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  • 1.
    Alm, Tove
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilvebrant, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Engineering bispecificityinto a single albumin-binding domain aimed for drug-targeting and extended in vivo half-life extension.Manuskript (preprint) (Övrigt vetenskapligt)
  • 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.
    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.

  • 8.
    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.

  • 9.
    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.

  • 10.
    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.

  • 11.
    Andersson, Ken G.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Sjöstrand, Nanna
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Coupled release and site-specific conjugation of Affibody molecules from the surface of E. coli using Sortase AManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

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

  • 12.
    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)
  • 13.
    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.

  • 14.
    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.

  • 15. 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)
  • 16.
    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.

  • 17. Deyev, S.
    et al.
    Vorobyeva, A.
    Schulga, A.
    Proshkina, G.
    Güler, Rezan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Mitran, B.
    Garousi, J.
    Altai, M.
    Buijs, J.
    Chernov, V.
    Orlova, A.
    Tolmachev, V.
    Comparative Evaluation of Two DARPin Variants: Effect of Affinity, Size, and Label on Tumor Targeting Properties2019Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 16, nr 3, s. 995-1008Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9-29-H 6 and G3-H 6 were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H 6 was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H 6 in comparison to 9-29-H 6 . Technetium-99m labeled G3-H 6 demonstrated a better biodistribution profile than 9-29-H 6 , with several-fold lower uptake in liver. Radioiodinated G3-H 6 showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H 6 with high clinical potential for imaging of HER2.

  • 18.
    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.

  • 19.
    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)
  • 20.
    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.

  • 21.
    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)
  • 22.
    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.

  • 23.
    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.

  • 24. 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.

  • 25. 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.

  • 26. Garousi, Javad
    et al.
    Honarvar, Hadis
    Andersson, Ken G.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Mitran, Bogdan
    Orlova, Anna
    Buijs, Jos
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, Fredrik Y.
    Tolmachev, Vladimir
    Comparative Evaluation of Affibody Molecules for Radionuclide Imaging of in Vivo Expression of Carbonic Anhydrase IX2016Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 13, nr 11, s. 3676-3687Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 27.
    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.

  • 28.
    Güler, Rezan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Thatikonda, Naresh
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Ghani, Hawraa Ali
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Artificial VEGFR2-Specific Growth Factors Demonstrate Agonistic Effects in Both Soluble Form and When Immobilized Via Spider SilkManuskript (preprint) (Övrigt vetenskapligt)
  • 29.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Fernandez, Carmen Diez
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Johannesson, Henrik
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase2010Ingår i: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, nr 2, s. 129-137Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a need to characterize the epitopes of affinity reagents to develop high quality affinity reagents for research, diagnostics and therapy. Here, we describe the analysis of epitopes of antibodies generated toward human tryptophanyl-tRNA synthetase (WARS) using both combinatorial bacterial display and suspension bead array. The bacterial display revealed that the polyclonal antibody binds to three separate epitopes and peptide scanning using 15-mers revealed binding to a 13 amino acid consensus sequence (ELINRIERATGQR). A mouse monoclonal antibody was generated and the mapping approach revealed binding toward a slightly shifted position of the same epitope. Structural analysis showed that the antibodies bind to a-helical regions on the surface of the target protein. An alanine-scanning experiment showed binding to four specific residues. The implications for the systematic analysis of antibody epitopes on the basis of these results are discussed.

  • 30.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Immunizations of inbred rabbits using the same antigen yield antibodies with similar, but not identical, epitopesManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties to obtain a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of in-bred rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen gene on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several in-bred rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 31.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 12, s. e45817-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 32.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Severa, Denise
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display2008Ingår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 278, nr 1, s. 128-136Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.

  • 33.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Jonsson, Andreas
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry2008Ingår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, nr 4, s. 247-255Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 109 variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (106 variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.

  • 34.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Göstring, Lovisa
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Gunneriusson, Elin
    Nilsson, Martin
    Affibody AB, Stockholm, Sweden.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Gedda, Lars
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala 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).
    Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules2011Ingår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 24, nr 4, s. 385-396Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.

  • 35.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies2010Ingår i: Recent Patents on Biotechnology, ISSN 1872-2083, Vol. 4, nr 3, s. 171-182Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.

  • 36.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    New Ways for Discovery of Biopharmaceuticals: Emerging Techniques using Surface Display on Gram-positive Bacteria for Combinatorial Protein Engineering and Characterization2009Ingår i: Bioforum Europe, ISSN 1611-597X, Vol. 13, nr 6-7, s. 022-Artikel i tidskrift (Refereegranskat)
  • 37.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Härd, Torleif
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity2015Ingår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, nr 11, s. 1707-1718Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.

