Ändra sökning
Avgränsa sökresultatet
123 1 - 50 av 136
RefereraExporteraLänk till träfflistan
Permanent länk
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Träffar per sida
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sortering
  • Standard (Relevans)
  • Författare A-Ö
  • Författare Ö-A
  • Titel A-Ö
  • Titel Ö-A
  • Publikationstyp A-Ö
  • Publikationstyp Ö-A
  • Äldst först
  • Nyast först
  • Skapad (Äldst först)
  • Skapad (Nyast först)
  • Senast uppdaterad (Äldst först)
  • Senast uppdaterad (Nyast först)
  • Disputationsdatum (tidigaste först)
  • Disputationsdatum (senaste först)
  • Standard (Relevans)
  • Författare A-Ö
  • Författare Ö-A
  • Titel A-Ö
  • Titel Ö-A
  • Publikationstyp A-Ö
  • Publikationstyp Ö-A
  • Äldst först
  • Nyast först
  • Skapad (Äldst först)
  • Skapad (Nyast först)
  • Senast uppdaterad (Äldst först)
  • Senast uppdaterad (Nyast först)
  • Disputationsdatum (tidigaste först)
  • Disputationsdatum (senaste först)
Markera
Maxantalet träffar du kan exportera från sökgränssnittet är 250. Vid större uttag använd dig av utsökningar.
  • 1. Agaton, C.
    et al.
    Galli, J.
    Guthenberg, I. H.
    Janzon, L.
    Hansson, M.
    Asplund, A.
    Brundell, E.
    Lindberg, S.
    Ruthberg, I.
    Wester, K.
    Wurtz, D.
    Hoog, C.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ponten, F.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues2003Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 2, nr 6, s. 405-414Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.

  • 2. Altai, M.
    et al.
    Honarvar, H.
    Wallberg, H.
    Strand, J.
    Varasteh, Z.
    Orlova, A.
    Dunas, F.
    Sandstrom, M.
    Rosestedt, M.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, V.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with Re-1882013Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, s. S219-S220Artikel i tidskrift (Övrigt vetenskapligt)
  • 3. Altai, M.
    et al.
    Wallberg, H.
    Honarvar, H.
    Strand, J.
    Orlova, A.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Varasteh, Z.
    Sandström, M.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, V.
    Re-188-Z(HER2: V2), a promising targeting agent against HER2-expressing tumors: in vitro and in vivo assessment2013Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, s. S119-S119Artikel i tidskrift (Övrigt vetenskapligt)
  • 4. Altai, Mohamed
    et al.
    Honarvar, Hadis
    Wållberg, Helena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Strand, Joanna
    Varasteh, Zohreh
    Rosestedt, Maria
    Orlova, Anna
    Dunås, Finn
    Sandström, Mattias
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, Vladimir
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.2014Ingår i: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 87, s. 519-28Artikel i tidskrift (Refereegranskat)
    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.

  • 5.
    Altai, Mohamed
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden.;Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Atterby, Christina
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, S-75237 Uppsala, Sweden..
    Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs2018Ingår i: CELLS, ISSN 2073-4409, Vol. 7, nr 10, artikel-id 164Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 6. Altai, Mohamed
    et al.
    Wållberg, Helena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Honarvar, Hadis
    Strand, Joanna
    Orlova, Anna
    Varasteh, Zohreh
    Sandström, Mattias
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Larsson, Erik
    Strand, Sven-Erik
    Lubberink, Mark
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. Uppsala University, Sweden.
    Tolmachev, Vladimir
    Re-188-Z(HER2:V2), a Promising Affibody-Based Targeting Agent Against HER2-Expressing Tumors: Preclinical Assessment2014Ingår i: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 55, nr 11, s. 1842-1848Artikel i tidskrift (Refereegranskat)
    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.

  • 7. Altai, Mohamed
    et al.
    Wållberg, Helena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Orlova, Anna
    Rosestedt, Maria
    Hosseinimehr, Seyed Jalal
    Tolmachev, Vladimir
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of Tc-99m-labeled recombinant Affibody molecules2012Ingår i: Amino Acids, ISSN 0939-4451, E-ISSN 1438-2199, Vol. 42, nr 5, s. 1975-1985Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 8. Andersson, C.
    et al.
    Hansson, M.
    Power, U.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column2001Ingår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 34, s. 25-32Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

  • 9. Andersson, C.
    et al.
    Liljestrom, P.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Power, U. F.
    Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen2000Ingår i: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 29, nr 4, s. 247-253Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.

