Affibody Molecules As Targeting Vectors for PET Imaging

cancers Review Affibody Molecules as Targeting Vectors for PET Imaging Vladimir Tolmachev 1,2,* and Anna Orlova 2,3,4 1 Department of Immunology, Genetics and Pathology, Uppsala University, 75185 Uppsala, Sweden 2 Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Tomsk Polytechnic University, 634050 Tomsk, Russia; [email protected] 3 Department of Medicinal Chemistry, Uppsala University, 75183 Uppsala, Sweden 4 Science for Life Laboratory, Uppsala University, 75237 Uppsala, Sweden * Correspondence: [email protected] Received: 14 February 2020; Accepted: 9 March 2020; Published: 11 March 2020 Abstract: Affibody molecules are small (58 amino acids) engineered scaffold proteins that can be selected to bind to a large variety of proteins with a high affinity. Their small size and high affinity make them attractive as targeting vectors for molecular imaging. High-affinity affibody binders have been selected for several cancer-associated molecular targets. Preclinical studies have shown that radiolabeled affibody molecules can provide highly specific and sensitive imaging on the day of injection; however, for a few targets, imaging on the next day further increased the imaging sensitivity. A phase I/II clinical trial showed that 68Ga-labeled affibody molecules permit an accurate and specific measurement of HER2 expression in breast cancer metastases. This paper provides an overview of the factors influencing the biodistribution and targeting properties of affibody molecules and the chemistry of their labeling using positron emitters. Keywords: PET; affibody molecules; HER2; EGFR; molecular imaging; radiolabeling 1. Introduction Radionuclide molecular imaging permitting non-invasive quantitative visualization of molecular targets is an attractive alternative to biopsy-based methods for stratifying patients for targeted therapies [1]. The use of positron emission tomography (PET) is a preferable method for molecular imaging because it provides a better spatial resolution, registration efficiency, and accuracy of activity quantification compared to single photon emission computed tomography (SPECT) [2]. Currently, the most common approach used to visualize molecular targets is immunoPET, which is a methodology based on the labeling of therapeutic monoclonal antibodies with long-lived positron 64 89 124 emitting nuclides, including Cu (T 1 = 12.7 h), Zr (T 1 = 78.4 h), and I (T 1 = 100.2 h) [3,4]. 2 2 2 The feasibility of such an approach has been demonstrated in a number of clinical studies [5–7]. Accumulated clinical experience has enabled the identification of several major issues in the routine clinical use of full-length immunoglobulin G (IgG) as imaging probes; these issues include slow extravasation and accumulation in tumors, slow clearance from blood and unspecific compartments, and unspecific uptake in target-negative tumors due to the enhanced permeability and retention (EPR) effect. To obtain an acceptable contrast and sensitivity, clinical imaging must be performed 4-7 days after injection [5–7]. Nevertheless, the unspecific tumor accumulation of IgG is associated with a risk of false-positive diagnostics [8]. Therefore, it is very likely that immunoPET will remain a valuable tool for the development of antibody-based therapeutics, but the prospects of its translation into day-by-day clinical practice are unclear. Cancers 2020, 12, 651; doi:10.3390/cancers12030651 www.mdpi.com/journal/cancers Cancers 2020, 12, 651 2 of 21 Empirical data suggest that a reduction in the size of targeting proteins permits the ability to obtainCancers a high 2019 imaging, 11, x contrast earlier than several days after the injection [9]. Indeed, the2 of use 21 of a single-domainEmpirical antibody data suggest (sdAb) that with a reduction a molecular in the weightsize of targeti of 12–15ng proteins kDa permits permits the the development ability to of imagingobtain probesa high imaging with a tumor-to-bloodcontrast earlier than ratio several of 10–30 days at after 1 h the after injection injection [9]. inIndeed, murine the models use of a [10]. Mathematicalsingle-domain modeling antibody predicts (sdAb) thatwith aa furthermolecular size weight reduction of 12–15 would kDa permits enable the an development increase in tumorof accumulationimaging probes if thea withffinity a tu ofmor imaging-to-blood probes ratio was of 10 high–30 enoughat 1 h after [11 ,injection12]. Thus in far,murine the developmentmodels [10]. of high-aMathematicalffinity immunoglobulin-based modeling predicts that targeting a further probes size reduction smaller than would a sdAb enable remains an increase very in challenging. tumor Nevertheless,accumulation this if is the possible affinity using of imaging engineered probes non-immunoglobulinwas high enough [11,12]. sca Thusffold far, proteins the development [13]. of high-affinity immunoglobulin-based targeting probes smaller than a sdAb remains very challenging. 