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  • 1. Altai, Mohamed
    et al.
    Strand, Joanna
    Rosik, Daniel
    Selvaraju, Ram Kumar
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Orlova, Anna
    Tolmachev, Vladimir
    Influence of Nuclides and Chelators on Imaging Using Affibody Molecules: Comparative Evaluation of Recombinant Affibody Molecules Site-Specifically Labeled with Ga-68 and In-111 via Maleimido Derivatives of DOTA and NODAGA2013In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 6, p. 1102-1109Article in journal (Refereed)
    Abstract [en]

    Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more of personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use should increase sensitivity of HER2 imaging. The chemical nature of the generator-produced positron-emitting radionuclide Ga-68 of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant Z(HER2:2395) HER2-binding Affibody molecule with Ga-68. DOTA and NODAGA were site-specifically conjugated to the Z(HER2:2395) Affibody molecule having a C-terminal cysteine and labeled with Ga-68 and In-111. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were dearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for Ga-68 than for In-111. The tumor uptake of Ga-68-NODAGA-Z(HER2:2395) and Ga-68-NODAGA-Z(HER2:2395) and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for Ga-68-NODAGA- Z(HER2:2395) (8 +/- 2 vs 5.0 +/- 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.

  • 2. Ekerljung, L.
    et al.
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Sohrabian, A.
    Andersson, K.
    Friedman, M.
    Frejd, F. Y.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gedda, L.
    Generation and evaluation of bispecific affibody molecules for simultaneous targeting of EGFR and HER22012In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 9, p. 1802-1811Article in journal (Refereed)
    Abstract [en]

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

  • 3. Garousi, Javad
    et al.
    Lindbo, Sarah
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, Hadis
    Velletta, Justin
    Mitran, Bogdan
    Altai, Mohamed
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Influence of the N -Terminal Composition on Targeting Properties of Radiometal-Labeled Anti-HER2 Scaffold Protein ADAPT62016In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, no 11, p. 2678-2688Article in journal (Refereed)
    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.

  • 4.
    Kanje, Sara
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    von Witting, Emma
    KTH, School of Biotechnology (BIO), Protein Technology.
    Chiang, Samuel
    Karolinska institutet, Stockholm.
    Bryceson, Yenan
    Karolinska institutet, Stockholm.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain2016In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812Article in journal (Refereed)
    Abstract [en]

    Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody’s binding to its target. Moreover, an emerging field for therapeutics is antibody–drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.

  • 5.
    Konrad, Anna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Covalent Immunoglobulin Labeling through a Photoactivable Synthetic Z Domain2011In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 22, no 12, p. 2395-2403Article in journal (Refereed)
    Abstract [en]

    Traditionally, labeling of antibodies has been performed by covalent conjugation to amine or carboxyl groups. These methods are efficient but suffer from nonspecificity, since all free and available amine/carboxyl groups have the possibility to react. This drawback may lead to uncontrolled levels and locations of the labeling. Hence, the labeled molecules might behave differently and, possibly, the binding site of the antibody will also be affected. In this project, we have developed a highly stringent method for labeling of antibodies by utilizing an immunoglobulin-binding domain from protein A, the Z domain. Domain Z has been synthesized with an amino acid analogue, benzoylphenylalanine, capable of forming covalent attachment to other amino acids upon UV-exposure. This feature has been used for directed labeling of immunoglobulins and subsequent use of these in different assays.

  • 6.
    Lindbo, Sarah
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Garousi, Javad
    Åstrand, Mikael
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, Hadis
    Orlova, Anna
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins.2016In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 27, no 3, p. 716-726Article in journal (Refereed)
    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.

  • 7. Liu, Li-Hong
    et al.
    Dietsch, Herve
    Schurtenberger, Peter
    Yan, Mingdi
    Department of Chemistry, Portland State University.
    Photoinitiated Coupling of Unmodified Monosaccharides to Iron Oxide Nanoparticles for Sensing Proteins and Bacteria2009In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 20, no 7, p. 1349-1355Article in journal (Refereed)
    Abstract [en]

    We report a versatile approach for the immobilization of unmodified monosaccharides onto iron oxide nanoparticles. Covalent coupling of the carbohydrate onto iron oxide nanoparticle surfaces was accomplished by the CH insertion reaction of photochemically activated phosphate-functionalized perfluorophenylazides (PFPAs), and the resulting glyconanoparticles were characterized by IR, TGA, and TEM. The surface-bound D-mannose showed the recognition ability toward Concanavalin A and Escherichia coli strain ORN178 that possesses mannose-specific receptor sites. Owing to the simplicity and versatility of the technique, together with the magnetic property of iron oxide nanoparticles, the methodology developed in this study serves as a general approach for the preparation of magnetic glyconanoparticles to be used in clinical diagnosis, sensing, and decontamination.

