali, Fahad
    Davodi, Belghis
    Nystrom, Thomas
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Rahbar, Afsar
    Norstedt, Gunnar
    Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme2016In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 48, p. 79558-79569Article in journal (Refereed)
    Abstract [en]

    Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans and is characterized with poor outcome. In this study, we investigated components of prolactin (Prl) system in cell models of GBM and in histological tissue sections obtained from GBM patients. Expression of Prolactin receptor (PrlR) was detected at high levels in U251-MG, at low levels in U87-MG and barely detectable in U373 cell lines and in 66% of brain tumor tissues from 32 GBM patients by immunohistochemical technique. In addition, stimulation of U251-MG and U87-MG cells but not U373 with Prl resulted in increased STAT5 phosphorylation and only in U251-MG cells with increased cellular invasion. Furthermore, STAT5 phosphorylation and cellular invasion induced in Prl stimulated cells were significantly reduced by using a Prl receptor antagonist that consists of Prl with four amino acid replacements. We conclude that Prl receptor is expressed at different levels in the majority of GBM tumors and that blocking of PrlR in U251-MG cells significantly reduce cellular invasion.

  • 5. Allerbring, Eva B.
    et al.
    Duru, Adil D.
    Chadderton, Jesseka
    Markov, Natalia
    Uchtenhagen, Hannes
    Popov, Alexander
    Madhurantakam, Chaithanya
    Sandalova, Tatyana
    Turner, Stephen J.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Protein Technology.
    Achour, Adnane
    Structural and thermodynamic basis underlying in vivo reestablishment of T-cell recognition of a viral escape mutant2015In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 68, no 2, p. 151-151Article in journal (Other academic)
  • 6. Altai, M.
    et al.
    Honarvar, H.
    Wallberg, H.
    Strand, J.
    Varasteh, Z.
    Orlova, A.
    Dunas, F.
    Sandstrom, M.
    Rosestedt, M.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, V.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with Re-1882013In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, p. S219-S220Article in journal (Other academic)
  • 7. Altai, M.
    et al.
    Tsourma, M.
    Mitran, B.
    Honarvar, H.
    Perols, Anna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Robillard, M.
    Rossin, R.
    ten Hoeve, W.
    Sandstrom, M.
    Orlova, A.
    Karlström, Amelie Eriksson
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, V.
    Affibody-based bioorthogonal chemistry-mediated radionuclide pretargeting: proof-of-principle.2015In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, p. S246-S246Article in journal (Refereed)
  • 8. Altai, M.
    et al.
    Wallberg, H.
    Honarvar, H.
    Strand, J.
    Orlova, A.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Varasteh, Z.
    Sandström, M.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, V.
    Re-188-Z(HER2: V2), a promising targeting agent against HER2-expressing tumors: in vitro and in vivo assessment2013In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, p. S119-S119Article in journal (Other academic)
  • 9. Altai, M.
    et al.
    Westerlund, Kristina
    KTH, School of Biotechnology (BIO), Protein Technology.
    Velletta, J.
    Honarvar, H.
    Orlova, A.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, V.
    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)
  • 10. Altai, M.
    et al.
    Westerlund, Kristina
    KTH, School of Biotechnology (BIO), Protein Technology.
    Velletta, J.
    Mitran, B.
    Honarvar, H.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    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, ZHER2:342-SR-HP1, and labeling the secondary agent, HP2, with the therapeutic radionuclide 177Lu. We also studied the biodistribution profile of 177Lu-HP2 in mice, and evaluated pretargeting with 177Lu-HP2 in vitro and in vivo. Methods The biodistribution profile of 177Lu-HP2 was evaluated in NMRI mice and compared to the previously studied 111In-HP2. Pretargeting using 177Lu-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 ZHER2:342-SR-HP1 the ligation time was reduced from 15 h to 30 min, and the yield increased from 45% to 70%. 177Lu-labeled HP2 binds specifically in vitro to BT474 and SKOV-3 cells pre-treated with ZHER2:342-SR-HP1. 177Lu-HP2 was shown to have a more rapid blood clearance compared to 111In-HP2 in NMRI mice, and the measured radioactivity in blood was 0.22 ± 0.1 and 0.68 ± 0.07%ID/g for 177Lu- and 111In-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 177Lu- and 111In-HP2, respectively, at 1 h p.i.). Co-injection with either Gelofusine or lysine significantly reduced the kidney uptake for 177Lu-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.). 177Lu-HP2 accumulated in SKOV-3 xenografts in BALB/C nu/nu mice when administered after injection of ZHER2:342-SR-HP1. Without pre-injection of ZHER2:342-SR-HP1, the uptake of 177Lu-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 ZHER2:342-SR-HP1. In conclusion, 177Lu-HP2 was shown to be a promising secondary agent for affibody-mediated tumor pretargeting in vivo.

  • 11. Altai, Mohamed
    et al.
    Honarvar, Hadis
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Protein Technology.
    Strand, Joanna
    Varasteh, Zohreh
    Rosestedt, Maria
    Orlova, Anna
    Dunås, Finn
    Sandström, Mattias
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.2014In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 87, p. 519-28Article in journal (Refereed)
    Abstract [en]

    Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

  • 12. Altai, Mohamed
    et al.
    Liu, Hao
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, Anna
    Tolmachev, Vladimir
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Influence of molecular design on biodistribution and targeting properties of an Affibody-fused HER2-recognising anticancer toxin2016In: International Journal of Oncology, ISSN 1019-6439, Vol. 49, no 3, p. 1185-1194Article in journal (Refereed)
    Abstract [en]

