Engineered Two-Helix Small Proteins for Molecular Recognition

Biosciences, Global Research, General Electric Company, 1 Research Circle, Niskayuna, NY 12309, USA.
ChemBioChem (Impact Factor: 3.09). 05/2009; 10(8):1293-6. DOI: 10.1002/cbic.200900062
Source: PubMed


Less is more: By starting with a high-affinity HER2-binding 3-helix affibody molecule, we successfully developed 2-helix small protein binders with 5 nM affinities by using a combination of several different strategies. Our efforts clearly suggest that 2-helix small proteins against important tumor targets can be obtained by rational protein design and engineering.

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    • "Affibody variant with affinity of 5 nM [14]. Although this affinity is appreciably lower than the affinity of parental Affibody molecules Z HER2:342 (22 pM, [4]), it is still sufficient for imaging applications [15]. "
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    ABSTRACT: Introduction: Affibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL). Methods: The HER2-targeting NCL-cyclized Affibody molecule ZHER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-ZHER2:342min was labeled with (111)In and (68)Ga. The binding affinity of DOTA-ZHER2:342min was evaluated in vitro. The targeting properties of (111)In- and (68)Ga-DOTA-ZHER2:342min were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of (111)In- and (68)Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge. Results: The dissociation constant (KD) for DOTA-ZHER2:342min binding to HER2 was 18nM according to SPR measurements. DOTA-ZHER2:342min was labeled with (111)In and (68)Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with KD1 in low nanomolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2h p.i. were 6.1±1.3 for (111)In- DOTA-ZHER2:342min and 4.6±0.7 for (68)Ga-DOTA-ZHER2:342min. However, the uptake of DOTA-ZHER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts. Conclusions: Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (ZHER2:342min) with low nanomolar target affinity and specific tumor uptake.
    Nuclear Medicine and Biology 01/2013; 40(3). DOI:10.1016/j.nucmedbio.2012.12.009 · 2.41 Impact Factor
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    • "Further clinical studies are warranted to assess the sensitivity and specificity of radiolabeled HER2-target- ing Affibody molecules. A two-helix variant of the Affibody molecule Z HER2:342 was recently produced by removal of helix 3, situated on the opposite site of the binding site and therefore not supposed to be involved in target binding [39]. The two alpha helices were stabilized by a disulfide bridge formed between two introduced homocysteines. "
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    ABSTRACT: Affibody molecules are a class of engineered affinity proteins with proven potential for therapeutic, diagnostic and biotechnological applications. Affibody molecules are small (6.5 kDa) single domain proteins that can be isolated for high affinity and specificity to any given protein target. Fifteen years after its discovery, the Affibody technology is gaining use in many groups as a tool for creating molecular specificity wherever a small, engineering compatible tool is warranted. Here we summarize recent results using this technology, propose an Affibody nomenclature and give an overview of different HER2-specific Affibody molecules. Cumulative evidence suggests that the three helical scaffold domain used as basis for these molecules is highly suited to create a molecular affinity handle for vastly different applications.
    FEBS letters 04/2010; 584(12):2670-80. DOI:10.1016/j.febslet.2010.04.014 · 3.17 Impact Factor
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    • "A number of both sequence mutations and synthetic strategies have been developed to optimize the affinity of the 2-helix small protein against HER2. Several constrained 2-helix constructs with high (low nM) HER2 affinity were successfully identified (Webster et al. 2009). Small animal PET imaging of HER2 expression has been achieved with a two-helix probe 68 Ga-DOTA-MUT-DS (Ren et al. 2009). "
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    ABSTRACT: Protein scaffold molecules are powerful reagents for targeting various cell signal receptors, enzymes, cytokines and other cancer-related molecules. They belong to the peptide and small protein platform with distinct properties. For the purpose of development of new generation molecular probes, various protein scaffold molecules have been labeled with imaging moieties and evaluated both in vitro and in vivo. Among the evaluated probes Affibody molecules and analogs, cystine knot peptides, and nanobodies have shown especially good characteristics as protein scaffold platforms for development of in vivo molecular probes. Quantitative data obtained from positron emission tomography, single photon emission computed tomography/CT, and optical imaging together with biodistribution studies have shown high tumor uptakes and high tumor-to-blood ratios for these probes. High tumor contrast imaging has been obtained within 1 h after injection. The success of those molecular probes demonstrates the adequacy of protein scaffold strategy as a general approach in molecular probe development.
    Amino Acids 02/2010; 41(5):1037-47. DOI:10.1007/s00726-010-0503-9 · 3.29 Impact Factor
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