The Cu-1,4,7,10-tetraazacyclododecane-1,4,7-β max tris(acetic acid)-10-acetate mono(-ethylmaleimide amide) (Mal-DOTA)-dimeric (Z) conjugate, abbreviated as Cu-DOTA-(Z), is an affibody derivative synthesized by Cheng et al. for positron emission tomography (PET) of HER2-expressing tumors (1). Affibody molecules are a group of nonimmunogenic scaffold proteins that derive from the B-domain of staphylococcal surface protein A (2, 3). In the past several years, affibodies have drawn significant attention for developing imaging and therapeutic agents because of their unique features (3, 4). First, affibodies are small, with only 58 amino acid residues (~7 kDa) (3, 5). The small size allows affibodies to be generated with solid-phase peptide synthesis and to be cleared quickly from kidneys. Second, affibodies have a high binding affinity and specificity to their targets. Their binding affinity can be further improved by generating multimeric constructs through the solvent-exposed termini of affibody Z-domain. The anti-HER2 monomeric affibody Z is an example that has a binding affinity of ~50 nM, but its dimeric form, (Z), exhibits an improved binding affinity up to ~3 nM (6). Third, affibodies lack cysteine residues and disulfide bridges in structure, and they fold rapidly. These features make it possible to chemically synthesize fully functional molecules and to introduce unique cysteine residues or chemical groups into affibodies for site-specific labeling. Several anti-HER2 affibody derivatives have been synthesized in this way. The imaging agent HPEM-His-(Z)-Cys was generated by radiobrominating the dimeric (Z) through the cysteine residues that were introduced to the C-terminus of (Z) (7). Several affibody derivatives (e.g., Ga-DOTA-Z, In-DOTA-Z, In-benzyl-DOTA-Z, and In-benzyl-DTPA-Z) were synthesized by coupling a chelating agent with a specifically protected site group of the Z peptide chain (3). Furthermore, these small affibody proteins can be selected and optimized with a strategy of sequence mutation and affinity maturation, and an example selected with this strategy is the anti-HER2 affibody Z, which has an increased affinity from 50 nM to 22 pM (8). The investigators at the Stanford University first tested the feasibility of the monomeric and dimeric forms of affibody Z for molecular imaging. Z is a commercially available anti-HER2 affibody. Both forms of the Z molecule were radiofluorinated with an F-labeled prosthetic group of 4-F-fluorobenzaldehyde (F-FBO-Z and F-FBO-(Z), respectively) (9). The investigators have also coupled Cu to the affibody through DOTA, leading to the development of imaging agents of Cu-DOTA- Z and Cu-DOTA-(Z) (1). Interestingly, these studies showed that smaller affibody constructs performed better in terms of tumor uptake and clearance. The investigators then generated a class of small proteins consisting of two α-helix bundles of the 3-helix affibody by deleting the helix 3 because the binding domain localizes in the α-helices 1 and 2 bundles (5). One of these 2-helix proteins is MUT-DS, which has α-helices 1 and 2 bundles, with a disulfide bridge being formed between the two inserted homocysteines (10-12). The helix conformation of MUT-DS has been shown to be improved with the placement of a disulfide bridge. MUT-DS showed a binding affinity to HER2 in the low-nM range. The radiolabeled MUT-DS derivatives exhibited favorable pharmacokinetics for both imaging and therapy of HER2-expressing tumors. This series of chapters summarizes the data obtained with the Z derivatives, and this chapter presents the data obtained with Cu-DOTA-(Z) (1).