    Targeted delivery of toxins is a promising way to treat disseminated cancer. The use of monoclonal antibodies as targeting moiety has provided proof-of-principle for this approach. However, extravasation and tissue penetration rates of antibody-based immunotoxins are limited due to antibody bulkiness. The use of a novel class of targeting probes, Affibody molecules, provides smaller toxin-conjugated constructs, which may improve targeting. Earlier, we have demonstrated that affitoxins containing a HER2-targeting Affibody moiety and a deimmunized and truncated exotoxin A from Pseudomonas aeruginosa, PE38X8, provide highly selective toxicity to HER2-expressing cancer cells. To evaluate the influence of molecular design on targeting and biodistribution properties, a series of novel affitoxins were labelled with the residualizing radionuclide In-111. In this study, we have shown that the novel conjugates are more rapidly internalized compared with the parental affitoxin. The use of a (HE)(3) purification tag instead of a hexahistidine tag enabled significant (p<0.05) reduction of the hepatic uptake of the affitoxin in a murine model. Fusion of the affitoxin with an albumin-binding domain (ABD) caused appreciable extension of the residence time in circulation and several-fold reduction of the renal uptake. The best variant, In-111-(HE)(3)-Z(HER2)-ABD-PE38X8, demonstrated receptor-specific accumulation in HER2-expressing SKOV-3 xenografts. In conclusion, a careful molecular design of scaffold protein based anticancer targeted toxins can appreciably improve their biodistribution and targeting properties.

  • 13. Altai, Mohamed
    et al.
    Perols, Anna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tsourma, Maria
    Mitran, Bogdan
    Honarvar, Hadis
    Robillard, Marc
    Rossin, Raffaella
    ten Hoeve, Wolter
    Lubberink, Mark
    Orlova, Anna
    Karlström, Amelie Eriksson
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    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 reabsorption of Affibody molecules prevents the use of residualizing radiometals, including several 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-human epidermal growth factor receptor 2 (HER2) Affibody molecule Z(2395). DOTA-tetrazine was labeled with In-111 and Lu-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: I-125-Z(2395)-TCO bound specifically to HER2-expressing cells in vitro with an affinity of 45 +/- 16 pM. In-111-tetrazine bound specifically and selectively to Z(2325)-TCO pretreated cells. In vivo studies demonstrated HER2-specific I-125-Z(2395)-TCO accumulation in xenografts. TCO-mediated In-111-tetrazine localization was shown in tumors, when the radiolabeled tracer was injected 4 h after an injection of Z(2395)-TCO. At 1 h after injection, the tumor uptake of In-111-tetrazine and Lu-177-tetrazine was approximately 2-fold higher than the renal uptake. Pretargeting provided more than a 56-fold reduction of renal uptake of In-111 in comparison with direct targeting. Conclusion: The feasibility of Affibody-based bioorthogonal chemistry-mediated pretargeting was demonstrated. The use of pre-targeting provides a substantial reduction of radiometal accumulation in kidneys, creating preconditions for palliative radionuclide therapy.

  • 14. Altai, Mohamed
    et al.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, Hadis
    Strand, Joanna
    Orlova, Anna
    Varasteh, Zohreh
    Sandström, Mattias
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Larsson, Erik
    Strand, Sven-Erik
    Lubberink, Mark
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology. Uppsala University, Sweden.
    Tolmachev, Vladimir
    Re-188-Z(HER2:V2), a Promising Affibody-Based Targeting Agent Against HER2-Expressing Tumors: Preclinical Assessment2014In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 55, no 11, p. 1842-1848Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of Tc-99m-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated Z(HER2:V2)) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate Re-188-Z(HER2:v2) as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors. Methods: Z(HER2:V2) was labeled with Re-188 using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment. Results: Binding of Re-188-Z(HER2:V2) to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 +/- 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 +/- 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 +/- 2, 12 +/- 2, 5 +/- 2, and 1.8 +/- 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 +/- 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that Re-188-Z(HER2:v2) would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible. Conclusion: (188)ReZ(HER2:v2) can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.

  • 15.
    Andersson, Ken G.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Directed evolution is today an established strategy for generation of new affinity proteins. This thesis describes the development of a cell-display method using Escherichia coli for directed evolution of Affibody molecules. Further, the thesis describes rational design of Affibody-based tracers, intended for future patient stratification using medical imaging. Fusing recombinant proteins to various autotransporters is a promising approach for efficient surface display on the surface of E. coli, as well as for construction of high-complexity libraries. In paper I, we successfully engineered an expression vector for display of Affibody molecules using the autotransporter AIDA-I. In paper II, a large Affibody library of 2.3x109 variants was constructed and screening using FACS resulted in new specific binders in the nanomolar range. In paper III, we demonstrated Sortase-mediated secretion and conjugation of binders directly from the E. coli surface. 

    The three following studies describe rational design of Affibody-based tracers against two cancer-associated targets for molecular imaging. First, anti-HER3 Affibody molecules were labelled with 111In, and SPECT imaging showed that the conjugates specifically targeted HER3-expressing xenografts. Furthermore, labeling with 68Ga for PET imaging showed that tumor uptake correlated with HER3 expression, suggesting that the tracers have potential for patient stratification. The last study describes the development and investigation of anti-EGFR Affibody-based imaging agents. Labeled with 89Zr, the Affibody tracer demonstrated higher tumor uptake at 3 h post injection than the anti-EGFR antibody cetuximab at 48 h post injection. 

    In conclusion, this thesis describes new tools and knowledge that will hopefully contribute to the development of affinity proteins for biotechnology, therapy and medical imaging in the future. 