  • 8.
    Norberg, Oscar
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Deng, Lingquan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Yan, Mingdi
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Photo-Click Immobilization of Carbohydrates on Polymeric Surfaces - A Quick Method to Functionalize Surfaces for Biomolecular Recognition Studies2009In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 20, no 12, p. 2364-2370Article in journal (Refereed)
    Abstract [en]

    Methods to rapidly functionalize specific polymeric surfaces with alkynes, which can subsequently be linked to azide-containing carbohydrates, are presented. The methods comprise two main concepts: azide photoligation and Cu-catalyzed azide-alkyne cycloaddition. 2-Azidoethyl-functionalized CL-D-mannopyranoside was synthesized and covalently attached to alkyne-functionalized polymeric surfaces using the techniques. The protein recognition properties of the carbollydrate-presenting surfaces were evaluated using quartz crystal microbalance biosensor instrumentation.

  • 9.
    Perols, Anna
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Site-Specific Photoconjugation of Antibodies Using Chemically Synthesized IgG-Binding Domains2014In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 25, no 3, p. 481-488Article in journal (Refereed)
    Abstract [en]

    Site-specific labeling of antibodies can be performed using the immunoglobulin-binding Z domain, derived from staphylococcal protein A (SpA), which has a well-characterized binding site in the Pc region of antibodies. By introducing a photoactivable probe in the Z domain, a covalent bond can be formed between the Z domain and the antibody by irradiation with UV light. The aim of this study was to improve the conjugation yield for labeling of different subclasses of IgG having different sequence composition, using a photoactivated Z domain variant. Four different variants of the Z domain (Z5BPA, Z5BBA, Z32BPA, and Z32BBA) were synthesized to investigate the influence of the position of the photoactivable probe and the presence of a flexible linker between the probe and the protein. For two of the variants, the photoreactive benzophenone group was introduced as part of an amino acid side chain by incorporation of the unnatural amino acid benzoylphenylalanine (BPA) during peptide synthesis. For the other two variants, the photoreactive benzophenone group was attached via a flexible linker by coupling of benzoylbenzoic acid (BBA) to the e-amino group of a selectively deprotected lysine residue. Photoconjugation experiments using human IgG1, mouse IgG I, and mouse IgG2A demonstrated efficient conjugation for all antibodies. It was shown that differences in linker length had a large impact on the conjugation efficiency for labeling of mouse IgG1, whereas the positioning of the photoactivable probe in the sequence of the protein had a larger effect for mouse IgG2A. Conjugation to human IgG1 was only to a minor extent affected by position or linker length. For each subclass of antibody, the best variant tested using a standard conjugation protocol resulted in conjugation efficiencies of 41-66%, which corresponds to on average approximately one Z domain attached to each antibody. As a combination of the two best performing variants, Z5BBA and Z32BPA, a Z domain variant with two photoactivable probes (Z5BBA32BPA) was also synthesized with the aim of targeting a wider panel of antibody subclasses and species. This new reagent could efficiently couple to all antibody subclasses that were targeted by the single benzophenone-labeled Z domain variants, with conjugation efficiencies of 26-41%.

