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  • 1. Garousi, J.
    et al.
    Lindbo, S.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Honarvar, H.
    Velletta, J.
    Mitran, B.
    Altai, M.
    Orlova, A.
    Tolmachev, V.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Influence of the N-terminal amino acid sequence on imaging properties of In-111-labeled anti-HER2 scaffold protein ADAPT62016Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, s. S55-S55Artikkel i tidsskrift (Fagfellevurdert)
  • 2.
    Garousi, J.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Mitran, B.
    Uppsala Univ, Uppsala, Sweden..
    Vorobyeva, A.
    Uppsala Univ, Uppsala, Sweden..
    Oroujeni, M.
    Uppsala Univ, Uppsala, Sweden..
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Selection of the most optimal ADAPT6-based probe for imaging of HER2 using PET and SPECT2018Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, s. S77-S78Artikkel i tidsskrift (Annet vitenskapelig)
  • 3. Garousi, J.
    et al.
    Lindbo, Sarah
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Orlova, A.
    Åstrand, Mikael
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Nilvebrant, Johan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Buijs, J.
    Sandstrom, M.
    Honarvar, H.
    Tolmachev, V.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Development of ADAPT6 as a new scaffold protein for radionuclide molecular imaging2014Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, s. S309-S309Artikkel i tidsskrift (Fagfellevurdert)
  • 4.
    Garousi, Javad
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Borin, Jesper
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    von Witting, Emma
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Oroujeni, Maryam
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Buijs, Jos
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours2019Inngår i: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 134, s. 37-48Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)(2)- (DiADAPT6L2), and -(SSSG)(3)- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with In-111 and I-125, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization.

  • 5. Garousi, Javad
    et al.
    Lindbo, Sarah
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Honarvar, Hadis
    Velletta, Justin
    Mitran, Bogdan
    Altai, Mohamed
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Influence of the N -Terminal Composition on Targeting Properties of Radiometal-Labeled Anti-HER2 Scaffold Protein ADAPT62016Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, nr 11, s. 2678-2688Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Radionuclide-imaging-based stratification of patients to targeted therapies makes cancer treatment more personalized and therefore more efficient. Albumin-binding domain derived affinity proteins (ADAPTs) constitute a novel group of imaging probes based on the scaffold of an albumin binding domain (ABD). To evaluate how different compositions of the N-terminal sequence of ADAPTS influence their biodistribution, a series of human epidermal growth factor receptor type 2 (HER2)-binding ADAPT6 derivatives with different N-terminal sequences were created: GCH(6)DANS (2), GC(HE)(3)DANS (3), GCDEAVDANS (4), and GCVD.ANS(5). These were compared with the parental variant: GCSS(HE)(3)DEAVDANS (1). All variants were site-specifically conjugated with a maleimido-derivative of a DOTA chelator and labeled with In-III. Binding to HER2-expressing cells in vitro, in vivo biodistribution as well as targeting properties of the new variants were compared with properties of the In-III-labeled parental ADAPT variant 1 (In-III-DOTA-1). The composition of the N-terminal sequence had an apparent influence on biodistribution of ADAPT6 in mice. The use of a hexahistidine tag in (InD)-In-III-OTA-2 was associated with elevated hepatic uptake compared to the (HE)(3)-containing counterpart, In-III-DOTA-3. All new variants without a hexahistidine tag demonstrated lower uptake in blood, lung, spleen, and muscle compared to uptake in the parental variant. The best new variants, In-III-DOTA-3 and In-III-DOTA-5, provided tumor uptakes of 14.6 +/- 2.4 and 12.5 +/- 1.3% ID/g at 4 h after injection, respectively. The tumor uptake of In-III-DOTA-3 was significantly higher than the uptake of the parental In-III-DOTA-1 (9.1 +/- 2.0% ID/g). The tumor-to-blood ratios of 395 +/- 75 and 419 +/- 91 at 4 h after injection were obtained for In-III-DOTA-5 and (IIII)n-DOTA-3, respectively. In conclusion, the N-terminal sequence composition affects the biodistribution and targeting properties of ADAPT-based imaging probes, and its optimization may improve imaging contrast.