  • 10. Andersson, C.
    et al.
    Sandberg, L.
    Wernérus, Henrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Johansson, M.
    Lovgren-Bengtsson, K.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation2000Ingår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 238, nr 02-jan, s. 181-193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-MS-M, ABP-MS-MI, ZZ-MS-M and ZZ-MS-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce MS-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.

  • 11.
    Andersson, Christin
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Wikman, Maria
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lövgren-Bengtsson, Karin
    Lundén, Anne
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation2001Ingår i: Journal of Immunological Methods, ISSN 0022-1759, Vol. 255, nr 1-2, s. 135-148Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment ΔSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-ΔSAG1 and His6-ABP-ΔSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-ΔSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, ΔSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that ΔSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

  • 12.
    Andersson, Ken G.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Oroujeni, Maryam
    Garousi, Javad
    Mitran, Bogdan
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Orlova, Anna
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    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 chelator2016Ingår i: International Journal of Oncology, ISSN 1019-6439, Vol. 49, nr 6, s. 2285-2293Artikel i tidskrift (Refereegranskat)
    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.

  • 13.
    Andersson, Ken G.
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Persson, Jonas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Autotransporter-Mediated Display of a Naive Affibody Library on the Outer Membrane of Escherichia coli2019Ingår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 14, nr 4, artikel-id 1800359Artikel i tidskrift (Refereegranskat)
    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 most frequently used, 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 has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naive affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 10(9) variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.

  • 14.
    Andersson, Ken G.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Persson, Jonas
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Autotransporter-mediated display of a naïve Affibody library on the outer membrane of E. coliManuskript (preprint) (Övrigt vetenskapligt)
    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.

  • 15.
    Andersson, Ken G
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Rosestedt, Maria
    Varasteh, Zohreh
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Sandström, Mattias
    Tolmachev, Vladimir
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Orlova, Anna
    Comparative evaluation of 111In-labeled NOTA‑conjugated affibody molecules for visualization of HER3 expression in malignant tumors2015Ingår i: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 34, nr 2, s. 1042-8Artikel i tidskrift (Refereegranskat)
    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.

  • 16.
    Andersson, Ken G.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Varasteh, Z.
    Rosenstedt, M.
    Rosestedt, M.
    Malm, M.
    KTH.
    Sandström, M.
    KTH.
    Tolmachev, V.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Orlova, A.
    111In-labeled NOTA-conjugated Affibody molecules for visualization of HER3 expression in malignant tumors2014Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, s. S311-S311Artikel i tidskrift (Övrigt vetenskapligt)
  • 17.
    Bass, Tarek
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Rosestedt, Maria
    Mitran, Bogdan
    Frejd, Fredrik Y.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, Vladimir
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Orlova, Anna
    In vivo evaluation of a novel format of a bivalent HER3-targeting and albumin- binding therapeutic affibody construct2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 43118Artikel i tidskrift (Refereegranskat)
    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.

  • 18. Binz, Hans
    et al.
    Ngoc, Thien Nguyen
    Ståhl, Stefan
    Uhlén, Mathias
    Nygren, Per-Åke
    Respiratory syncytial virus protein g expressed on bacterial membrane1994Patent (Övrig (populärvetenskap, debatt, mm))
    Abstract [en]

    A method for preparing a peptide or protein, wherein (a) a DNA sequence coding for a heterologous polypeptide on a peptide sequence between amino acid residues 130 and 230 of respiratory syncytial virus protein G, sub-groups A and B, or a peptide sequence at least 80 % homologous thereto, and (b) means enabling the expression of the polypeptide on the bacterial membrane surface, are inserted into a bacterium which is not pathogenic for mammals. The resulting conjugate polypeptide and a live bacterium expressing same, pharmaceutical compositions containing them and their use for preparing a vaccine, as well as a DNA sequence coding for said polypeptide, are also disclosed.