  • 10.
    Perols, Anna
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Honarvar, Hadis
    Strand, Joanna
    Selvaraju, Ramkumar
    Orlova, Anna
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Tolmachev, Vladimir
    Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of In-111-Labeled Synthetic Anti-HER2 Affibody Molecules2012In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 8, p. 1661-1670Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with Tc-99m at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K-D values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1) K50-DOTA showed a slightly lower melting point (57 degrees C) compared to DOTA-A1-Z(HER2:S1) (64 degrees C) and DOTA-K5S-Z(HER2:S1) (62 degrees C), but all variants showed good refolding properties after heat treatment All conjugates were successfully labeled with In-III resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in L5174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1) and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

  • 11.
    Rosik, Daniel
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Thibblin, Alf
    Antoni, Gunnar
    Orlova, Anna
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    Incorporation of a Triglutamyl Spacer Improves the Biodistribution of Synthetic Affibody Molecules Radiofluorinated at the N-Terminus via Oxime Formation with F-18-4-Fluorobenzaldehyde2014In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 25, no 1, p. 82-92Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are a class of affinity agents for molecular imaging based on a non-immunoglobulin protein scaffold. Previous studies have demonstrated high contrast for in vivo imaging of cancer-associated molecular abnormalities using Affibody molecules. Using the radionuclide F-18 for labeling and PET as the imaging modality, the sensitivity of molecular imaging using Affibody molecules can be further increased. The use of oxime formation between an aminooxy-functionalized peptide and F-18-fluorobenzaldehyde (F-18-FBA) is a promising way of radiolabeling of targeting peptides. However, previous studies demonstrated that application of this method to Affibody molecules is associated with high liver uptake. We hypothesized that incorporation of a triglutamyl spacer between the aminooxy moiety and the N-terminus of a synthetic Affibody molecule would decrease the hepatic uptake of the F-18-N-(4-fluorobenzylidine)oxime) (F-18-FBO)-labeled tracer. To verify this, we have produced two variants of the HER2-targeting Z(HER2:342) Affibody molecule by peptide synthesis: OA-PEP4313, where aminooxyacetic acid was conjugated directly to the N-terminal alanine, and OA-E-3-PEP4313, where a triglutamyl spacer was introduced between the aminooxy moiety and the N-terminus. We have found that the use of the spacer is associated with a minor decrease of affinity, from K-D = 49 pM to K-D = 180 pM. Radiolabeled F-18-FBO-E-3-PEP4313 demonstrated specific binding to HER2-expressing ovarian carcinoma SKOV-3 cells and slow internalization. Biodistribution studies in mice demonstrated that the use of a triglutamyl linker decreased uptake of radioactivity in liver 2.7-fold at 2 h after injection. Interestingly, radioactivity uptake in kidneys was also reduced (2.4-fold). Experiments in BALB/C nu/nu mice bearing SKOV-3 xenografts demonstrated HER2-specific uptake of F-18-FBO-E-3-PEP4313 in tumors. At 2 h pi, the tumor uptake (20 +/- 2% ID/g) exceeded uptake in liver 5-fold and uptake in kidneys 3.6-fold. The tumor-to-blood ratio was 21 +/- 3. The microPET/CT imaging experiment confirmed the biodistribution data. In conclusion, the use of a triglutamyl spacer is a convenient way to improve the biodistribution profile of Affibody molecules labeled at the N-terminus using F-18-FBA. It provides a tracer capable of producing high-contrast images of HER2-expressing tumors.

  • 12.
    Stiller, Christiane
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Aghelpasand, Hooman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Frick, Tobias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Westerlund, Kristina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Ahmadian, Afshin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Eriksson Karlström, Amelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Fast and Efficient Fc-Specific Photoaffinity Labeling To Produce Antibody-DNA Conjugates2019In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 30, no 11, p. 2790-2798Article in journal (Refereed)
    Abstract [en]

    Antibody DNA conjugates are powerful tools for DNA-assisted protein analysis. Growing usage of these methods demands efficient production of high-quality conjugates. We developed an easy and fast synthesis route yielding covalent antibody-DNA conjugates with a defined conjugation site and low batch-to-batch variability. We utilize the Z domain from protein A, containing the unnatural amino acid 4-benzoylphenylalanine (BPA) for photoaffinity labeling of the antibodies' Fc region. Z(xBPA) domains are C-terminally modified with triple-glycine (G(3))-modified DNA-oligonucleotides enzymatic Sortase A coupling. We reliable modification of the most commonly used IgG's. To prove our conjugates' functionality, we detected antibody-antigen binding events in an assay called Droplet Barcode Sequencing for Protein analysis (DBS-Pro). It confirms not only retained functionality for both conjugate parts but also the potential of using DBS-Pro for quantifying protein abundances. As intermediates are easily storable and our approach is modular, it offers a convenient strategy for screening various antibody-DNA conjugates using the same starting material.