     

  • 16.
    Andersson, Ken G.
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Oroujeni, Maryam
    Garousi, Javad
    Mitran, Bogdan
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, Anna
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with Tc-99m using a peptide-based cysteine-containing chelator2016In: International Journal of Oncology, ISSN 1019-6439, Vol. 49, no 6, p. 2285-2293Article in journal (Refereed)
    Abstract [en]

    The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide Tc-99m should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with Tc-99m using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, Tc-99m-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that Tc-99m-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6 1 and 2.5 0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8 0.4 and 8 3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of Tc-99m-labeled ZEGFR:2377 for imaging of EGFR in vivo.

  • 17.
    Andersson, Ken G.
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Persson, Jonas
    KTH, School of Biotechnology (BIO), Protein Technology. Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Autotransporter-mediated display of a naïve Affibody library on the outer membrane of E. coliManuscript (preprint) (Other academic)
    Abstract [en]

    Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is the most frequently used method, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli have several properties that are valuable for library applications and then in particular the high transformation efficiency. Although the first studies on display of recombinant peptides and proteins on E. coli were reported over 25 years ago, the method is still not fully established for directed evolution of affinity proteins. More recently, the use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and in particular for directed evolution of different enzymes. Here, we report on display of a large naïve Affibody library on the outer membrane of E. coli using the autotransporter AIDA-I. The expression cassette was first engineered by removing non-essential sequences, followed by introduction of an Affibody library, comprising more than 109 variants, into the new display vector. Selections by FACS against five different target molecules resulted in a panel of binders with down to nanomolar affinities.

  • 18.
    Andersson, Ken G
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Rosestedt, Maria
    Varasteh, Zohreh
    Malm, Magdalena
    KTH, School of Biotechnology (BIO), Protein Technology.
    Sandström, Mattias
    Tolmachev, Vladimir
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, Anna
    Comparative evaluation of 111In-labeled NOTA‑conjugated affibody molecules for visualization of HER3 expression in malignant tumors2015In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 34, no 2, p. 1042-8Article in journal (Refereed)
    Abstract [en]

    Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium‑111 (111In) and assessed invitro and invivo for imaging properties using single photon emission computed tomography (SPECT). Labeling of HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA with 111In provided stable conjugates. Invitro cell tests demonstrated specific binding of the two conjugates to HER3-expressing BT‑474 breast carcinoma cells. In mice bearing BT‑474 xenografts, the tumor uptake of the two conjugates was receptor‑specific. Direct invivo comparison of 111In-HEHEHE-Z08698-NOTA and 111In-HEHEHE-Z08699‑NOTA demonstrated that the two conjugates provided equal radioactivity uptake in tumors, although the tumor-to-blood ratio was improved for 111In-HEHEHE-Z08698-NOTA [12±3 vs. 8±1, 4h post injection (p.i.)] due to more efficient blood clearance. 111In-HEHEHE-Z08698-NOTA is a promising candidate for imaging of HER3-expression in malignant tumors using SPECT. Results of the present study indicate that this conjugate could be used for patient stratification for anti-HER3 therapy.

  • 19.
    Andersson, Ken G.
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Sjöstrand, Nanna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Coupled release and site-specific conjugation of Affibody molecules from the surface of E. coli using Sortase AManuscript (preprint) (Other academic)
    Abstract [en]

    Combinatorial protein engineering using libraries displayed on various microorganisms is a powerful method forgeneration of new affinity proteins. Successful efforts often result in broad panels of isolated binders, which are thentypically subcloned, produced, purified and characterized in various assays. Many such assays also require conjugation tofor example reporters or other functional molecules and the downstream production and modification thus tends to be verylaborious and limits the number of candidates that can be screened. Staphylococcal sortase A is a natural transpeptidasethat catalyzes the ligation between a LPXTG motif and N-terminal glycines and is today used in a variety of applicationsfor site-specific conjugation of different molecules to recombinant proteins. We have previously developed a surfacedisplay method for combinatorial protein engineering of Affibody molecules on the outer membrane of E. coli usingautodisplay. Here, we introduced a sortase-A recognition motif into the displayed recombinant proteins and evaluatedsortase-mediated release and specific conjugation of various reporters to Affibody molecules. The approach has potentialto significantly increase the flexibility and throughput of downstream characterization of affinity proteins after directedevolution using cell display and FACS.

  • 20.
    Andersson, Ken G.
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Varasteh, Z.
    Rosenstedt, M.
    Rosestedt, M.
    Malm, M.
    KTH.
    Sandström, M.
    KTH.
    Tolmachev, V.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, A.
    111In-labeled NOTA-conjugated Affibody molecules for visualization of HER3 expression in malignant tumors2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, p. S311-S311Article in journal (Other academic)
  • 21. Andersson, Sandra
    et al.
    Konrad, Anna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ashok, Nikhil
    Pontén, Fredrik
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Asplund, Anna
    Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection2013In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 61, no 11, p. 773-784Article in journal (Refereed)
    Abstract [en]

    Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.

  • 22. Armbrecht, L.
    et al.
    Dincer, C.
    Kling, A.
    Horak, Josef
    KTH, School of Biotechnology (BIO), Protein Technology. University of Freiburg, Germany.
    Kieninger, J.
    Urban, G.
    Self-assembled magnetic bead chains for sensitivity enhancement of microfluidic electrochemical biosensor platforms2015In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 15, no 22, p. 4314-4321Article in journal (Refereed)
    Abstract [en]

    In this paper, we present a novel approach to enhance the sensitivity of microfluidic biosensor platforms with self-assembled magnetic bead chains. An adjustable, more than 5-fold sensitivity enhancement is achieved by introducing a magnetic field gradient along a microfluidic channel by means of a soft-magnetic lattice with a 350 mu m spacing. The alternating magnetic field induces the self-assembly of the magnetic beads in chains or clusters and thus improves the perfusion and active contact between the analyte and the beads. The soft-magnetic lattices can be applied independent of the channel geometry or chip material to any microfluidic biosensing platform. At the same time, the bead-based approach achieves chip reusability and shortened measurement times. The bead chain properties and the maximum flow velocity for bead retention were validated by optical microscopy in a glass capillary. The magnetic actuation system was successfully validated with a biotin-streptavidin model assay on a low-cost electrochemical microfluidic chip, fabricated by dry-film photoresist technology (DFR). Labelling with glucose oxidase (GOx) permits rapid electrochemical detection of enzymatically produced H2O2.