  • 19.
    Boutajangout, Allal
    et al.
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Psychiat, Langone Hlth, 550 1St Ave, New York, NY 10016 USA.;NYU, Langone Med Ctr, Dept Physiol & Neurosci, New York, NY USA..
    Lindberg, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Awwad, Abdulaziz
    King Abdulaziz Univ, Sch Med, Jeddah, Saudi Arabia..
    Paul, Arun
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA..
    Baitalmal, Rabaa
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA..
    Almokyad, Ismail
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA..
    Hoiden-Guthenberg, Ingmarie
    Affibody AB, Solna, Sweden..
    Gunneriusson, Elin
    Affibody AB, Solna, Sweden..
    Frejd, Fredrik Y.
    Affibody AB, Solna, Sweden..
    Hard, Torleif
    Swedish Univ Agr Sci SLU, Dept Chem & Biotechnol, Uppsala, Sweden..
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Wisniewski, Thomas
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Psychiat, Langone Hlth, 550 1St Ave, New York, NY 10016 USA.;NYU, Sch Med, Dept Pathol, New York, NY 10016 USA..
    Affibody-Mediated Sequestration of Amyloid beta Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model2019Ingår i: Frontiers in Aging Neuroscience, ISSN 1663-4365, E-ISSN 1663-4365, Vol. 11, artikel-id 64Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Different strategies for treatment and prevention of Alzheimer's disease (AD) are currently under investigation, including passive immunization with anti-amyloid beta (anti-A beta) monoclonal antibodies (mAbs). Here, we investigate the therapeutic potential of a novel type of A beta-targeting agent based on an affibody molecule with fundamentally different properties to mAbs. We generated a therapeutic candidate, denoted Z(SYM73)-albumin-binding domain (ABD; 16.8 kDa), by genetic linkage of the dimeric Z(SYM73) affibody for sequestering of monomeric A beta-peptides and an ABD for extension of its in vivo half-life. Amyloid precursor protein (APP)/PS1 transgenic AD mice were administered with Z(SYM73)-ABD, followed by behavioral examination and immunohistochemistry. Results demonstrated rescued cognitive functions and significantly lower amyloid burden in the treated animals compared to controls. No toxicological symptoms or immunology-related side-effects were observed. To our knowledge, this is the first reported in vivo investigation of a systemically delivered scaffold protein against monomeric A beta, demonstrating a therapeutic potential for prevention of AD.

  • 20. Boutajangout, Allal
    et al.
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Awwad, Abdulaziz
    Paul, Arun
    Wahlberg, Elisabet
    Gudmundsdotter, Hanna
    Härd, Torleif
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Wisniewski, Thomas
    Affibody-mediated Reduction of Amyloid Burden and Improvement of Cognitive Decline in an Animal Model of Alzheimer’s diseaseManuskript (preprint) (Övrigt vetenskapligt)
  • 21. Cano, F.
    et al.
    Plotnicky-Gilquin, H.
    Nguyen, T. N.
    Liljeqvist, S.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Bonnefoy, J. Y.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Robert, A.
    Partial protection to respiratory syncytial virus (RSV) elicited in mice by intranasal immunization using live staphylococci with surface-displayed RSV-peptides2000Ingår i: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 18, nr 24, s. 2743-2752Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A live bacterial vaccine-delivery system based on the food-grade bacterium Staphylococcus carnosus was used for delivery of peptides from the G glycoprotein of human respiratory syncytial virus, subtype A (RSV-A). Three peptides, corresponding to the G protein amino acids, 144-159 (denoted G5), 190-203 (G9) and 171-188 (G4 S), the latter with four cysteine residues substituted for serines, were expressed by recombinant means as surface-exposed on three different bacteria, and their surface accessibility on the bacteria was verified by fluorescence-activated cell sorting (FACS). Intranasal immunization of mice with the live recombinant staphylococci elicited significant anti-peptide as well as anti-virus serum IgG responses of balanced IgG1/IgG2a isotype profiles, and upon viral challenge with 10(5) tissue culture infectious doses(50) (TCID50), lung protection was demonstrated for approximately half of the mice in the G9 and G4 S immunization groups. To our knowledge, this is the first study in which protective immunity to a viral pathogen has been evoked using food-grade bacteria as vaccine-delivery vehicles.