  • 6. Garousi, Javad
    et al.
    Lindbo, Sarah
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Mitran, Bogdan
    Buijs, Jos
    Vorobyeva, Anzhelika
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Comparative evaluation of tumor targeting using the anti-HER2 ADAPT scaffold protein labeled at the C-terminus with indium-111 or technetium-99m2017Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikkel-id 14780Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    ABD-Derived Affinity Proteins (ADAPTs) is a novel class of engineered scaffold proteins derived from an albumin-binding domain of protein G. The use of ADAPT6 derivatives as targeting moiety have provided excellent preclinical radionuclide imaging of human epidermal growth factor 2 (HER2) tumor xenografts. Previous studies have demonstrated that selection of nuclide and chelator for its conjugation has an appreciable effect on imaging properties of scaffold proteins. In this study we performed a comparative evaluation of the anti-HER2 ADAPT having an aspartate-glutamate-alanine-valine-aspartate-alanine-asparagine-serine (DEAVDANS) N-terminal sequence and labeled at C-terminus with (99)mTc using a cysteine-containing peptide based chelator, glycine-serine-serine-cysteine (GSSC), and a similar variant labeled with In-111 using a maleimido derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Both (99)mTc-DEAVDANS-ADAPT6-GSSC and In-111-DEAVDANS-ADAPT6-GSSC-DOTA accumulated specifically in HER2-expressing SKOV3 xenografts. The tumor uptake of both variants did not differ significantly and average values were in the range of 19-21% ID/g. However, there was an appreciable variation in uptake of conjugates in normal tissues that resulted in a notable difference in the tumor-to-organ ratios. The In-111-DOTA label provided 2-6 fold higher tumor-to-organ ratios than (99)mTc-GSSC and is therefore the preferable label for ADAPTs.

  • 7. Garousi, Javad
    et al.
    Lindbo, Sarah
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Nilvebrant, Johan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Åstrand, Mikael
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Buijs, Jos
    Sandstrom, Mattias
    Honarvar, Hadis
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers2015Inngår i: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 75, nr 20, s. 4364-4371Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, In-111 for SPECT imaging and Ga-68 for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule In-111/Ga-68-DOTA(HE) 3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.

  • 8.
    Hober, Sophia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Nilvebrant, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Bispecific applications of non-immunoglobulin scaffold binders2019Inngår i: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 154, s. 143-152Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Non-immunoglobulin scaffolds represent a proven group of small affinity proteins that can be engineered in vitro to similar affinity and potency as monoclonal antibodies. Several novel candidate biotherapeutics that exploit the potential advantages scaffold proteins hold over larger and more complex antibodies have been developed over the past decade. The ease of using small and robust binding proteins as flexible and modular building blocks has led to the development of a wide range of innovative approaches to combine them in various bi- and multispecific formats. This progress is expected to aid the ongoing challenge of identifying niche applications where clear differentiation from antibody-based molecules will be key to success. Given the many engineering options that are available for non-immunoglobulin scaffold proteins, they have potential to not only complement but probably also surpass antibodies in certain applications.

  • 9.
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Generation and engineering of ABD-derived affinity proteins for clinical applications2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Proteins that specifically recognize and bind to other molecules or structures are important tools in industrial and medical applications. Binding proteins engineered from small stable scaffold proteins have been utilized for several purposes due to their favorable biophysical properties, tolerance to mutagenesis, efficient tissue penetration and ease of production. The 46 amino acid long albumin-binding domain (ABD) derived from the bacterial receptor Protein G is a promising scaffold that has been explored in this thesis. The scaffold was subjected to combinatorial protein engineering for generation of ABD-derived binding proteins with novel specificities. Furthermore, the medical potential of engineered ABD- derived affinity proteins (ADAPTs) was evaluated in a series of pre-clinical studies.