  • 13. Tolmachev, Vladimir
    et al.
    Altai, Mohamed
    Sandstrom, Mattias
    Perols, Anna
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Boschetti, Frederic
    Orlova, Anna
    Evaluation of a Maleimido Derivative of NOTA for Site-Specific Labeling of Affibody Molecules2011In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 22, no 5, p. 894-902Article in journal (Refereed)
    Abstract [en]

    Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z(HER2:239S) resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys(61)]-Z(HER2:239S) with In-111 gave a yield of >95% after 20 min at 60 degrees C. In vitro cell tests demonstrated specific binding of [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) was 8.2 +/- 0.9% IA/g and the tumor-to-blood ratio was 31 +/- 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) and [In-111-MMA-DOTA-Cys(61)]-Z(HER2:239S) demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [In-111-MMA-NOTA-Cys(61)]-Z(HER2:239S) due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.

  • 14. Tolmachev, Vladimir
    et al.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Malmberg, Jennie
    Ahlgren, Sara
    Hosseinimehr, Seyed Jalal
    Sandström, Mattias
    Abrahmsen, Lars
    Orlova, Anna
    Gräslund, Torbjorn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    HEHEHE-Tagged Affibody Molecule May Be Purified by IMAC, Is Conveniently Labeled with [Tc-99m(CO)(3)](+), and Shows Improved Biodistribution with Reduced Hepatic Radioactivity Accumulation2010In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 21, no 11, p. 2013-2022Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [Tc-99m(CO)(3)](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER2:342) were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [Tc-99m(CO)(3)](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER2:342)-H-6 and (HE)(3)-Z(HER2:342), respectively, could be efficiently purified using IMAC and stably labeled with [Tc-99m(CO)(3)](+) and were subsequently compared with the parental H-6-Z(HER2:342) having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [Tc-99m(CO)(3)](+)-Z(HER2:342)-H-6 compared to [Tc-99m(CO)(3)](+)-H-6-Z(HER2:342), and more than 10-fold lower with [Tc-99m(CO)(3)](+)-(HE)(3)-Z(HER2:342). These differences translated into appreciably superior tumor-to-liver ratio for [Tc-99m(CO)(3)](+)-(HE)(3)-Z(HER2:342) compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.

  • 15. Tran, Thuy A.
    et al.
    Ekblad, Torun
    Orlova, Anna
    Sandstrom, Mattias
    Feldwisch, Joachim
    Wennborg, Anders
    Abrahmsen, Lars
    Tolmachev, Vladimir
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Effects of Lysine-Containing Mercaptoacetyl-Based Chelators on the Biodistribution of Tc-99m-Labeled Anti-HER2 Affibody Molecules2008In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, no 12, p. 2568-2576Article in journal (Refereed)
    Abstract [en]

    The effects of polar (mercaptoacetyl-triseryl) and negatively charged (mercaptoacetyl-triglumatyl) chelators on the biodistribution of Tc-99m-labeled anti-HER2 Affibody molecules were previously investigated. With glycine, serine, and glutamate, we demonstrated that substitution with a single amino acid in the chelator can significantly influence the biodistribution properties and the excretion pathways. Here, we have taken this investigation further, by analyzing the effects of introduction of a positive amino acid residue on the in vivo properties of the Tc-99m-labeled Affibody molecule. The Affibody molecules with mercaptoacetyl-seryl-lysyl-seryl (maSKS) and mercaptoacetyl-trilysyl (maKKK) extensions were produced by peptide synthesis and labeled with Tc-99m in alkaline conditions. A comparative biodistribution was performed in normal mice to evaluate the excretion pathway. A shift toward renal excretion was obtained when serine was substituted with lysine in the chelatin sequence. The radioactivity in the gastrointestinal tract was reduced 3-fold for the Tc-99m-maSKS-Z(HER2:342) and Tc-99m-maKKK-Z(HER2:342) in comparison with the Tc-99m-maSSS-Z(HER2:342) conjugate 4 h post injection (p.i.). The radioactivity in the liver was elevated when a triple substitution of positively charged lysine was used. The tumor targeting properties of Tc-99m-maSKS-Z(HER2:342) were further investigated in SKOV-3 xenografts. The tumor uptake of Tc-99m-maSKS-Z(HER2:342) was 17 +/- 7% IA/g 4 h p.i. Tumor xenografts were well-visualized by gamma scintigraphy. In conclusion, the substitution with one single lysine in the chelator results in better tumor imaging properties of the Affibody molecule Z(HER2:342) and is favorable for imaging of tumors and metastases in the abdominal area. Multiple lysine residues in the chelator are, however, undesirable due to elevated uptake both in the liver and kidneys.