  • 22. Carlsson, Jörgen
    et al.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Eriksson, Tove
    Gunnariusson, Elin
    Nilsson, Fredrik
    Polypeptides having binding affinity for HER22003Patent (Övrig (populärvetenskap, debatt, mm))
    Abstract [en]

    A polypeptide is provided, which has a binding affinity for HER2 and which is related to a domain of staphylococcal protein A (SPA) in that the sequence of the polypeptide corresponds to the sequence of the SPA domain having from 1 to about 20 substitution mutations. Nucleic acid encoding the polypeptide, as well as expression vector and host cell for expressing the nucleic acid, are also provided. Also provided is the use of such a polypeptide as a medicament, and as a targeting agent for directing substances conjugated thereto to cells overexpressing HER2. Methods, and kits for performing the methods, are also provided, which methods and kits rely on the binding of the polypeptide to HER2.

  • 23. 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, Skolan för bioteknologi (BIO), Proteinteknologi.
    Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake2016Ingår i: EJNMMI Research, ISSN 2191-219X, E-ISSN 2191-219X, Vol. 6, artikel-id 58Artikel i tidskrift (Refereegranskat)
    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.

  • 24.
    Dahlsson Leitao, Charles
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Rinne, S. S.
    Mitran, B.
    Vorobyeva, A.
    Andersson, Ken Gösta
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Tolmachev, V.
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orlova, A.
    Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68 Ga-Labeled Tracers2019Ingår i: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, nr 5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)₃-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)₃-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)₃-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)₃-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)₃-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.

  • 25. Ekerljung, L.
    et al.
    Wållberg, Helena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Sohrabian, A.
    Andersson, K.
    Friedman, M.
    Frejd, F. Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Gedda, L.
    Generation and evaluation of bispecific affibody molecules for simultaneous targeting of EGFR and HER22012Ingår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, nr 9, s. 1802-1811Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 26.
    Falk, Ronny
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Agaton, Charlotta
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Kiesler, E.
    Jin, S.
    Wieslander, L.
    Visa, N.
    Hober, Sophia
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    An improved dual-expression concept, generating high-quality antibodies for proteomics research2003Ingår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 38, s. 231-239Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.

  • 27.
    Falk, Ronny
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Gräslund, Susanne
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Brundell, Eva
    Höög, Christer
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    A high-stringency proteomics concept aimed for generation of antibodies specific for cDNAencoded proteins2002Ingår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 35, nr 2, s. 75-82Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A novel dual bacterial expression system, designed for high-throughput generation of antibodies specific for cDNA-encoded proteins, is presented. The concept involves parallel expression of cDNA-encoded proteins, in two vector systems, as fusions with two different tags, both enabling single-step affinity purification under denaturing conditions. One of the fusion tags includes a portion with documented immunopotentiating effect to stimulate antibody production, and the generated fusion proteins are used to elicit antibodies. The second fusion protein is used in an immobilized form as an affinity ligand to enrich, from the generated antisera, antibodies with selective reactivity to the cDNA-encoded part. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The five antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the five antibodies gave specific staining in Western-blot screening of various cell types and tissue homogenates. When the same five cDNAs were processed and analysed using a single-vector method, antibodies with a more non-specific staining were generated. We thus conclude that the presented dual-vector method offers a highly stringent strategy for generation of monospecific polyclonal antibodies.

  • 28. Falk, Ronny
    et al.
    Ramstrom, Margareta
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Approaches for systematic proteome exploration2007Ingår i: Biomolecular Engineering, ISSN 1389-0344, E-ISSN 1878-559X, Vol. 24, nr 2, s. 155-168Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    With the completion of the human genome project (HUGO) during recent years, gene function, protein abundance and expression patterns in tissues and cell types have emerged as central areas for the scientific community. A mapped human proteome will extend the value of the genome sequence and large-scale efforts aiming at elucidating protein localization, abundance and function are invaluable for biomarker and drug discovery. This research area, termed proteomics, is more demanding than any genome sequencing effort and to perform this on a wide scale is a highly diverse task. Therefore, the proteornics field employs a range of methods to examine different aspects of proteomics including protein localization, protein-protein interactions, posttranslational modifications and alteration of protein composition (e.g. differential expression) in tissues and body fluids. Here, some of the most commonly used methods, including chromatographic separations together with mass spectrometry and a number of affinity proteomics concepts are discussed and exemplified.