    In the first studies, ADAPTs suitability as tracers for radionuclide molecular imaging was evaluated. Factors influencing biodistribution and tumor targeting properties were assessed in mice models bearing HER2 positive xenografts. All tested ADAPT constructs demonstrated high and specific targeting of HER2-expressing tumor cells as well as fast clearance from circulation. The results also showed that the size and character of the N- terminus affected the biodistribution profile of ADAPTs. Moreover, the targeting properties of ADAPTs proved to be highly influenced by the residualizing properties of the attached radionuclide label. Taken together, the results provided the first evidence that tumor imaging can be performed using ADAPTs and the favorable pharmacokinetic profiles in the studied mice models suggest that the scaffold is a promising candidate for clinical applications.

    In the last study, a platform for generation of stable ABD-derived affinity proteins with novel binding specificities was established using a multi-step approach combining directed evolution and rational protein design. A broad combinatorial protein library with 20 randomized positions in ABD was designed and binders against three distinct targets were selected using phage display. Characterization of the selected binders provided information regarding optimal positions to randomize in a final library. In addition, the isolated binders were subjected to mutagenesis in certain surface exposed positions and mutations that provided increased stability were introduced into the original scaffold. Finally, a more focused combinatorial protein library consisting of 11 randomized positions was designed and constructed. The library was validated by selections against the same set of targets as for the first, broad library. The isolation of highly stable affinity ligands confirms that the library can be used for generation of diverse and stable affinity molecules.

  • 10.
    Lindbo, Sarah
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Garousi, J.
    Åstrand, Mikael
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Honarvar, H.
    Orlova, A.
    Hober, S.
    Tolmachev, V.
    Influence of Histidine-Containing Tags on Biodistribution of Radiolabelled ADAPT-Based Imaging Probes2015Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, s. S100-S100Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aim. ADAPTs are a class of small ( ∼ 5 kDa) robust scaffold proteins suitable as probes for radionuclide molecular imaging in vivo. The attachment of a histidine-containing tags to the scaffold proteins allows efficient purification by immobilized metal ion affinity chromatography (IMAC) and permits labelling with 99mTc(CO)3. Earlier, we have demonstrated that replacement of the hexahistidine (H6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HE3)-tag reduces hepatic uptake of radiolabelled affibody molecules. The same effect has been confirmed for other scaffold proteins, DARPins, and short peptides. The aim of this study was to evaluate effect of histidine-containing tag composition on biodistribution of ADAPTs. Material and methods. A series of anti-HER2 ADAPT6 probes having DEAVDANS lead sequence and H6- or HE3-tags at N-termini has been prepared. In two variants, maleimido derivative of DOTA was conjugated to a unique cysteine was incorporated at N-terminus. DOTA-C-HE3-ADAPT6 and DOTA-C-H6-ADAPT6 were labelled with 111In. HE3-ADAPT6 and H6-ADAPT6 were labelled with 99mTc(CO)3using IsoLink kit. Binding specificity, affinity, and cellular processing of new conjugates was evaluated using HER2-expressing SKOV-3 ovarian carcinoma cells. Biodistribution at 1,4 and 24 h p.i. was evaluated in normal NMRI mice. Tumour-targeting properties of the best 99mTc(CO) 3-labelled variant, 99mTc(CO)3-H6-ADAPT6 were evaluated in BALB/C nu/nu mice bearing SKOV-3 xenografts. Results. All radiolabeled ADAPTs demonstrated specific binding to SKOV-3 cells with affinities in the range of 1.1-2.8 nM. The internalization by SKOV-3 cells was slow. In vivo, all conjugates cleared rapidly from blood via kidneys with subsequent renal re-absorption. The hepatic uptake of 111In-DOTA-C-H6-ADAPT6 was slightly but significantly (p<0.05) higher that the uptake of 111In-DOTA-C-HE3-ADAPT6 at 1 h pi, but biodistribution was very similar at later time points. Surprisingly, the uptake of 99mTc(CO)3-HE3-ADAPT6 was significantly (p<0.05) higher than uptake of 99mTc(CO)3-H6-ADAPT6 in liver, blood, and bone at 1h p.i. At 4 h p.i., hepatic uptakes were equal, but 99mTc(CO)3-H6-ADAPT6 provided lower uptake in blood and bone. 99mTc(CO)3-H6-ADAPT6 demonstrated high (19? 3 %ID/g at 4 h p.i.) and specific tumour uptake. Tumour-to-blood and tumour-to-liver ratios were 102? 29 and 12? 3, respectively. Conclusion. Influence of histidine-containing tag on biodistribution of scaffold proteins depends on composition of a scaffold protein and might differ appreciably. This should be take into account in molecular design of imaging probes based on engineered scaffold proteins