  • 23. Armbrecht, L.
    et al.
    Dincer, C.
    Kling, A.
    Horak, Josef
    KTH, School of Biotechnology (BIO), Protein Technology. Department of Microsystems Engineering - IMTEK, University of Freiburg, Freiburg, Germany.
    Kieninger, J.
    Urban, G.
    Signal amplification using magnetic bead chains in microfluidic electrochemical biosensors2015In: 2015 Transducers - 2015 18th International Conference on Solid-State Sensors, Actuators and Microsystems, IEEE , 2015, p. 1601-1604Conference paper (Refereed)
    Abstract [en]

    We present a novel approach to increase the sensitivity of microfluidic biosensor platforms using magnetic micro-bead chains. An almost 2-fold sensitivity enhancement is achieved by introducing a magnetic field gradient along a microfluidic channel by means of a soft-magnetic lattice with lattice spacings down to 100 μm. The magnetic field gradient induces self-assembly of the magnetic beads in chains or clusters and thus improves the active contact between analyte and beads. This facile strategy significantly increases the active bead surface while allowing for complete independence of traditional biosensor materials and channel geometries, chip-reusability and shortened measurement times. Bead chain properties were validated with optical microscopy in a glass capillary and with electrochemical measurements via glucose oxidase (GOx) labels on an integrated microfluidic chip fabricated in dry-film photo resist technology (DFR).

  • 24.
    Bass, Tarek
    KTH, School of Biotechnology (BIO), Protein Technology.
    Affibody molecules targeting HER3 for cancer therapy2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The development of targeted therapy has contributed tremendously to the treatment of patients with cancer. The use of highly specific affinity proteins to target cancer cells has become a standard in treatment strategies for several different cancers. In light of this, many cancer cell markers are investigated for their potential use in diagnostics and therapy. One such marker is the human epidermal growth factor receptor 3, HER3. It has been established as an important contributor to many cancer types. The function of HER3 is to relay cell growth signals from outside of the cell to the inside. Interfering with- and inhibit- ing the function of HER3 has emerged as an interesting strategy for cancer therapeutics. The studies presented in this thesis aim to target HER3 with small, engineered affinity domain proteins for therapeutic purposes. Monomeric affibody molecules have previously been engineered to bind and inhibit HER3 in vitro. Due to the relatively low expression of HER3, an increase in valency appears promising to strengthen the therapeutic potential. Affibody molecules targeting the receptor were thus linked to form bivalent and bispecific constructs and evaluated both in vitro and in vivo. In the first study of this thesis affibody molecules specific for HER3 and HER2 were fused to an albumin binding domain to form bivalent and bispecific construct. The constructs inhibited ligand-induced receptor phos- phorylation of both HER2 and HER3 more efficiently than monomeric affibody molecules. A second approach to enhance the potential of affibody molecules in tumor targeting is described in the second study, where monomeric HER3-binding affibody molecules were engineered to increase their affinity for HER3. The resulting variants showed a 20-fold in- creased affinity and higher capacity to inhibit cancer cell growth. Combining the findings of the first two studies, the third study describes the evaluation of a HER3-targeting bivalent affibody construct for potential application as a therapeutic. Here, the bivalent construct inhibited cancer cell growth in vitro and was found to slow down tumor growth in mice, while being well tolerated and showing no visible toxicity. The fourth study built upon these findings and compares a very similar bivalent construct to the clinically-investigated HER3-specific monoclonal antibody seribantumab. The affibody construct showed very comparable efficacy with the antibody in terms of decreasing tumor growth rate and ex- tending mouse survival. Collectively, these works describe for the first time the use of alternative affinity protein constructs with therapeutic potential targeting HER3.

  • 25.
    Bass, Tarek
    KTH, School of Biotechnology (BIO), Protein Technology.
    Evaluating the therapeutic potential of a dimeric HER3-binding affibody construct in comparison with a monoclonal antibody, seribantumab.Manuscript (preprint) (Other academic)
    Abstract [en]

    A number of monoclonal antibodies targeting HER3 are currently under clinical investigation as potential cancer therapeutics. We have earlier generated high affinity (low picomolar) affibody molecules targeting HER3. These are small, 58 amino acid, non-immunoglobulin based scaffold proteins that have proved suitable for tumor targeting applications, previously primarily for molecular imaging purposes. Our high affinity HER3-binding affibody molecule has demonstrated to have anti-proliferative capacity on HER3-positive tumor cells. When formatted as a bivalent construct, in which the two affibody moieties are flanking a small albumin-binding domain (ABD), we have recently demonstrated that tumor growth could be delayed in mice for HER3-positive xenografts. In this study, we have modified the construct further and reduced the size. In a comparative study, we evaluated safety, the capacity to delay tumor growth in mice with BxPC-3 xenografts, and mouse survival. Our novel construct was compared to the HER3-specific monoclonal antibody seribantumab (MM-121), presently in clinical development. They were found to be equally potent in their therapeutic effects and in their safety profile. We conclude that this format of bivalent HER3-binding affibody molecules seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.

