| 1. |
- Feldwisch, Joachim, et al.
(författare)
-
Design of an optimized scaffold for affibody molecules.
- 2010
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 398:2, s. 232-247
-
Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z(HER2:342). Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 degrees C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z(HER2:2891)-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K(D), of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 degrees C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor alpha, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor beta) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.
|
|
| 2. |
- Altai, Mohamed, et al.
(författare)
-
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 NODAGA
- 2013
-
Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 24:6, s. 1102-1109
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 3. |
- Rosik, Daniel, 1978-
(författare)
-
On the Design of Affibody Molecules for Radiolabeling and In Vivo Molecular Imaging
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Affibody molecules have lately shown great potential as tools for in vivo molecular imaging. These small, 3-helical bundles, with their highly stable protein scaffold, are well suited for the often harsh conditions of radiolabeling. Their small size allows for rapid clearance from the blood circulation which permits the collection of images already within hours after injection. This thesis includes four papers aimed at engineering different variants of a HER2-binding Affibody molecule to enable effective and flexible radiolabeling and enhancing the molecular imaging in terms of imaging contrast and resolution.In paper I an Affibody molecule was engineered to function as a multifunctional platform for site-specific labeling with different nuclides for radionuclide imaging. This was done using only natural amino acids, thereby allowing for both synthetic and recombinant production. By grafting the amino acid sequence -GSECG to the C-terminal of our model-protein, a HER2-binding Affibody molecule, we enabled site specific labeling with both trivalent radiometals and with 99m Tc. Maleim-ide-DOTA was conjugated to the cysteine residue for labeling with 111 In, while the peptide sequence was able to chelate 99m Tc directly. This approach can also be used for site-specific labeling with other probes available for thiol-chemistry, and is applicable also to other protein scaffolds.In paper II we investigated the impact of size and affinity of radiolabeled Affibody molecules on tumor targeting and image contrast. Two HER2-targeting Affibody molecules, a two-helix (~5 kDa) and a three-helix (~7 kDa) counterpart, were synthetically produced, labeled with 111 In via chelation by DOTA and directly compared in terms of biodistribution and targeting properties. Results showed that the smaller variant can provide higher contrast images, at the cost of lower tumor uptake, in high-expressing HER2-tumors. However, neither the tumor uptake nor the contrast of the two-helix variant is sufficient to compete with the three-helix molecule in tumors with low expression of HER2.In paper III and IV we were aiming to find methods to improve the labeling of Affibody molecules with 18 F for PET imaging. Current methods are either complex, time-consuming or generate heavily lipophilic conjugates. This results in low yields of radiolabeled tracer, low specific activity left for imaging, undesirable biodistribution or a combination thereof. In paper III we demonstrate a swift and efficient 2-step, 1-pot method for labeling HER2-binding Affibody molecules by the formation of aluminum 18 F-fluoride (Al 18 F) and its chelation by NOTA, all in 30 min. The results show that the 18 F-NOTA-approach is a very promising method of labeling Affibody molecules with 18 F and further investigation of this scheme is highly motivated. In the last paper we pursued the possibility of decreasing the high kidney retention that is common among small radiotracers with residual-izing radiometabolites. In this work 18 F-4-fluorobenzaldehyde (FBA) was conjugated to a synthetic HER2-targeting Affibody molecule via oxime ligation. However, to avoid elevated liver retention, as seen in previous studies with this kind of label, a hydrophilic triglutamyl spacer between the aminooxy moiety and the N-terminal was introduced. A comparison of the two constructs (with and without the triglutamyl spacer) showed a clear reduction of retention in both kidney and liver in NMRI mice at 2 h p.i. when the spacer was included. In the light of these promising results, further studies including tumor-bearing mice, are in preparation.
|
|
| 4. |
- Tolmachev, Vladimir, et al.
(författare)
-
Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR In-111-DOTA-Z(EGFR:2377) Affibody molecule : aspect of the injected tracer amount
- 2010
-
Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 37:3, s. 613-622
-
Tidskriftsartikel (refereegranskat)abstract
- Overexpression of epidermal growth factor receptor (EGFR) is a prognostic and predictive biomarker in a number of malignant tumours. Radionuclide molecular imaging of EGFR expression in cancer could influence patient management. However, EGFR expression in normal tissues might complicate in vivo imaging. The aim of this study was to evaluate if optimization of the injected protein dose might improve imaging of EGFR expression in tumours using a novel EGFR-targeting protein, the DOTA-Z(EGFR:2377) Affibody molecule. An anti-EGFR Affibody molecule, Z(EGFR:2377), was labelled with In-111 via the DOTA chelator site-specifically conjugated to a C-terminal cysteine. The affinity of DOTA-Z(EGFR:2377) for murine and human EGFR was measured by surface plasmon resonance. The cellular processing of In-111-DOTA-Z(EGFR:2377) was evaluated in vitro. The biodistribution of radiolabelled Affibody molecules injected in a broad range of injected Affibody protein doses was evaluated in mice bearing EGFR-expressing A431 xenografts. Site-specific coupling of DOTA provided a uniform conjugate possessing equal affinity for human and murine EGFR. The internalization of In-111-DOTA-Z(EGFR:2377) by A431 cells was slow. In vivo, the conjugate accumulated specifically in xenografts and in EGFR-expressing tissues. The curve representing the dependence of tumour uptake on the injected Affibody protein dose was bell-shaped. The highest specific radioactivity (lowest injected protein dose) provided a suboptimal tumour-to-blood ratio. The results of the biodistribution study were confirmed by gamma-camera imaging. The In-111-DOTA-Z(EGFR:2377) Affibody molecule is a promising tracer for radionuclide molecular imaging of EGFR expression in malignant tumours. Careful optimization of protein dose is required for high-contrast imaging of EGFR expression in vivo.
|
|