  • 11.
    Lindbo, Sarah
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Garousi, Javad
    Mitran, Bogdan
    Altai, Mohamed
    Buijs, Jos
    Orlova, Anna
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, Vladimir
    Radionuclide Tumor Targeting Using ADAPT Scaffold Proteins: Aspects of Label Positioning and Residualizing Properties of the Label2018Inngår i: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 59, nr 1, s. 93-99Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Visualization of cancer-associated alterations of molecular phenotype using radionuclide imaging is a noninvasive approach to stratifying patients for targeted therapies. The engineered albumin-binding domain-derived affinity protein (ADAPT) is a promising tracer for radionuclide molecular imaging because of its small size (6.5 kDa), which satisfies the precondition for efficient tumor penetration and rapid clearance. Previous studies demonstrated that the human epidermal growth factor receptor type 2 (HER2)-targeting ADAPT6 labeled with radiometals at the N terminus is able to image HER2 expression in xenografts a few hours after injection. The aim of this study was to evaluate whether the use of a non-residualizing label or placement of the labels at the C terminus would further improve the targeting properties of ADAPT6. Methods: Two constructs, Cys(2)-ADAPT6 and Cys(59)-ADAPT6, having the (HE)(3)DANS sequence at the N terminus were produced and site-specifically labeled using In-111-DOTA or I-125-iodo-((4-hydroxyphenyl) ethyl) maleimide (HPEM). The conjugates were compared in vitro and in vivo. HER2-targeting properties and biodistribution were evaluated in BALB/C nu/nu mice bearing ovarian carcinoma cell (SKOV-3) xenografts. Results: Specific HER2 binding and high affinity were preserved after labeling. Both Cys(2)-ADAPT6 and Cys59-ADAPT6 were internalized slowly by HER2-expressing cancer cells. Depending on the label position, uptake at 4 h after injection varied from 10% to 22% of the injected dose per gram of tumor tissue. Regardless of terminus position, the I-125-HPEM label provided more than 140-fold lower renal uptake than the In-111-DOTA label at 4 after injection. The tumor-to-organ ratios were, in contrast, higher for both of the (111)InDOTA- labeled ADAPT variants in other organs. Tumor-to-blood ratios for In-111-labeled Cys(2)-ADAPT6 and Cys(59)-ADAPT6 did not differ significantly (250-280), but In-111-DOTA-Cys(59)-ADAPT6 provided significantly higher tumor-to-lung, tumor-to-liver, tumor-to-spleen, and tumor-to-muscle ratios. Radioiodinated variants had similar tumor-to-organ ratios, but I-125-HPEM-Cys(59)-ADAPT6 had significantly higher tumor uptake and a higher tumor-to-kidney ratio. Conclusion: Residualizing properties of the label strongly influence the targeting properties of ADAPT6. The position of the radiolabel influences targeting as well, although to a lesser extent. Placement of a label at the C terminus yields the best biodistribution features for both radiometal and radiohalogen labels. Low renal retention of the radioiodine label creates a precondition for radionuclide therapy using I-131-labeled HPEM-Cys(59)-ADAPT6.