  • 26.
    Bass, Tarek
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Rosestedt, Maria
    Mitran, Bogdan
    Frejd, Fredrik Y.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, Anna
    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.

  • 27.
    Boström, Tove
    KTH, School of Biotechnology (BIO), Protein Technology.
    High-throughput protein analysis using mass spectrometry-based methods2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers.

    The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format.

  • 28.
    Boström, Tove
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Danielsson, Frida
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Johansson, Henrik J.
    Karlinska Institute, Cancer Proteomics Mass Spectrometry, Dep. of Oncology-Pathology.
    Tegel, Hanna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lehtiö, Janne
    Karolinska Institute, Cancer Proteomics Mass Spectrometry, Dep. of Oncology-Pathology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ottosson Takanen, Jenny
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Investigating the correlation of protein and mRNA levels in human cell lines using quantitative proteomics and transcriptomicsManuscript (preprint) (Other academic)
    Abstract [en]

    An important topic of discussion in proteomics is the degree of correlation of RNA and protein levels in cells, tissues and organs. In this study, the difference in protein and mRNA levels for a number of selected gene targets were investigated across six human cell lines using quantitative proteomics and next generation sequencing-based transcriptomics. The copy numbers of 32 proteins were determined using an absolute quantitative proteomics approach (PrEST-SILAC), where heavy isotope-labeled protein fragments were used as internal standards. A cross evaluation of protein copy numbers determined by mass spectrometry and staining profiles using immunohistochemistry showed good correlation. The mRNA levels were determined using RNA sequencing based on digital counting of sequencing reads and the levels determined as FPKM values. Comparison of the relative variations in mRNA and protein levels for individual genes across the six cell lines showed correlation between protein and mRNA levels, including six genes with high variability in expression levels in the six cell lines resulting in an average correlation of 0.9 (Spearman's rank coefficient). In summary, the analysis of the selected protein targets supports the conclusion that the translation rate across cell lines correlates for a particular gene, suggesting that individual protein levels can be predicted from the respective mRNA levels by defining the relation between protein and mRNA, specific for each human gene.

  • 29.
    Boström, Tove
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Johansson, Henrik J.
    Karolinska Institute.
    Lehtiö, Janne
    Karolinska Institute.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Investigating the Applicability of Antibodies Generated within the Human Protein Atlas as Capture Agents in Immunoenrichment Coupled to Mass Spectrometry2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 10, p. 4424-4435Article in journal (Refereed)
    Abstract [en]

    For identification and characterization of proteins in complex samples, immunoenrichment coupled to mass spectrometry is a good alternative due to the sensitivity of the affinity enrichment and the specificity of mass spectrometry analysis. Antibodies are commonly used affinity agents; however, for high-throughput analysis, antibody availability is usually a bottleneck. Here we present a protocol for immunoenrichment coupled to mass spectrometry in a high-throughput setup, where all steps from bead coupling to mass spectrometry sample preparation are performed in parallel in a 96-well format. Antibodies generated within the Human Protein Atlas project were tested for applicability as capture agents. The antibodies were covalently attached to protein A beads, making it possible to reuse the coupled beads at least three times without destroying the antibody binding efficiency. Target proteins were captured from a U251 MG cell lysate, eluted, digested, and analyzed using mass spectrometry. Of 30 investigated antibodies, around 50% could successfully capture the corresponding native target protein, making the available library of more than 21 000 antibodies a valuable resource for immunoenrichment assays. Due to the diversity of different antibodies regarding affinity and specificity, analyzing antibodies in a high-throughput format is challenging. Even though protocol optimization for individual antibodies can be advantageous for future studies, our method enables a fast screening strategy to determine the usefulness of antibodies in immunoenrichment setups. In addition, we show that the specificity of the antibodies can be investigated by using label-free quantification.

  • 30.
    Boström, Tove
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ottosson Takanen, Jenny
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Antibodies as means for selective mass spectrometry2015In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376XArticle in journal (Refereed)
    Abstract [en]

    For protein analysis of biological samples, two major strategies are used today; mass spectrometry (MS) and antibody-based methods. Each strategy offers advantages and drawbacks. However, combining the two using an immunoenrichment step with MS analysis brings together the benefits of each method resulting in increased sensitivity, faster analysis and possibility of higher degrees of multiplexing. The immunoenrichment can be performed either on protein or peptide level and quantification standards can be added in order to enable determination of the absolute protein concentration in the sample. The combination of immunoenrichment and MS holds great promise for the future in both proteomics and clinical diagnostics. This review describes different setups of immunoenrichment coupled to mass spectrometry and how these can be utilized in various applications.

  • 31. Boutajangout, Allal
    et al.
    Lindberg, Hanna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Awwad, Abdulaziz
    Paul, Arun
    Wahlberg, Elisabet
    Gudmundsdotter, Hanna
    Härd, Torleif
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Wisniewski, Thomas
    Affibody-mediated Reduction of Amyloid Burden and Improvement of Cognitive Decline in an Animal Model of Alzheimer’s diseaseManuscript (preprint) (Other academic)
  • 32. Chen, G.
    et al.
    Sidhu, S. S.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Protein Technology. University of Toronto, Canada.
    Synthetic antibodies in infectious disease2017In: Recombinant Antibodies for Infectious Diseases, Springer-Verlag New York, 2017, p. 79-98Chapter in book (Refereed)
    Abstract [en]

    Rapid spread of microbial resistance and recent outbreaks of viral disease have led to renewed interest in antibody-based therapies for infectious diseases. Synthetic antibody libraries displayed on phage offer unique advantages over traditional immunization-based antibody generation, including full control over library design and selection conditions. The technology has matured beyond natural antibodies and is capable of providing novel insights into infectious disease and can generate novel antibodies that cannot be produced by the natural immune system. This chapter gives an overview of recombinant antibody library technology with an emphasis on our work using a highly successful synthetic single framework Fab library. We demonstrate its utility in targeting viruses and bacterial toxins in five case studies.