  • 29.
    Fleetwood, Filippa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Andersson, Ken A.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    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 strains2014Ingår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 13, s. 179-Artikel i tidskrift (Refereegranskat)
    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.

  • 30.
    Fleetwood, Filippa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Andersson, Ken
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Development And Optimization Of An e.Coli-based Display Platform For Selection Of Affinity Proteins2014Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 23, s. 135-135Artikel i tidskrift (Övrigt vetenskapligt)
  • 31.
    Fleetwood, Filippa
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Devoogdt, Nick
    Pellis, Mireille
    Wernery, Ulrich
    Muyldermans, Serge
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Surface display of a single-domain antibody library on Gram-positive bacteria2013Ingår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 70, nr 6, s. 1081-1093Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

  • 32.
    Fleetwood, Filippa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, Fredrik
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Efficient blocking of VEGFR2-mediated signaling using biparatopic Affibody constructsManuskript (preprint) (Övrigt vetenskapligt)
  • 33.
    Fleetwood, Filippa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Güler, Rezan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gordon, Emma
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Claesson-Welsh, Lena
    Löfblom, John
    Novel affinity binders for neutralization of vascular endothelial growth factor (VEGF) signaling2016Ingår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 73, nr 8, s. 1671-1683Artikel i tidskrift (Refereegranskat)
    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.

  • 34.
    Fleetwood, Filippa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Klint, Susanne
    Hanze, Martin
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gunneriusson, Elin
    Frejd, Fredrik
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity2014Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 4, s. 7518-Artikel i tidskrift (Refereegranskat)
    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.

  • 35.
    Friedman, Mikaela
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Lindström, Sara
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule2009Ingår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, s. 121-131Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

  • 36.
    Friedman, Mikaela
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Nordberg, Erika
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Adams, Gregory P.
    Fox Chase Canc Ctr, Dept Med Oncol, Philadelphia.
    Nilsson, Fredrik Y.
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Carlsson, Jörgen
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor2007Ingår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, nr 4, s. 189-199Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by Phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 2550 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents; for radionuclide based imaging applications in various carcinomas ils discussed.

  • 37.
    Friedman, Mikaela
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Orlova, Anna
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Johansson, Eva
    Affibody AB, Bromma.
    Eriksson, Tove L. J.
    Affibody AB, Bromma.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Tolmachev, Vladimir
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Nilsson, Fredrik Y.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding Affibody molecule2008Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, nr 5, s. 1388-1402Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K-d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, Kd, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K-d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.

  • 38.
    Friedman, Mikaela
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Engineered affinity proteins for tumour-targeting applications2009Ingår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 53, s. 1-29Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Targeting of tumour-associated antigens is an expanding treatment modality in clinical oncology as an alternative to, or in combination with, conventional treatments, such as chemotherapy, external-radiation therapy and surgery. Targeting of antigens that are unique or more highly expressed in tumours than in normal tissues can be used to increase the specificity and reduce the cytotoxic effect on normal tissues. Several targeting agents have been studied for clinical use, where monoclonal antibodies have been the ones most widely used. More than 20 monoclonal antibodies are approved for therapy today and the largest field is oncology. Advances in genetic engineering and in vitro selection technology has enabled the feasible high-throughput generation of monoclonal antibodies, antibody derivatives [e.g. scFvs, Fab molecules, dAbs (single-domain antibodies), diabodies and minibodies] and more recently also non-immunoglobulin scaffold proteins. Several of these affinity proteins have been investigated for both in vivo diagnostics and therapy. Affinity proteins in tumour-targeted therapy can affect tumour progression by altering signal transduction or by delivering a payload of toxin, drug or radionuclide. The ErbB receptor family has been extensively studied as biomarkers in tumour targeting, primarily for therapy using monoclonal antibodies. Two receptors in the ErbB family, EGFR (epidermal growth factor receptor) and HER2 (epidermal growth factor receptor 2), are over-expressed in various malignancies and associated with poor patient prognosis and are therefore interesting targets for solid turnours. In the present review, strategies are described for tumour targeting of solid turnours using affinity proteins to deliver radionuclides, either for molecular imaging or radiotherapy. Antibodies, antibody derivatives and non-immunoglobulin scaffold proteins are discussed with a certain focus on the affibody (Affibody (R)) molecule.