  • 12.
    Lindbo, Sarah
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Garousi, Javad
    Uppsala university.
    Mitran, Bogdan
    Uppsala university.
    Vorobyeva, Anzhelika
    Uppsala university.
    Oroujeni, Maryam
    Uppsala university.
    Orlova, Anna
    Uppsala university.
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH). KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Tolmachev, Vladimir
    Uppsala university.
    Optimized molecular design of ADAPT-based HER2-imaging probes labelled with 111In and 68Ga2018Inngår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, nr 7, s. 2674-2683Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of 111In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE)3DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C59-DEAVDANS-ADAPT6-GSSC and DOTA-C61-(HE)3DANS-ADAPT6-GSSC) were stably labeled with 111In for SPECT and 68Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. 111In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to 68Ga-labeled counterparts. The best performing variant was DOTA-C61-(HE)3DANS-ADAPT6-GSSC, which provided tumor-to-blood ratios of 208 ± 36 and 109 ± 17 at 3 h for 111In and 68Ga labels, respectively.

  • 13.
    Lindbo, Sarah
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Garousi, Javad
    Åstrand, Mikael
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Honarvar, Hadis
    Orlova, Anna
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, Vladimir
    Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins.2016Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, nr 3, s. 716-726Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with (99m)Tc(CO)3. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)3-tag instead of the commonly used hexahistidine (H6)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H6- or (HE)3-tags in the N-termini were prepared. The constructs, (HE)3-ADAPT6 and H6-ADAPT6, were labeled with two different nuclides, (99m)Tc or (111)In. The labeling with (99m)Tc(CO)3 utilized the histidine-containing tags, while (111)In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For (111)In-labeled ADAPTs, the use of (HE)3 provided a significantly (p < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of (111)In-(HE)3-ADAPT6 and H6-ADAPT6 at later time points. Interestingly, in the case of (99m)Tc, (99m)Tc(CO)3-H6-ADAPT6 provided significantly (p < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP.

  • 14.
    Lindbo, Sarah
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Gunneriusson, Elin
    Affibody AB.
    Ekblad, Caroline
    Affibody AB.
    Fant, Gunilla
    Affibody AB.
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Design, construction and characterization of an ABD-based library with improved stabilityManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Recombinant affinity proteins binding specifically to other molecules are important tools for many clinical and industrial applications. Small robust protein scaffolds have proven to be well suited as frameworks for generation of novel affinity binders due to their stability. Here we used the albumin-binding domain (ABD) of protein G from Streptococcus G148 as scaffold to design a new combinatorial library capable of generating stable binders to various target proteins with high affinity and specificity. To create a robust framework able to generate highly stable binders, mutations in the non-binding region were evaluated and residues providing increased stability were introduced into the scaffold. By combining rational design with combinatorial protein engineering we also evaluated the surface exposed amino acids at the albumin-binding interface and identified 11 residues suitable for randomization. The potency of the novel scaffold library was assessed by screening for binders using phage display against three distinct targets; complement factor 4, (C4), insulin and interleukin-6 (IL-6). Binders in the nanomolar range with melting temperature above 57°C were selected for all three targets. Notably, the identified IL-6 binders were characterized by extreme thermal stability with variants demonstrating organized structures even at 90°C. This demonstrates that stable binders with distinct specificities can be generated and thus proves the high potential of the library.

  • 15.
    Liu, Hao
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. ltai, Mohamed; Garousi, Javad; Tolmachev, Vladimir.
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Ding, Haozhong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Altai, Mohamed
    Garousi, Javad
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A2019Inngår i: International Journal of Oncology, ISSN 1019-6439, Vol. 55, nr 1, s. 309-319Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain-derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA-derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half-life extension, can be used for specific killing of HER2-expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (K-D 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (K-D) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50-values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA-derivatives are promising agents for targeted cancer therapy.