  • 33. Cheng, Qing
    et al.
    Wallberg, Helena
    Grafstrom, Jonas
    Lu, Li
    Thorell, Jan-Olov
    Olofsson, Maria Hagg
    Linder, Stig
    Johansson, Katarina
    Tegnebratt, Tetyana
    Arner, Elias S. J.
    Stone-Elander, Sharon
    Ahlzen, Hanna-Stina Martinsson
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake2016In: EJNMMI Research, ISSN 2191-219X, E-ISSN 2191-219X, Vol. 6, article id 58Article in journal (Refereed)
    Abstract [en]

    Background: Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors. Methods: The EGFR-binding Affibody molecule Z(EGFR:2377) and its size-matched non-binding control Z(Taq:3638) were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (Z(EGFR:2377)-ST and Z(Taq:3638)-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with C-11 for in vivo PET studies. Kinetic scans with the C-11-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions. Results: Flow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZE(GFR:2377)-ST-DyLight488. [Methyl-C-11]-labeled Z(EGFR:2377)-ST-CH3 and Z(Taq:3638)-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-C-11]-Z(EGFR:2377)-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since Z(EGFR:2377) binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels. Conclusions: Same-day in vivo dual tracer imaging enabled by the Sel-tag technology and C-11-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand.

  • 34.
    Dekki Shalaly, Nancy
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ria, Massimiliano
    Johansson, Ulrika
    KTH, School of Biotechnology (BIO), Protein Technology.
    Avall, Karin
    Berggren, Per-Olof
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Protein Technology.
    Silk matrices promote formation of insulin-secreting islet-like clusters2016In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 90, p. 50-61Article in journal (Refereed)
    Abstract [en]

    Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro. Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation.

  • 35.
    Dezfouli, Mahya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Redin, David
    KTH, School of Biotechnology (BIO), Protein Technology.
    Borgström, Erik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Edfors, Fredrik
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Droplet-based Immuno-Sequencing to Deconvolute Affinity Recognition EventsManuscript (preprint) (Other academic)
  • 36.
    Edfors, Fredrik
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Boström, Tove
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Zeiler, Marlis
    Johansson, Henrik J.
    Karlinska Institute.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lehtiö, Janne
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mann, Matthias
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins2014In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, p. 1611-1624Article in journal (Refereed)
    Abstract [en]

    AThe combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  • 37. Elgstrand Wettergren, E.
    et al.
    Seijsing, Johan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Sest, M.
    Quintino, L.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lundberg, C.
    Up-regulation of endogenous GAD67 expression in vivo using designed zinc finger-based transcription factorsManuscript (preprint) (Other academic)
  • 38.
    Fagerland, Jenny
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Pappalardo, Daniela
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology. University of Sannio, Italy.
    Schmidt, Björn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Syrén, Per-Olof
    KTH, School of Biotechnology (BIO), Protein Technology.
    Finne-Wistrand, Anna
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Template-assisted enzymatic synthesis of oligopeptides from a polylactide chain2017In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 18, no 12, p. 4271-4280Article in journal (Refereed)
    Abstract [en]

    Peptides are often attached to polymer materials, as bioactive components, for the control of interactions between the material and its surrounding proteins and cells. However, synthesizing peptides and attaching them to polymers can be challenging and laborious. Herein, we describe the grafting of oligopeptides to an aliphatic polyester, using a one-step chemo-enzymatic synthesis with papain as the biocatalySt. To enable enzyme-mediated functionalization of the polyester, ethyl hept-6-enoylalaninate (grafter) was synthesized and attached to polylactide chains using thiol-ene click reactions. The oligopeptides were grafted onto the polylactide chains using two different synthetic routes: the grafting from strategy, in which the grafter was attached to the polyester prior to oligopeptide synthesis, or the grafting to strategy, in which oligopeptides were synthesized on the grafter first, then attached to the polymer chain. The final products were analyzed and their structures were confirmed using nuclear magnetic resonance (NMR). The peptide attachment was evaluated using size exclusion chromatography (SEC), contact angle measurement and energy-dispersive X-ray spectroscopy scanning electron microscopy (EDS-SEM). Furthermore, the mechanistic aspects of the synthesis of the oligopeptides on the grafter were studied using molecular dynamics (MD) simulations. The simulation revealed that hydrogen bonding (between the P1 amide nitrogen of the grafter backbone and the carbonyl oxygen of D158 in the papain) maintain the grafter in a productive conformation to stabilize the transition state of nitrogen inversion, a key step of the biocatalytic mechanism. Apart from being biologically relevant, both experimental and computational results suggest that the designed grafter is a good template for initiating chemo-enzymatic synthesis. The results also showed that the grafting to strategy was more successful compared to the grafting from strategy. Overall, a successful synthesis of predefined peptide functionalized polylactide was prepared, where the oligopeptides were grafted in an easy, time efficient, and environmentally friendly way.

  • 39.
    Fleetwood, Filippa
    KTH, School of Biotechnology (BIO), Protein Technology.
    Bacterial display systems for engineering of affinity proteins2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Directed evolution is a powerful method for engineering of specific affinity proteins such as antibodies and alternative scaffold proteins. For selections from combinatorial protein libraries, robust and high-throughput selection platforms are needed. An attractive technology for this purpose is cell surface display, offering many advantages, such as the quantitative isolation of high-affinity library members using flow-cytometric cell sorting. This thesis describes the development, evaluation and use of bacterial display technologies for the engineering of affinity proteins.