  • 38.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Johansson, Anna
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Härd, Torleif
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide2013Ingår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 8, nr 1, s. 139-145Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have previously generated an affibody molecule for the disease-associated amyloid beta (A beta) peptide, which has been shown to inhibit the formation of various A beta aggregates and revert the neurotoxicity of A beta in a fruit fly model of Alzheimer's disease. In this study, we have investigated a new bacterial display system for combinatorial protein engineering of the A beta-binder as a head-to-tail dimeric construct for future optimization efforts, e.g. affinity maturation. Using the bacterial display platform, we have: (i) demonstrated functional expression of the dimeric binder on the cell surface, (ii) determined the affinity and investigated the pH sensitivity of the interaction, (iii) demonstrated the importance of an intramolecular disulfide bond through selections from a cell-displayed combinatorial library, as well as (iv) investigated the effects from rational truncation of the N-terminal part of the affibody molecule on surface expression level and A beta binding. Overall, the detailed engineering and characterization of this promising A beta-specific affibody molecule have yielded valuable insights concerning its unusual binding mechanism. The results also demonstrated that our bacterial display system is a suitable technology for future protein engineering and characterization efforts of homo- or heterodimeric affinity proteins.

  • 39.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Sandersjöö, Lisa
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Meister, S. W.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method2017Ingår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 12, nr 1, artikel-id 1600364Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aggregation of misfolded peptides and proteins is a key event in several neurodegenerative diseases. Suggested treatments of such disorders aim to inhibit the initial aggregation process. Here, we have developed an intracellular, function-based screening method, intended for isolation of aggregation-inhibitors from combinatorial protein libraries by flow-cytometric cell sorting. The method is based on fusion of aggregation-prone peptides to a fluorescent protein, functioning as a solubility reporter. Co-expression of a protein-based aggregation-inhibitor should prevent aggregation and thus increase the whole-cell fluorescence. We evaluated the method using the aggregation-prone Alzheimer's-related amyloid-β (Aβ) peptide in fusion to green-fluorescent protein (GFP), and an Aβ aggregation-inhibiting Affibody molecule. To adapt the method for library applications, the inhibitor was linked to an mCherry reporter for normalization of protein expression levels. We found that aggregation propensity correlates with fluorescence intensity, as co-expression of the Affibody-inhibitor increased the whole-cell fluorescence relative to a non-inhibitor. Employing improved cultivation parameters, we furthermore demonstrated efficient rescue from aggregation of an α-synuclein-derived protein using a different type of aggregation-inhibitor. Importantly, we also showed that the Aβ aggregation-inhibiting Affibody molecule could be isolated from a 1:10,000 background of non-inhibitors, with around 3,500-fold enrichment, in one cycle of fluorescence-based cell sorting. In conclusion, our new method represents a promising approach for generation of novel protein-based aggregation-inhibitors.

  • 40.
    Lindberg, Hanna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Sandersjöö, Lisa
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Development of a fluorescence-based intracellular method for function-based isolation of protein-based aggregation inhibitorsManuskript (preprint) (Övrigt vetenskapligt)
  • 41.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Affibody molecules binding to the ErbB3 receptorPatent (Övrig (populärvetenskap, debatt, mm))
  • 42.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Bacterial display in combinatorial protein engineering2011Ingår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, nr 9, s. 1115-1129Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Technologies for display of recombinant protein libraries are today essential tools in many research-intensive fields, such as in the drug discovery processes of biopharmaceutical development. Phage display is still the most widely used method, but alternative systems are available and are becoming increasingly popular. The most rapidly expanding of the alternative systems are the cell display-based technologies, offering innovative strategies for selection and characterization of affinity proteins. Most investigations have focused on eukaryotic yeast for display of protein libraries, but similar systems are also being developed using prokaryotic hosts. This review summarizes the field of bacterial surface display with a strong emphasis on library applications for generation of new affinity proteins. The main focus will be on the most recent progress of the work on primarily Escherichia coli, but also on studies using a recently developed system for display on Gram-positive Staphylococcus carnosus. In addition, general strategies for combinatorial protein engineering using cell display are discussed along with the latest developments of new methodologies with comparisons to mainly phage display technology.

  • 43.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Bacterial display in directed evolution for generation of new biopharmaceuticals2011Ingår i: Biotech International, ISSN 2032-2887, Vol. 23, nr June, s. 26-29Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    The successful use of monoclonal antibodies and antibody derivatives for therapeutic and in vivo diagnostic applications has resulted in a rapid progression in the fields surrounding biopharmaceutical drug discovery and combinatorial protein engineering. Consequently, investigations on new and improved technologies for both generation and characterisation of specific antibodies and alternative protein scaffolds are continuously being reported in the literature. This review summarises the recent efforts in development and application of bacterial display platforms for such purposes.