2. EngineeredNevertheless, Sca thisffold is Protein-Basedpossible using engineered Imaging Probes:non-immunoglobulin Affibody Molecules scaffold proteins [13]. Antibodies2. Engineered spectacularly Scaffold Protein exemplify-Based naturalImaging a ffiProbesnity: proteins Affibody capable Molecules of specific binding to a large variety of molecular motifs. Nevertheless, there is a broad repertoire of natural binding proteins, Antibodies spectacularly exemplify natural affinity proteins capable of specific binding to a large which might be utilized to develop novel affinity ligands. The key feature of such binders is the variety of molecular motifs. Nevertheless, there is a broad repertoire of natural binding proteins, presencewhich of amight robust be skeletonutilized to called develop a sca novelffold, affinity which ligands. ensures The the stablekey feature positioning of such ofbinders variable is the amino acidspresence and minimizes of a robust the skeleton entropy called penalty. a scaffold, Large which combinatorial ensures the libraries, stable positioning created by of the variable randomization amino of variableacids and amino minimizes acids, permitthe entropy the selectionpenalty. Large of proteins combinatorial binding librarie withs, a created high a ffibynity the randomi and specificityzation to desirableof variable molecular amino structures. acids, permit Such the binders selection are of calledproteins engineered binding with sca aff highold proteins affinity and (ESPs). specificity ESPs are increasinglyto desirable applied molecular as a molecular structures. recognition Such binders moiety are called for engineered targeting therapeutics scaffold proteins and (ESPs). are considered ESPs a complementare increasingly to or substitution applied as a of molecular monoclonal recognition antibodies. moiety We recommendfor targeting severaltherapeutics excellent and revieware papersconsidered for a more a detailedcomplement description to or substitution of ESPs [14 of– 17monoclonal]. antibodies. We recommend several Toexcellent the best review of our papers knowledge, for a more the detailed first type description of scaff oldof ESPs protein, [14–17 which]. was evaluated in vivo for To the best of our knowledge, the first type of scaffold protein, which was evaluated in vivo for radionuclide imaging, was that of affibody molecules [18,19]. Affibody molecules utilize a cysteine-free radionuclide imaging, was that of affibody molecules [18,19]. Affibody molecules utilize a cysteine- three-helical scaffold (Figure1) of a modified B-domain of protein A (58 amino acids, 7 kDa) [ 20]. free three-helical scaffold (Figure 1) of a modified B-domain of protein A (58 amino acids, 7 kDa) [20]. A combinatorialA combinator libraryial library has has been been created created by by the the randomization randomization of of thirteen thirteen amino amino acids acids located located on on heliceshelices one andone and two two and and normally normally involved involved in in thethe bindingbinding of of the the Fc Fc domain domain of ofimmunoglobulins. immunoglobulins. Phage-displayPhage-display selection selection enables enables the the generation generation of of a affibodyffibody molecules with with aff ainitiesffinities in inthe the nanomolar nanomolar rangerange [21]. [21]. Affi nityAffinity maturation maturation permits permits a a further further increaseincrease of the the affinity affinity from from a few a few nM nM to a to few a fewpM. pM. High-aHighffinity-affinity affibody affibody binders binders have have been been selected selected for potentialfor potential cancer-associated cancer-associated molecular molecular targets targets such as humansuch epidermalas human growthepidermal factor growt receptorh factor type receptor 2 (HER2) type [22 2], (HER2) epidermal [22], growth epidermal factor growth receptor factor (EGFR or HER1)receptor [23], (EGFR human or epidermalHER1) [23], growth human factorepidermal receptor growth type factor 3 (HER3) receptor [24 type], insulin-like 3 (HER3) [24], growth insulin factor-1- receptorlike (IGF-1R)growth factor [25],-1 platelet-derived receptor (IGF-1R) growth [25], platelet factor-derived receptor growthβ (PDGFR factor βreceptor)[26], vascular β (PDGFRβ) endothelial [26], vascular endothelial growth factor receptor 2 (VEGFR2) [27], programmed death-ligand 1 (PD-L1) growth factor receptor 2 (VEGFR2) [27], programmed death-ligand 1 (PD-L1) [28], and carbonic [28], and carbonic anhydrase IX (CAIX) [29]. anhydrase IX (CAIX) [29]. Figure 1. Structure of an affibody scaffold. Asterisks mark the segments where randomized

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