## 3 Metal-Based Protein, Peptide and Antibody Drug Conjugates
Usually, in bioconjugated complexes, BFC is coordinated to the radiometal and simultaneously bonded to the amino acid residue of the antibody. The most common BFCs for \({}^{64}\)Cu metal centre are 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), but recently, they have been declared unstable in vivo. In 2021, [PERSON] and collaborators optimized a new category of propylene cross-bridged BFCs with a PEG-based linker to conjugate trastuzumab antibody in \({}^{64}\)Cu complexes (**47** and **48**, Figure 9A) [94]. Both new platforms have shown in vivo stability and a higher uptake in HER2-positive cancer cell lines NIH3T6.7 and SKOV3 with respect to HER2-negative cells HEK293 and CT26 [94]. The strategy of self-assembling synthesis has been developed to prepare cationic [Pd\({}_{2}\)L\({}_{4}\)]\({}^{4+}\) metallacages tethered to a blood-brain barrier (BBB)-penetrating peptide, PepH3, in order to overcome the well-known difficulty of ensuring drugs reach their brain targets. Complex **49**, whose structure is depicted in Figure 9B, has been investigated by [PERSON], [PERSON] and [PERSON] using bEnd.3 cells as an in vitro model to evaluate the ability of the metallacages to translocate the BBB [95]. In addition, the radiolabelling of the cage with [99 mTc]\({}^{-}\), the purification of the resulting radioactive cage and the assessment of its brain penetration properties were investigated in vivo. It is noteworthy to mention that the water-soluble system can cross the BBB only once conjugated to PepH3 moiety, and therefore, the radiolabelled [99 mTc]\({}^{-}\) derivative presents a brain accumulation decidedly higher than that of the non-bioconjugated system [95]. Neuroendocrine tumours (NETs) are diagnosticated by imaging techniques such as PET and SPECT, which take advantage of the use of radiolabelled somatostatin analogues bearing radionuclides. Somatostatin receptors (SSTRs) are overexpressed in NETs, and they may be bound to the tracer that is able to internalize the agonist-receptor system into tumour cells. Radiabelled tracers are usually designed using a bifunctional chelator introduced via the peptide \(N\) terminus. More rarely, radiolabelled agents can be prepared via incorporation of the radiometal as an integral part of the peptide. The most employed radiometals are \({}^{99m}\)Tc and \({}^{186}\)Re. In 2021, [PERSON] and collaborators proposed the first \({}^{99m}\)Tc/\({}^{186}\)Re-cyclized SSTR antagonist peptide using the potent acetylated SSTR2 antagonist, Ac-4-NO\({}_{2}\)-Phe-c(\({}_{\text{p}}\)Cys-Tyr-pTrp-Lys-Thr-Cys)-DTyr-NH\({}_{2}\)[96]. The non-radioactive standard complex **50a**, depicted in Figure 9C, has demonstrated good in vitro binding affinity on SSTR2-expressing AR42] cells (IC\({}_{50}\) = 43 nM). Among the \({}^{186}\)Re/\({}^{99m}\)Tc radiotracers **50b**-**c**, only complex **50c** exhibited good in vitro stability, and for this reason, it is a promising agent for diagnosis [97]. The same authors have also proposed GRPR-specific [99 mTc/\({}^{186}\)Re]Tc/Re-tricarbonyl complexes using hydrophilic NOTA and NODAGA chelators conjugated via a 6-aminohexanoic acid linker (6-Ahx) to the powerful GRPR-targeting peptide antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH\({}_{2}\) (Figure 9D). A high hydrophilicity and excellent in vitro and in vivo stability were observed for all complexes **51-53a**-**c**. Moreover, a favourable pharmacokinetics in PC-3 murine tumour and a good receptor-mediated tumour uptake was noticed. Promising results have been reported by high-quality micro-SPECT/CT images for compound **53b**. The reasons for its effectiveness could be attributed to the charge of the complex  
Figure 9: (**A**) \({}^{64}\)Cu trastuzumab radiolabelled complexes; (**B**) Palladium metallacages; (**C,D**) Re and Tc complexes synthesized by Hennkens.