    Affinity proteins used in therapeutic and diagnostic applications commonly aim to specifically bind to disease-related drug targets. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical process in various types of cancer and vascular eye disorders. Vascular Growth Factor Receptor 2 (VEGFR2) is one of the main regulators of angiogenesis. The first two studies presented in this thesis describe the engineering of a biparatopic Affibody molecule targeting VEGFR2, intended for therapeutic and in vivo imaging applications. Monomeric VEGFR2-specific Affibody molecules were generated by combining phage and staphylococcal display technologies, and the engineering of two Affibody molecules, targeting distinct epitopes on VEGFR2 into a biparatopic construct, resulted in a dramatic increase in affinity. The biparatopic construct was able to block the ligand VEGF-A from binding to VEGFR2-expressing cells, resulting in an efficient inhibition of VEGFR2 phosphorylation and angiogenesis-like tube formation in vitro.

    In the third study, the staphylococcal display system was evaluated for the selection from a single-domain antibody library. This was the first demonstration of successful selection from an antibody-based library on Gram-positive bacteria. A direct comparison to the selection from the same library displayed on phage resulted in different sets of binders, and higher affinities among the clones selected by staphylococcal display. These results highlight the importance of choosing a display system that is suitable for the intended application.

    The last study describes the development and evaluation of an autotransporter-based display system intended for display of Affibody libraries on E. coli. A dual-purpose expression vector was designed, allowing efficient display of Affibody molecules, as well as small-scale protein production and purification of selected candidates without the need for sub-cloning. The use of E. coli would allow the display of large Affibody libraries due to a high transformation frequency. In combination with the facilitated means for protein production, this system has potential to improve the throughput of the engineering process of Affibody molecules.

    In summary, this thesis describes the development, evaluation and use of bacterial display systems for engineering of affinity proteins. The results demonstrate great potential of these display systems and the generated affinity proteins for future biotechnological and therapeutic use.

  • 40.
    Fleetwood, Filippa
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Andersson, Ken A.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains2014In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 13, p. 179-Article in journal (Refereed)
    Abstract [en]

    Background: Cell display technologies (e.g. bacterial display) are attractive in directed evolution as they provide the option to use flow-cytometric cell sorting for selection from combinatorial libraries. The aim of this study was to engineer and investigate an expression vector system with dual functionalities: i) recombinant display of Affibody libraries on Escherichia coli for directed evolution and ii) small scale secreted production of candidate affinity proteins, allowing initial downstream characterizations prior to subcloning. Autotransporters form a class of surface proteins in Gram-negative bacteria that have potential for efficient translocation and tethering of recombinant passenger proteins to the outer membrane. We engineered a bacterial display vector based on the E. coli AIDA-I autotransporter for anchoring to the bacterial surface. Potential advantages of employing autotransporters combined with E. coli as host include: high surface expression level, high transformation frequency, alternative promoter systems available, efficient translocation to the outer membrane and tolerance for large multi-domain passenger proteins. Results: The new vector was designed to comprise an expression cassette encoding for an Affibody molecule, three albumin binding domains for monitoring of surface expression levels, an Outer membrane Protease T (OmpT) recognition site for potential protease-mediated secretion of displayed affinity proteins and a histidine-tag for purification. A panel of vectors with different promoters were generated and evaluated, and suitable cultivation conditions were investigated. The results demonstrated a high surface expression level of the different evaluated Affibody molecules, high correlation between target binding and surface expression level, high signal-to-background ratio, efficient secretion and purification of binders in OmpT-positive hosts as well as tight regulation of surface expression for the titratable promoters. Importantly, a mock selection using FACS from a 1: 100,000 background yielded around 20,000-fold enrichment in a single round and high viability of the isolated bacteria after sorting. Conclusions: The new expression vectors are promising for combinatorial engineering of Affibody molecules and the strategy for small-scale production of soluble recombinant proteins has the potential to increase throughput of the entire discovery process.

  • 41.
    Fleetwood, Filippa
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Andersson, Ken
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Development And Optimization Of An e.Coli-based Display Platform For Selection Of Affinity Proteins2014In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 23, p. 135-135Article in journal (Other academic)
  • 42.
    Fleetwood, Filippa
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Frejd, Fredrik
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Efficient blocking of VEGFR2-mediated signaling using biparatopic Affibody constructsManuscript (preprint) (Other academic)
  • 43.
    Fleetwood, Filippa
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Güler, Rezan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Gordon, Emma
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Claesson-Welsh, Lena
    Löfblom, John
    Novel affinity binders for neutralization of vascular endothelial growth factor (VEGF) signaling2016In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 73, no 8, p. 1671-1683Article in journal (Refereed)
    Abstract [en]

    Angiogenesis denotes the formation of new blood vessels from pre-existing vasculature. Progression of diseases such as cancer and several ophthalmological disorders may be promoted by excess angiogenesis. Novel therapeutics to inhibit angiogenesis and diagnostic tools for monitoring angiogenesis during therapy, hold great potential for improving treatment of such diseases. We have previously generated so-called biparatopic Affibody constructs with high affinity for the vascular endothelial growth factor receptor-2 (VEGFR2), which recognize two non-overlapping epitopes in the ligand-binding site on the receptor. Affibody molecules have previously been demonstrated suitable for imaging purposes. Their small size also makes them attractive for applications where an alternative route of administration is beneficial, such as topical delivery using eye drops. In this study, we show that decreasing linker length between the two Affibody domains resulted in even slower dissociation from the receptor. The new variants of the biparatopic Affibody bound to VEGFR2-expressing cells, blocked VEGFA binding, and inhibited VEGFA-induced signaling of VEGFR2 over expressing cells. Moreover, the biparatopic Affibody inhibited sprout formation of endothelial cells in an in vitro angiogenesis assay with similar potency as the bivalent monoclonal antibody ramucirumab. This study demonstrates that the biparatopic Affibody constructs show promise for future therapeutic as well as in vivo imaging applications.