  • 16.
    Tolmachev, Vladimir
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Altai, Mohamed
    Uppsala Univ, Uppsala, Sweden..
    von Witting, Emma
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Vorobyeva, Anzhelika
    Uppsala Univ, Uppsala, Sweden..
    Oroujeni, Maryam
    Uppsala Univ, Uppsala, Sweden..
    Mitran, Bogdan
    Uppsala Univ, Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Uppsala, Sweden..
    Garousi, Javad
    Uppsala Univ, Uppsala, Sweden..
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Selection of the optimal macrocyclic chelators for labelling with In-111 and Ga-68 improves contrast of HER2 imaging using engineered scaffold protein ADAPT62019Inngår i: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 62, s. S100-S101Artikkel i tidsskrift (Annet vitenskapelig)
  • 17.
    Wu, Yu-Tang
    et al.
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Qiu, Xue
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Susumu, Kimihiro
    US Naval Res Lab, Opt Sci Div, Code 5600, Washington, DC USA.;KeyW Corp, Hanover, MD 21076 USA..
    Medintz, Igor L.
    US Naval Res Lab, Ctr Bio Mol Sci & Engn, Code 6900, Washington, DC USA..
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hildebrandt, Niko
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins2018Inngår i: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 14, nr 35, artikkel-id 1802266Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Engineered scaffold affinity proteins are used in many biological applications with the aim of replacing natural antibodies. Although their very small sizes are beneficial for multivalent nanoparticle conjugation and efficient Forster resonance energy transfer (FRET), the application of engineered affinity proteins in such nanobiosensing formats has been largely neglected. Here, it is shown that very small (approximate to 6.5 kDa) histidine-tagged albumin-binding domain-derived affinity proteins (ADAPTs) can efficiently self-assemble to zwitterionic ligand-coated quantum dots (QDs). These ADAPT-QD conjugates are significantly smaller than QD-conjugates based on IgG, Fab', or single-domain antibodies. Immediate applicability by the quantification of the human epidermal growth factor receptor 2 (HER2) in serum-containing samples using time-gated Tb-to-QD FRET detection on the clinical benchtop immunoassay analyzer KRYPTOR is demonstrated here. Limits of detection down to 40 x 10(-12)m (approximate to 8 ng mL(-1)) are in a relevant clinical concentration range and outperform previously tested assays with antibodies, antibody fragments, and nanobodies.

  • 18.
    Åstrand, Mikael
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Nilvebrant, Johan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. Centre for Cellular and Biomolecular Research, Donnelly Centre, University of Toronto, Toronto, ON, Canada.
    Björnmalm, Mattias
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. Department of Chemical and Biomolecular Engineering, University of Melbourne, Parkville, VIC, Australia.
    Lindbo, Sarah
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Investigating affinity-maturation strategies and reproducibility of fluorescence-activated cell sorting using a recombinant ADAPT library displayed on staphylococci2016Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 29, nr 5, s. 187-195Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    During the past decades, advances in protein engineering have resulted in the development of various in vitro selection techniques (e.g. phage display) to facilitate discovery of new and improved proteins. The methods are based on linkage between genotype and phenotype and are often performed in successive rounds of selection. Since the resulting output depends on the selection pressures used and the applied strategy, parameters in each round must be carefully considered. In addition, studies have reported biases that can cause enrichment of unwanted clones and/or low correlation between abundance in output and affinity. We have recently developed a selection method based on display of protein libraries on Staphylococcus carnosus and isolation of affinity proteins by fluorescence-activated cell sorting. Here, we compared duplicate selections for affinity maturation using equilibrium binding at different target concentrations and kinetic off-rate selection. The results showed that kinetic selection is efficient for isolation of high-affinity binders and that equilibrium selection at subnanomolar concentrations should be avoided. Furthermore, the reproducibility of the selection was high and a clear correlation was observed between enrichment and affinity. This work reports on the reproducibility of bacterial display in combination with FACS and provides insights into selection design to help guide the development of new affinity proteins.

1 - 18 of 18
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