  • 39. Garousi, J.
    et al.
    Anderson, Ken
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Dam, J. H.
    Olsen, B. B.
    Orlova, A.
    Buijs, J.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Thisgaard, H.
    Tolmachev, V.
    The use of radiocobalt as a label improves PET imaging of EGFR using DOTA-conjugated affibody molecules2015Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, s. S244-S244Artikel i tidskrift (Refereegranskat)
  • 40. Garousi, Javad
    et al.
    Anderson, Ken G.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Mitran, Bogdan
    Pichl, Marie-Louise
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Orlova, Anna
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, Vladimir
    PET imaging of epidermal growth factor receptor expression in tumours using Zr-89-labelled ZEGFR:2377 affibody molecules2016Ingår i: International Journal of Oncology, ISSN 1019-6439, Vol. 48, nr 4, s. 1325-1332Artikel i tidskrift (Refereegranskat)
    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.

  • 41. Garousi, Javad
    et al.
    Andersson, Ken G.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Dam, Johan H.
    Olsen, Birgitte B.
    Mitran, Bogdan
    Orlova, Anna
    Buijs, Jos
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Thisgaard, Helge
    Tolmachev, Vladimir
    The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 5961Artikel i tidskrift (Refereegranskat)
    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.

  • 42. Goetsch, L.
    et al.
    Plotnicky-Gilquin, H.
    Champion, T.
    Beck, A.
    Corvaia, N.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Bonnefoy, J. Y.
    Nguyen, T. N.
    Power, U. F.
    Influence of administration dose and route on the immunogenicity and protective efficacy of BBG2Na, a recombinant respiratory syncytial virus subunit vaccine candidate2000Ingår i: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 18, nr 24, s. 2735-2742Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The immunogenicity and protective efficacy of BBG2Na, a novel recombinant respiratory syncytial virus subunit vaccine candidate, was assessed in BALB/c mice under various conditions of dose, administration route and number of immunisations. A single intra-peritoneal (i.p.) dose of 2 mu g, or two doses of 0.2 mu g, were sufficient to induce elevated RSV-A serum antibodies and sterilising lung protective immunity. Serum antibody titres were significantly boosted following second immunisations, but not a third. Of three routes of immunisation, i.p. induced the highest RSV-A antibody titres, followed in efficacy by the intramuscular (i.m.) and subcutaneous (s.c.) routes. Nonetheless, all three routes induced comparable and sterilising lung protection. In contrast, upper respiratory tract protection was observed only after i.p. vaccination, although significant viral titre reductions were evident following i.m. or s.c. immunisations. Interestingly, Pepscan analyses indicated that antibody epitope usage was highest in i.p. and lowest in i.m. immunised mice, respectively. Nonetheless, all routes resulted in antibody responses to known lung protective epitopes (protectopes). Thus, the prevention of serious lower respiratory tract disease, the principle goal of a RSV vaccine, but not URT infection, is dose dependent but unlikely to be influenced by the route of BBG2Na administration.

  • 43. Graslund, S.
    et al.
    Larsson, M.
    Falk, R.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hoog, C.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Single-vector three-frame expression systems for affinity-tagged proteins2002Ingår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 215, nr 1, s. 139-147Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.

  • 44.
    Gräslund, Susanne
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Eklund, Malin
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Falk, Ronny
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygen, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products2002Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Journal of biotechnology, Vol. 99, nr 1, s. 41-50Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.