  • 16.
    Tran, Thuy
    et al.
    Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Engfeldt, Torun
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Sandström, Mattias
    Medical Radiation Physics, Uppsala University Hospital, Uppsala University.
    Feldwisch, Joachim
    Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Abrahmsén, Lars
    Affibody AB, Bromma.
    Wennborg, Anders
    Affibody AB, Bromma.
    Tolmachev, Vladimir
    Division of Biomedical Radiation Sciences, Rudbeck Laboratory.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
     99mTc-maEEE-ZHER2:342, an Affibody Molecule-Based Tracer for the Detection of HER2 Expression in Malignant Tumors2007In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 18, no 6, p. 1956-1964Article in journal (Refereed)
    Abstract [en]

    Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of 99mTc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule ZHER2:342 with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of 99mTc-maGEG-ZHER2:342 and 99mTc-maEEE-ZHER2:342 was compared with 99mTc-maGGG-ZHER2:342 in normal mice, and the tumor targeting properties of 99mTc-maEEE-ZHER2:342 were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for 99mTc-labeling of ZHER2:342 reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. 99mTc-maEEE-ZHER2:342 showed a receptor-specific tumor uptake of 7.9 ± 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with 99mTc-maEEE-ZHER2:342 could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for 99mTc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making 99mTc-maEEE-ZHER2:342 a promising tracer for clinical imaging of HER2 overexpression in tumors.

  • 17. Tran, Thuy
    et al.
    Engfeldt, Torun
    Orlova, Anna
    Widstrom, Charles
    Bruskin, Alexander
    Tolmachev, Vladimir
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    In vivo evaluation of cysteine-based chelators for attachment of Tc-99m to tumor-targeting affibody molecules2007In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 18, no 2, p. 549-558Article in journal (Refereed)
    Abstract [en]

    Affibody molecules present a new class of affinity proteins, which utilizes a scaffold based on a 58-amino acid domain derived from protein A. The small (7 kDa) Affibody molecule can be selected to bind to cell-surface targets with high affinity. An Affibody molecule (Z(HER2:342)) with a dissociation constant (K-d) of 22 pM for binding to the HER2 receptor has been reported earlier. Preclinical and pilot clinical studies have demonstrated the utility of radiolabeled Z(HER2:342) in imaging of HER2-expressing tumors. The small size and cysteine-free structure of Affibody molecules enable complete peptide synthesis and direct incorporation of radionuclide chelators. The goal of this study was to evaluate if incorporation of the natural peptide sequences cysteine-diglycine (CGG) and cysteine-triglycine (CGGG) sequences would enable labeling of Affibody molecules with Tc-99m. In a model monomeric form, the chelating sequences were incorporated by peptide synthesis. The HER2-binding affinity was 280 and 250 pM for CGG-Z(HER2:342) and CGGG-Z(HER2:342,) respectively. Conjugates were directly labeled with Tc-99m with 90% efficiency and preserved the capacity to bind specifically to HER2-expressing cells. The biodistribution in normal mice showed a rapid clearance from the blood and the majority of organs (except kidneys). In the mice bearing SKOV-3 xenografts, tumor uptake of Tc-99m-CGG-Z(HER2:342) was HER2-specific and a tumor-to-blood ratio of 9.2 was obtained at 6 h postinjection. Gamma-camera imaging with Tc-99m-CGG-Z(HER2:342) clearly visualized tumors at 6 h postinjection. The results show that the use of a cysteine-based chelator enables Tc-99m-labeling of Affibody molecules for imaging.