  • 44.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO).
    Staphylococcal surface display for protein engineering and characterization2007Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution.

    Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering.

    In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins.

    Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization.

  • 45.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Feldwisch, J.
    Tolmachev, V.
    Carlsson, J.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Frejd, F. Y.
    Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications2010Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 584, nr 12, s. 2670-2680Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

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

  • 46.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Feldwisch, J.
    Affibody AB, Stockholm, Sweden..
    Tolmachev, V.
    Uppsala Univ, Rudbeck Lab, Dept Oncol Radiol & Clin Immunol, Uppsala, Sweden..
    Carlsson, J.
    Uppsala Univ, Rudbeck Lab, Dept Oncol Radiol & Clin Immunol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, F. Y.
    Affibody AB, Stockholm, Sweden..
    Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, s. 31-31Artikel i tidskrift (Övrigt vetenskapligt)
  • 47.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Frejd, Fredrik
    Alternative Scaffolds as Bispecific Antibody Mimetics2011Ingår i: Bispecific Antibodies / [ed] Roland Kontermann, Berlin Heidelberg: Springer-Verlag , 2011Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    The use of non-immunoglobulin-based protein scaffolds for engineering of specific recognition was first described some 15 years ago and has matured as a discipline in parallel with the rapidly expanding monoclonal antibody field. As bispecific antibodies and antibody fragments have come into focus lately, the corresponding development of bispecific alternative scaffolds is also emerging. Here, the concept of alternative scaffold proteins is introduced along with comparisons to the antibody and antibody derivatives counterparts. Although the field of bispecifics is anticipated to grow rapidly in the near future, relatively few examples of bispecific binders based on alternative protein scaffolds are reported in the literature. This chapter will present an overview of work that have been published, including a brief introduction to the particular molecular scaffolds with the structural basis and some of the biophysical and biochemical properties before describing the bispecific application.

  • 48.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Frejd, Fredrik Y.
    Uppsala University.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Non-immunoglobulin based protein scaffolds2011Ingår i: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 22, nr 6, s. 843-848Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Non-immunoglobulin based protein scaffolds have been reported as promising alternatives to traditional monoclonal antibodies for over a decade and are often mentioned as part of the next-generation immunotherapeutics. Today, this class of biologics is beginning to demonstrate its potential for therapeutic applications and several are currently in preclinical or clinical development. A common denominator for most of these new scaffolds is the attractive properties that differentiate them from monoclonal antibodies including small size, cysteine-free sequence, flexible pharmacokinetic properties, and ease of generating multispecific molecules. In addition to therapeutic applications, substantial evidence point to superior performance of several of these scaffolds in molecular imaging compared to full-length antibodies. Here we review the most recent progress using alternative protein scaffolds for therapy and medical imaging.

  • 49.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Kronqvist, Nina
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Wernérus, Henrik
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism2007Ingår i: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 102, nr 3, s. 736-747Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The study was conducted with an aim to optimize the transformation efficiency of the Gram-positive bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface displayed combinatorial protein libraries.

    Methods and Results: We have thoroughly investigated a number of different parameters for: (i) the preparation of electrocompetent cells; (ii) the treatment of cells before electroporation; (iii) the electroporation step itself; and (iv) improved recovery of transformed cells. Furthermore, a method for heat-induced inactivation of the host cell restriction system was devised to allow efficient transformation of the staphylococci with DNA prepared from other species, such as Escherichia coli. Previously described protocols for S. carnosus, giving transformation frequencies of approximately 10(2) transformants per transformation could be improved to reproducible procedures giving around 10(6) transformants for a single electroporation event, using plasmid DNA prepared from either S. carnosus or E. coli. The transformed staphylococcal cells were analysed using flow cytometry to verify that the entire cell population retained the introduced plasmid DNA and expressed the recombinant protein in a functional form on the cell surface at the same level as the positive control population.

    Conclusions: The results demonstrate that the transformation frequency for S. carnosus could be dramatically increased through optimization of the entire electroporation process, and that the restriction barrier for interspecies DNA transfer, could be inactivated by heat treatment of the cells prior to electroporation.

    Significance and Impact of the Study: The generation of large combinatorial protein libraries, displayed on the surface of S. carnosus can be envisioned in the near future, thus dramatically improving the selection compared with the traditional biopanning procedure used in phage display.

  • 50.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Rosenstein, Ralf
    Nguyen, Minh-Thu
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Götz, Friedrich
    Staphylococcus carnosus: from starter culture to protein engineering platform2017Ingår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 101, nr 23-24, s. 8293-8307Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and "food grade" species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus.

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