  • 45.
    Grönwall, Caroline
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Jonsson, Andreas
    Affibody AB, Bromma.
    Lindström, Sara
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Gunneriusson, Elin
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Herne, Nina
    Affibody AB, Bromma.
    Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides2007Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 128, nr 1, s. 162-183Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.

  • 46.
    Grönwall, Caroline
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi. KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sjöberg, Anna
    Affibody AB, Bromma.
    Ramström, Margareta
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi. KTH, Skolan för bioteknologi (BIO), Proteomik.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi. KTH, Skolan för bioteknologi (BIO), Proteomik.
    Jonasson, Per
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi. KTH, Skolan för bioteknologi (BIO), Proteomik.
    Affibody-mediated transferrin depletion for proteomics applications2007Ingår i: Biotechnology Journal, ISSN 1860-6768, Vol. 2, nr 11, s. 1389-1398Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An Affibody® (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.

  • 47.
    Grönwall, Caroline
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Snelders, Eveline
    Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Hospital.
    Jarelöv Palm, Anna
    Eriksson, Fredrik
    Department of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Hospital.
    Herne, Nina
    Affibody AB, Bromma.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Generation of Affibody (R) ligands binding interieukin-2 receptor alpha/CD252008Ingår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 50, nr 2, s. 97-112Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affibody (R) molecules specific for human IL-2R alpha, the IL-2 (interieukin-2) receptor a subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody (R) molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody (R) molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody (R) molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody (R) molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody (R) molecules bound to CD4(+) CD25(+) PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody (R) molecules as targeting agents for medical imaging and for therapeutic applications is discussed.

  • 48.
    Grönwall, Caroline
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Engineered affinity proteins-Generation and applications2009Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 140, nr 3-4, s. 254-269Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The use of combinatorial protein engineering to design proteins with novel binding specificities and desired properties has evolved into a powerful technology, resulting in the recent advances in protein library selection strategies and the emerge of a variety of new engineered affinity proteins. The need for different protein library selection methods is due to that each target protein pose different challenges in terms of its availability and inherent properties. At present, alternative engineered affinity proteins are starting to complement and even challenge the classical immunoglobulins in different applications in biotechnology and potentially also for in vivo use as imaging agents or as biotherapeutics. This review article covers the generation and use of affinity proteins generated through combinatorial protein engineering. The most commonly used selection techniques for isolation of desired variants from large protein libraries are described. Different antibody derivatives, as well as a variety of the most validated engineered protein scaffolds, are discussed. In addition, we provide an overview of some of the major present and future applications for these engineered affinity proteins in biotechnology and medicine.

  • 49. Gulich, S.
    et al.
    Linhult, M.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Engineering streptococcal protein G for increased alkaline stability2002Ingår i: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 15, nr 10, s. 835-842Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most protein-based affinity chromatography media are very sensitive towards alkaline treatment, which is a preferred method for regeneration and removal of contaminants from the purification devices in industrial applications. In a previous study, we concluded that a simple and straightforward strategy consisting of replacing asparagine residues could improve the stability towards alkaline conditions. In this study, we have shown the potential of this rationale by stabilizing an IgG-binding domain of streptococcal protein G, i.e. the C2 domain. In order to analyze the contribution of the different amino acids to the alkaline sensitivity of the domain we used a single point mutation strategy. Amino acids known to be susceptible towards high pH, asparagine and glutamine, were substituted for less-alkali-susceptible residues. In addition, aspartic acid residues were mutated to evaluate if the stability could be further increased. The stability of the different C2 variants was subsequently analyzed by exposing them to NaOH. The obtained results reveal that the most sensitive amino acid towards alkaline conditions in the structure of C2 is Asn36. The double mutant, C2(N7,36A), was found to be the most stable mutant constructed. In addition to the increased alkaline stability and also very important for potential use as an affinity ligand, this mutated variant also retains the secondary structure, as well as the affinity to the Fc fragment of IgG.

  • 50.
    Göstring, Lovisa
    et al.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Frejd, Fredrik Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Gedda, Lars
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala Sweden.
    Cellular Effects of HER3-Specific Affibody Molecules2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 6, s. e40023-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recent studies 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. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.

123 1 - 50 av 136
RefereraExporteraLänk till träfflistan
Permanent länk
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf