In recent years, numerous studies have examined the selection of Affibody proteins for molecular targeting. Currently, phage display technology has been introduced for all reported de novo selections of Affibody-binding proteins [12]. This technology was based on selection protocols and standard phage mid vectors. Other selection systems are also being investigated as alternative methods, including staphylococcal display, ribosomal display, microbead display, and b-lactamase-based protein fragment complementation [13]. The affinities obtained from Affibody proteins for different targets are similar to the affinities of antibody fragments or alternative scaffolds from similar native libraries. Different target structure characteristics and library complexity resulted in different dissociation constants (K_D), ranging from micromolar to low picomolar. For example, the highest affinity for an Affibody target to the breast cancer marker ErbB2 was recently reported as only 22 pm [14]. The epidermal growth factor receptor (EGFR)-binding Affibody molecules with moderate affinity have also been isolated using phase-display selection technology. A study found that the molecules bind specifically to EGFR on EGFR-expressing cells in biosensors [15, 16]. Protein scaffolds can be prepared using either chemical or biological methods. For some small protein scaffolds, both methods can be used. After display selection, high-affinity scaffold molecules can be well established from the bulk of regular DNA templates. These DNA templates are produced using regular recombinant techniques. The high-affinity molecules exhibited high specificity and targeting capacity in protein engineering. For example, Affibodies can be prepared in E. coli, which makes the procedure easy and inexpensive compared with antibody production [17, 18]. In purification using a nickel column, a His-6 tag is typically added to the sequence of a scaffold protein when it is denatured [19]. This added His-6 can not only help purification but also achieve site-specific labeling with radionuclide ^99mTc. More complicated post-transcription modifications can be either a disadvantage or advantage as is the case with glycosylation [19].

Small Affibody molecules can also be prepared directly by solid-phase peptide synthesis (SPPS), which is a desirable way to produce Affibody molecules with function groups for site-specifically labeling with a variety of reporters such as fluorophores, radionuclides, and nanoparticles [20, 21]. Synthesis of Affibodies using SPPS provides the freedom to label the protein with more than one reporter, and it facilitates the isolation of a homogeneous product when compared to recombinant production [21]. Furthermore, this approach could avoid the biological contaminants which largely exist in recombinant production. However, a limitation on this chemical approach is that the synthetic efficiency highly depends on the sequence and length of the Affibody proteins [22]. But the yield in organic synthesis of small proteins could be improved using a approach which involves the parallel synthesis of fragments and then assembling of them [23]. Importantly, it is feasible to perform chemical synthesis of the scaffold proteins without affecting the active amino acids or other functional group (Fig. 12.2) [20]. Manual modifications have been extensively applied to the synthetic Affibody proteins. For example, the resin-bound proteins have been manually modified by the introduction of thiol and biotin groups for surface immobilization (Fig. 12.2) [20].

For the promotion the applications of Affibody, in addition to SPPS, combinatorial assembled Affibody molecules have been designed and prepared by native chemical ligation (NCL) [21, 22]. In Affibody molecules, the amino acids located in the N-terminal part of the protein (helices 1 and 2) determine the target-binding ability and specificity, whereas the C-terminal part (the third helix and its flanking regions) is preserved in all variants [24]. The distinct division of the protein sequence between variable and constant segment makes Affibody high tolerance toward scaffold modification. And then it makes a fragment condensation strategy very appealing. Thus, the concept of combinatorial assembling has been introduced for the synthesis of three different Affibody molecules. Specifically, the helice 3 of Affibody containing a cysteine residue located at the site of ligation is prepared and can be used for directed immobilization, and it has no unintentional interference to the function groups of the investigated proteins. Moreover, different C-terminal modifications to helice 3 are performed to improve yield in preparation of Affibody proteins [24]. Three different 2-helix Affibodies with specificity for IgG, insulin, or human epidermal growth factor receptor 2 (HER2) can then be coupled with helice 3 by NCL to produce the full Affibodies (Fig. 12.3) [23].

The most widely used molecular imaging agents in translational medicine are based on the radiolabeled molecules. It is very important to use appropriate labeling chemistry and find suitable radionuclides when developing protein based imaging probes or radiotherapeutics. Labeling chemistry, chosen for different radionuclides, is one of the most important factors in the successful engineering of new diagnostic or therapeutic proteins. It plays a substantial role in efficient radiolabeling and maintains the bioactivity of the molecules. For example, radionuclides can be used to label proteins and molecules directly by in situ oxidation [25–27]. This method is straightforward and typically results in efficient labeling. However, the function groups such as tyrosines may be critical for binding, and the directly labeling could results in low-binding affinity and targeting efficiency [26–28]. In some reports, anti-HER2 Affibody molecules labeled with radionuclides directly indeed show a loss of binding capacity in relation to the tyrosine modification in binding sites [25]. The inactivation of the HER2 Affibody molecule could be prevented using indirect labeling methods, such as site-specific modification with chelator or other moieties. Indirect labeling methods can consequently avoid the direct oxidization of protein and reduce the modification to the active amino groups of lysines and cisterns [25, 29]. Radionuclides selected for diagnosis and therapy highly depend on the properties of the targeting agent and its application. Physical half-life, the energy type of the emitted radiation, and the availability and cost of the nuclide must be considered. In addition, the requirements for matching the binder’s biological properties and targeting mechanisms with radionuclide’s physical properties should be considered. Different applications also require different radiation. For example, ¹⁸F, ¹¹C, ¹³N, and ¹⁵O are the most common radionuclides for PET, whereas ^99mTc and ¹¹¹In are most often used for single-photon emission computed tomography (SPECT) [30, 31]. In these radionuclides, the physical half-lives of ¹¹C, ¹³N, and ¹⁵O are too short to label peptides and proteins. Radionuclides with relatively longer half-lives, such as ⁶⁸Ga and ⁸⁹Zr, are used for PET imaging for targeted peptide or protein radiopharmaceuticals.

The cellular uptake and retention of radiolabeled molecules by cells and excretory organs are important to consider for the optimal design of an imaging probe. Different radionuclides (such as ¹²⁵I and ¹¹¹In) could impact targeting efficacy, imaging contrast, cellular protein degradation, and catabolism [30, 31]. For radio halogen-labeled biomolecules, after lysosomal degradation radionuclide catabolites are often quickly released from cells as uncharged lipophilic molecules. In contrast, radionuclides trapped in chelators are positively charged and typically hydrophilic and stay in cytoplasm for a long time. In addition, catabolism may occur in blood, excretory organs and even in tumor sites. As a result, the radio catabolites could re-enter the circulation and be redistributed [32]. This non-specific radioactivity would contribute to a high image background. Thus, the properties of radio catabolites should be considered when selecting a radionuclide for translational medicine engineering. Depending on their chemical nature, radio catabolites accumulated in the liver may be trapped inside hepatocytes or excreted into bile. This hepatobiliary excretion results in an increased background in the lower abdomen. Consequently, the behavior of a radiolabeled probe in excretory organs plays an important role in clinical performance and contributes to different imaging and therapy applications [33]. Increased uptake in the excretory organs generally indicates an undesirable elevated intake in healthy tissues. It has also been shown those labeled molecules smaller than 60 kDa are associated with high tumor retention of radioactivity but accompanied by a high accumulation of radioactivity in kidneys, because 60 kDa is the cutoff for glomerular filtration, and smaller proteins are mostly reabsorbed by the kidney [34, 35].