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  • 1.
    Andersson, Ken G.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

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

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

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

     

  • 2.
    Andersson, Ken G.
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Oroujeni, Maryam
    Garousi, Javad
    Mitran, Bogdan
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, Anna
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with Tc-99m using a peptide-based cysteine-containing chelator2016In: International Journal of Oncology, ISSN 1019-6439, Vol. 49, no 6, p. 2285-2293Article in journal (Refereed)
    Abstract [en]

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

  • 3.
    Andersson, Ken G.
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Persson, Jonas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Ståhl, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Autotransporter-Mediated Display of a Naive Affibody Library on the Outer Membrane of Escherichia coli2019In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 14, no 4, article id 1800359Article in journal (Refereed)
    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.

  • 4.
    Andersson, Ken G.
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Persson, Jonas
    KTH, School of Biotechnology (BIO), Protein Technology. Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Autotransporter-mediated display of a naïve Affibody library on the outer membrane of E. coliManuscript (preprint) (Other academic)
    Abstract [en]

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

  • 5.
    Andersson, Ken G.
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Sjöstrand, Nanna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Coupled release and site-specific conjugation of Affibody molecules from the surface of E. coli using Sortase AManuscript (preprint) (Other academic)
    Abstract [en]

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

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

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

  • 7. Garousi, Javad
    et al.
    Honarvar, Hadis
    Andersson, Ken G.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Mitran, Bogdan
    Orlova, Anna
    Buijs, Jos
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Frejd, Fredrik Y.
    Tolmachev, Vladimir
    Comparative Evaluation of Affibody Molecules for Radionuclide Imaging of in Vivo Expression of Carbonic Anhydrase IX2016In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 13, no 11, p. 3676-3687Article in journal (Refereed)
    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.

  • 8. Nosrati, Masoumeh
    et al.
    Solbak, Sara
    Nordesjö, Olle
    Nissbeck, Mikael
    Dourado, Daniel F A R
    Andersson, Ken G.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Housaindokht, Mohammad Reza
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Virtanen, Anders
    Danielson, U Helena
    Flores, Samuel Coulbourn
    Insights from engineering the Affibody-Fc interaction with a computational-experimental method2017In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 30, no 9, p. 593-601Article in journal (Refereed)
    Abstract [en]

    The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes. We used a variation of FoldX to predict changes in protein-protein-binding affinity, and produce a list of 41 single-amino acid substitutions on the SpA molecule, of which four are near wild type (wt) and five are at most a factor of four from wt affinity. The nine substitutions include one which removes lysine, and several others which change charge. Subtle modulations in affinity may be useful for modifying column elution conditions. The method is applicable to other protein-protein systems, providing molecular insights with lower workload than existing experimental techniques.

  • 9.
    Orlova, A.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Rosestedt, M.
    Uppsala Univ, Uppsala, Sweden..
    Rinne, S. S.
    Uppsala Univ, Uppsala, Sweden..
    Mitran, B.
    Uppsala Univ, Uppsala, Sweden..
    Vorobyeva, A.
    Uppsala Univ, Uppsala, Sweden..
    Andersson, Ken G.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Imaging contrast of HER3 expression using monomeric affibody-based imaging probe can be improved by co-injection of affibody trimer2018In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, p. S673-S673Article in journal (Other academic)
  • 10. Oroujeni, M.
    et al.
    Andersson, K.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Garousi, J.
    Mitran, B.
    Orlova, A.
    Löfblom, J.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, V.
    Imaging of EGFR Expression Using 99mTC-Labelled ZEGFR:2377 Affibody Molecule2016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S238-S238Article in journal (Refereed)
  • 11.
    Oroujeni, M.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Garousi, J.
    Uppsala Univ, Uppsala, Sweden..
    Andersson, Ken G.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Mitran, B.
    Uppsala Univ, Uppsala, Sweden..
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Comparative evaluation of anti-EFGR affibody molecules labelled with gallium-68 and zirconium-89 using desferrioxamine B as a chelator2018In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, p. S674-S675Article in journal (Other academic)
  • 12.
    Oroujeni, Maryam
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Garousi, Javad
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Andersson, Ken G.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, SE-75123 Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, SE-75123 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75003 Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Preclinical Evaluation of [Ga-68]Ga-DFO-ZEGFR:2377: A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors2018In: CELLS, ISSN 2073-4409, Vol. 7, no 9, article id 141Article in journal (Refereed)
    Abstract [en]