  • 44.
    Fleetwood, Filippa
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Klint, Susanne
    Hanze, Martin
    KTH, School of Biotechnology (BIO), Protein Technology.
    Gunneriusson, Elin
    Frejd, Fredrik
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity2014In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 4, p. 7518-Article in journal (Refereed)
    Abstract [en]

    Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases.

  • 45. Fuentes, L.
    et al.
    Gomez-Cid, L.
    Fernandez-Santos, M. E.
    Suarez-Sancho, S.
    Plasencia, V.
    Climent, A.
    Sanz-Ruiz, R.
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Protein Technology. Protein-based materials.
    Atienza, F.
    Fernandez-Aviles, F.
    Biocompatibility of mesenchymal stem cells with a spider silk matrix and its potential use as scaffold for cardiac tissue regeneration2016In: Cardiovascular Research, ISSN 0008-6363, E-ISSN 1755-3245, Vol. 111, p. S93-S93Article in journal (Other academic)
  • 46. Garousi, J.
    et al.
    Anderson, Ken
    KTH, School of Biotechnology (BIO), Protein Technology.
    Dam, J. H.
    Olsen, B. B.
    Orlova, A.
    Buijs, J.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Thisgaard, H.
    Tolmachev, V.
    The use of radiocobalt as a label improves PET imaging of EGFR using DOTA-conjugated affibody molecules2015In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, p. S244-S244Article in journal (Refereed)
  • 47. Garousi, J.
    et al.
    Lindbo, S.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, H.
    Velletta, J.
    Mitran, B.
    Altai, M.
    Orlova, A.
    Tolmachev, V.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Influence of the N-terminal amino acid sequence on imaging properties of In-111-labeled anti-HER2 scaffold protein ADAPT62016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S55-S55Article in journal (Refereed)
  • 48. Garousi, J.
    et al.
    Lindbo, Sarah
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Orlova, A.
    Åstrand, Mikael
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Buijs, J.
    Sandstrom, M.
    Honarvar, H.
    Tolmachev, V.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Development of ADAPT6 as a new scaffold protein for radionuclide molecular imaging2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, p. S309-S309Article in journal (Refereed)
  • 49. Garousi, Javad
    et al.
    Anderson, Ken G.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Mitran, Bogdan
    Pichl, Marie-Louise
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, Anna
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    PET imaging of epidermal growth factor receptor expression in tumours using Zr-89-labelled ZEGFR:2377 affibody molecules2016In: International Journal of Oncology, ISSN 1019-6439, Vol. 48, no 4, p. 1325-1332Article in journal (Refereed)
    Abstract [en]

    Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which is overexpressed in many types of cancer. The use of EGFR-targeting monoclonal antibodies and tyrosine-kinase inhibitors improves significantly survival of patients with colorectal, non-small cell lung cancer and head and neck squamous cell carcinoma. Detection of EGFR overexpression provides important prognostic and predictive information influencing management of the patients. The use of radionuclide molecular imaging would enable non-invasive repeatable determination of EGFR expression in disseminated cancer. Moreover, positron emission tomography (PET) would provide superior sensitivity and quantitation accuracy in EGFR expression imaging. Affibody molecules are a new type of imaging probes, providing high contrast in molecular imaging. In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a radionuclide Zr-89. The Zr-89-DFO-ZEGFR:2377 tracer demonstrated specific high affinity (160 +/- 60 pM) binding to EGFR-expressing A431 epidermoid carcinoma cell line. In mice bearing A431 xenografts, Zr-89-DFO-ZEGFR: 2377 demonstrated specific uptake in tumours and EGFR-expressing tissues. The tracer provided tumour uptake of 2.6 +/- 0.5% ID/g and tumour-to-blood ratio of 3.7 +/- 0.6 at 24 h after injection. Zr-89-DFO-ZEGFR: 2377 provides higher tumour-to-organ ratios than anti-EGFR antibody Zr-89-DFO-cetuximab at 48 h after injection. EGFR-expressing tumours were clearly visualized by microPET using Zr-89-DFO-ZEGFR: 2377 at both 3 and 24 h after injection. In conclusion, Zr-89-DFO-ZEGFR: 2377 is a potential probe for PET imaging of EGFR-expression in vivo.

  • 50. Garousi, Javad
    et al.
    Andersson, Ken G.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Dam, Johan H.
    Olsen, Birgitte B.
    Mitran, Bogdan
    Orlova, Anna
    Buijs, Jos
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Thisgaard, Helge
    Tolmachev, Vladimir
    The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 5961Article in journal (Refereed)
    Abstract [en]

    Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The In-111-labelled DOTA-conjugated Z(EGFR:2377) Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-Z(EGFR:2377). DOTA-Z(EGFR:2377) was labelled with Co-57 (T-1/2 = 271.8 d), Co-55 (T-1/2 = 17.5 h), and, for comparison, with the positron-emitting radionuclide Ga-68 (T-1/2 = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope Co-57 was used in animal studies. Both Co-57-DOTA-Z(EGFR:2377) and Ga-68-DOTA-Z(EGFR:2377) demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-Z(EGFR:2377) were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, Co-57-DOTA-Z(EGFR:2377) demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for (68)GaDOTA-Z(EGFR:2377). The results of this study suggest that the positron-emitting cobalt isotope 55Co would be an optimal label for DOTA-Z(EGFR:2377) and further development should concentrate on this radionuclide as a label.

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