[⁶⁴Cu]-1,4,7,10-Tetra-azacyclododecane- N, N’, N’’, N’’’-tetraacetic acid conjugated knottin 2.5F

Integrin receptors that mediate tumor angiogenesis, growth, and metastasis through a complex network of signaling pathways are known to be expressed on the surface of cancerous tumor cells and neovasculature (1-3). Because of their role in tumor development and progression, inhibition of integrin receptor activity is being actively investigated in clinical trials for the treatment and imaging of various cancers (4, 5). Non-invasive imaging probes can be used to determine not only the efficacy of integrin-targeted anti-cancer drugs, but also to monitor disease progression and metastasis (6, 7). Although integrin receptors usually bind through the arginine-glycine-aspartic acid (RGD) motif of the extracellular matrix protein ligands, it is the amino acid residues surrounding the RGD motif that determine the receptor specificity and affinity for the ligand (4, 8, 9). As a result of the small size of the drugs, mainly peptides, that target the integrin receptor, it has been challenging to generate peptides containing the RGD motif that have an improved pharmacokinetic behavior, receptor affinity, and tumor uptake for in vivo imaging purposes. Any modification of the peptide structure has yielded integrin-targeted imaging agents that have only a limited advancement and application in the clinics (10, 11). Only one imaging compound with an RGD motif, a radioactive fluorine-labeled cyclic pentapeptide ([¹⁸F]-galacto-RGD), was determined to be suitable to identify integrin-positive tumors and to investigate α_vβ₃ integrin expression in humans. However, [¹⁸F]-galacto-RGD has been shown to have a low tumor uptake, and it generated a high background signal due to accumulation in the liver (12, 13).

In an effort to develop imaging agents for the detection of integrin receptors expressed on tumor cell surface and neovasculature, Kimura et al. (14) used the directed evolution technique (15, 16) to place the integrin-binding RGD motif into a cystine knot peptide (also known as knottin) trypsin inhibitor of the squash plant (Ecballium elaterium) (17). The peptide was reported to have a high affinity for the α_vβ₃ and α_vβ₅ or the α_vβ₃, α_vβ₅, and α₅β₁ integrin receptors. In general, knottins have a core structure containing a disulfide bond, are resistant to proteolysis, have a high thermodynamic stability, and are nonimmunogenic (14). The knottin peptide containing an RGD motif (designated as knottin 2.5F) was labeled with radioactive copper (⁶⁴Cu) to obtain [⁶⁴Cu]knottin 2.5F (⁶⁴Cu-1,4,7,10-tetra-azacyclododecane-N,N’,N’’,N’’’-tetraacetic acid-2.5F (⁶⁴Cu-DOTA-2.5F)) and used to image xenograft tumors in mice with positron emission tomography (PET). It is pertinent to mention here that the amino acid sequence and structure of knottins 2.5D and 2.5F (also studied by Kimura et. al. (14)) are discussed elsewhere (14).