    Radionuclide imaging of epidermal growth factor receptor (EGFR) expression in tumors may stratify patients for EGFR-targeting therapies and predict response or resistance to certain treatments. Affibody molecules, which are nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging. In this study, maleimido derivative of desferrioxamine B (DFO) chelator was site-specifically coupled to the C-terminal cysteine of the anti-EGFR affibody molecule ZEGFR:2377, and the DFO-ZEGFR:2377 conjugate was labeled with the generator-produced positron-emitting radionuclide Ga-68. Stability, specificity of binding to EGFR-expressing cells, and processing of [Ga-68]Ga-DFO-ZEGFR:2377 by cancer cells after binding were evaluated in vitro. In vivo studies were performed in nude mice bearing human EGFR-expressing A431 epidermoid cancer xenografts. The biodistribution of [Ga-68]Ga-DFO-ZEGFR:2377 was directly compared with the biodistribution of [Zr-89]Zr-DFO-ZEGFR:2377. DFO-ZEGFR:2377 was efficiently (isolated yield of 73 +/- 3%) and stably labeled with Ga-68. Binding of [Ga-68]Ga-DFO-ZEGFR:2377 to EGFR-expressing cells in vitro was receptor-specific and proportional to the EGFR expression level. In vivo saturation experiment demonstrated EGFR-specific accumulation of [Ga-68]Ga-DFO-ZEGFR:2377 in A431 xenografts. Compared to [Zr-89]Zr-DFO-ZEGFR:2377, [Ga-68]Ga-DFO-ZEGFR:2377 demonstrated significantly (p < 0.05) higher uptake in tumors and lower uptake in spleen and bones. This resulted in significantly higher tumor-to-organ ratios for [Ga-68]Ga-DFO-ZEGFR:2377. In conclusion, [Ga-68]Ga-DFO-ZEGFR:2377 is a promising probe for imaging of EGFR expression.

  • 13.
    Rinne, Sara
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Mitran, Bogdan
    Uppsala Univ, Uppsala, Sweden..
    Gentry, Joshua
    Uppsala Univ, Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Uppsala Univ, Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH Royal Inst Technol, Stockholm, Sweden..
    Andersson, Ken G.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH Royal Inst Technol, Stockholm, Sweden..
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Tolmachev, Vladimir
    Uppsala Univ, Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Uppsala, Sweden..
    Optimizing the molecular design of Ga-68-labeled affibody molecules for in vivo PET imaging of HER3 expression2019In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 62, p. S468-S470Article in journal (Other academic)
  • 14.
    Rinne, Sara S.
    et al.
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Bass, Tarek
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Andersson, Ken G.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Optimization of HER3 expression imaging using affibody molecules: Influence of chelator for labeling with indium-1112019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 655Article in journal (Refereed)
    Abstract [en]

    Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N',N ''-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid), and DOTAGA (1,4,7,10-tetraazacyclododececane, 1-(glutaric acid)-4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z(08698) and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged In-111-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.

  • 15. Rosestedt, M.
    et al.
    Andersson, Ken G.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Mitran, B.
    Rinne, S. S.
    Tolmachev, V.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, A.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Evaluation of a radiocobalt-labelled affibody molecule for imaging of human epidermal growth factor receptor 3 expression2017In: International Journal of Oncology, ISSN 1019-6439, Vol. 51, no 6, p. 1765-1774Article in journal (Refereed)
    Abstract [en]

    The human epidermal growth factor receptor 3 (HER3) is involved in the development of cancer resistance towards tyrosine kinase-Targeted therapies. Several HER3-Targeting therapeutics are currently under clinical evaluation. Non-invasive imaging of HER3 expression could improve patient management. Affibody molecules are small engineered scaffold proteins demonstrating superior properties as targeting probes for molecular imaging compared with monoclonal antibodies. Feasibility of in vivo HER3 imaging using affibody molecules has been previously demonstrated. Preclinical studies have shown that the contrast when imaging using anti-HER3 affibody molecules can be improved over time. We aim to develop an agent for PET imaging of HER3 expression using the long-lived positron-emitting radionuclide cobalt-55 (55Co) (T1/2=17.5 h). A long-lived cobalt isotope 57Co was used as a surrogate for 55Co in this study. The anti-HER3 affibody molecule HEHEHE-ZHER3-NOTA was labelled with radiocobalt with high yield, purity and stability. Biodistribution of 57Co-HEHEHE-ZHER3-NOTA was measured in mice bearing DU145 (prostate carcinoma) and LS174T (colorectal carcinoma) xenografts at 3 and 24 h post injection (p.i.). Tumour-To-blood ratios significantly increased between 3 and 24 h p.i. (p&lt;0.05). At 24 h p.i., tumour-To-blood ratios were 6 for DU145 and 8 for LS174T xenografts, respectively. HER3-expressing xenografts were clearly visualized in a preclinical imaging setting already 3 h p.i., and contrast further improved at 24 h p.i. In conclusion, the radiocobalt-labelled anti-HER3 affibody molecule, HEHEHE-ZHER3-NOTA, is a promising tracer for imaging of HER3 expression in tumours.

  • 16. Rosestedt, M.
    et al.
    Mitran, B.
    Andersson, K. G.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, J.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, S.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, V.
    Orlova, A.
    Development and Evaluation of Radiocobalt-labelled Affibody Molecule for Next Day PET Imaging of HER3 Expression2016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S37-S38Article in journal (Refereed)
1 - 16 of 16
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