  • 18. Wang, Huibin
    et al.
    Zhang, Yiming
    Yuan, Xun
    Chen, Yi
    Yan, Mingdi
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    A Universal Protocol for Photochemical Covalent Immobilization of Intact Carbohydrates for the Preparation of Carbohydrate Microarrays2011In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 22, no 1, p. 26-32Article in journal (Refereed)
    Abstract [en]

    A universal photochemical method has been established for the immobilization of intact carbohydrates and their analogues, and for the fabrication of carbohydrate microarrays. The method features the use of perfluorophenyl azide (PFPA)-modified substrates and the photochemical reaction of surface azido groups with printed carbohydrates. Various aldoses, ketoses, nonreducing sugars such as alditols, and their derivatives can be directly arrayed on the PFPA-modified chips. The lectin-recognition ability of arrayed mannose, glucose, and their oligo- and polysaccharides were confirmed using surface-plasmon resonance imaging and laser-induced fluorescence imaging.

  • 19.
    Westerlund, Kristina
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, H.
    Tolmachev, V.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting2015In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 26, no 8, p. 1724-1736Article in journal (Refereed)
    Abstract [en]

    In radioimmunotherapy, the contrast between tumor and normal tissue can be improved by using a pretargeting strategy with a primary targeting agent, which is conjugated to a recognition tag, and a secondary radiolabeled molecule binding specifically to the recognition tag. The secondary molecule is injected after the targeting agent has accumulated in the tumor and is designed to have a favorable biodistribution profile, with fast clearance from blood and low uptake in normal tissues. In this study, we have designed and evaluated two complementary peptide nucleic acid (PNA)-based probes for specific and high-affinity association in vivo. An anti-HER2 Affibody-PNA chimera, Z<inf>HER2:342</inf>-SR-HP1, was produced by a semisynthetic approach using sortase A catalyzed ligation of a recombinantly produced Affibody molecule to a PNA-based HP1-probe assembled using solid-phase chemistry. A complementary HP2 probe carrying a DOTA chelator and a tyrosine for dual radiolabeling was prepared by solid-phase synthesis. Circular dichroism (CD) spectroscopy and UV thermal melts showed that the probes can hybridize to form a structured duplex with a very high melting temperature (T<inf>m</inf>), both in HP1:HP2 and in Z<inf>HER2:342</inf>-SR-HP1:HP2 (T<inf>m</inf> = 86-88 °C), and the high binding affinity between Z<inf>HER2:342</inf>-SR-HP1 and HP2 was confirmed in a surface plasmon resonance (SPR)-based binding study. Following a moderately fast association (1.7 × 105 M-1 s-1), the dissociation of the probes was extremely slow and <5% dissociation was observed after 17 h. The equilibrium dissociation constant (K<inf>D</inf>) for Z<inf>HER2:342</inf>-SR-HP1:HP2 binding to HER2 was estimated by SPR to be 212 pM, suggesting that the conjugation to PNA does not impair Affibody binding to HER2. The biodistribution profiles of 111In- and 125I-labeled HP2 were measured in NMRI mice, showing very fast blood clearance rates and low accumulation of radioactivity in kidneys and other organs. The measured radioactivity in blood was 0.63 ± 0.15 and 0.41 ± 0.15%ID/g for 125I- and 111In-HP2, respectively, at 1 h p.i., and at 4 h p.i., the kidney accumulation of radioactivity was 0.17 ± 0.04%ID/g for 125I-HP2 and 3.83 ± 0.39%ID/g for 111In-HP2. Taken together, the results suggest that a PNA-based system has suitable biophysical and in vivo properties and is a promising approach for pretargeting of Affibody molecules.

  • 20.
    Zhou, Juan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Butchosa, Nuria
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Jayawardena, H. Surangi N.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Yan, Mingdi
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Glycan-Functionalized Fluorescent Chitin Nanocrystals for Biorecognition Applications2014In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 25, no 4, p. 640-643Article in journal (Refereed)
    Abstract [en]

    A new platform based on chitin nanocrystals has been developed for biorecognition applications. TEMPO-oxidized chitin nanocrystals (TCNs) were labeled with a fluorescent imidazoisoquinolinone dye, and simultaneously conjugated with carbohydrate ligands, resulting in dually functionalized TCNs. The biorecognition properties of the nanocrystals were probed with lectins and bacteria, resulting in selective interactions with their corresponding cognate carbohydrate-binding proteins, as visualized by optical, fluorescence, STEM, and TEM imaging. This represents a new approach to multifunctional nanomaterials based on naturally occurring polymers, holding high potential for biomedical applications.

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